**Sex Hormones and Infertility**

Iptisam Ipek Muderris and Gokalp Oner *Erciyes University / Department of Obstetric and Gynecology Turkey* 

#### **1. Introduction**

80 Sex Hormones

Slabbekoorn, D., van Goozen, S.H.M., Megens, J., Gppren, L.J.G., & Cohen-Kettenis, P. T. (1999).

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Tan, U. Tan, M., Polat, P., et al. (1999). Magnetic resonance imaging brainsize/IQ relations in

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Tierney, M.C. (2000). Oestradiol concentrations in prediction of cognitive decline in women.

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Van Goozen, S.H.M., Cohen-Kettenis, P.T., Gooren, L.J.G. et al. (1995). Gender differences in

Wolf, O.T., Preut, R., Hellhammer, D.H. et al., (2000). Testosterone and cognition in elderly

Wolf, O.T. (2003). Cogntive functions and sex steroids. *Annales d' endocrinologie*, Vol. 64, pp.

Wolf, O.T. & Kirschbaum, C. (2002). Endogenous estradiol and testosterone levels are

Yaffe, K., Lui, L-Y., Grady, D. , et al. (2000). Cognitive decline in women in relation to nonprotein-bound oestradiol concentrations. *Lancet*, Vol. 356, pp. 708-712. Yaffe, K., Haan, M., Byers, A., Tangen, C., & Kuller, L. (2000). Estrogen use, APOE, and

Yaffe, K., Lui, L.Y., Zmuda, J. & Cauley, J. (2002). Sex hormones and cognitive function in older men. *Journal of American Geriatry Society*, Vol. 50, No. 4, pp. 707-712.

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transsexuals. *Neuropsychologia,* Vol. 32, No. 10, pp. 1153-1157.

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The Lancet, Vol. 356, pp. 694-695.

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1949-1953.

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and hand skill in right-handed young adults. *International Journal of Neuroscience,*

and serum testosterone level in young men. *International Journal of Neuroscience,*

intelligence in men and women. *International Journal of Neuroscience*, Vol. 95, pp. 77-83.

speed, and serum testosterone level in left-handed male subjects. *International* 

ability reconsidered in relation to body size, lung volume, and sex hormones.

androgens on cognitive performance: causal evidence in a group of female-to-male

behavior: activating effects of cross-sex hormones. *Psychoneuroendocrinology,* Vol.

men: a single testosterone injection blocks the practice effect in verbal fluency, but has no effecton spatial or verbal memory. *Biological Psychiatry,* Vol. 47, No. 1, pp. 650-654.

associated with cognitive performance in older women and men. *Hormone and* 

cognitive decline. Evidence of gene-environment interaction. *Neurology*, Vol. 54, pp.

The normal physiology of the female reproductive system involves a hypothalamus that secretes gonadotropin-releasing hormone (GnRH)in a pulsatile manner, a pituitary gland that can be stimulated by the hypothalamus to regularly secrete both luteinizing hormone (LH) and follicle-stimulating hormone (FSH), an ovary that has both methodical enzymatic system and steroidogenesis for producing the sex hormones such as estrogen and progesteron, and a functional uterus that can be responded by these hormones.

Sex hormones play a crucial role in reproductive biology as well as in general physiology. The most important aim of sex hormones is to design the cycle and to produce an optimal environment for pregnancy according to form ovarian physiology including follicular growth, ovulation, and corpus luteum formation and endometrial response including proliferative and secretuar phase for implantation. Among the various functions, sex hormones influence pregnancy, cardiovascular function, bone metabolism, and an individual's sense of general well-being. The action of sex hormones is mediated via extracellular signals to the nucleus to affect a physiologic response.

#### **2. Gonadotropin-releasing hormone (GnRH)**

Gonadotropin-releasing hormone (GnRH) is a decapeptide pulsatile produced by neurons with cell bodies primarily in the arcuate nucleus of the hypothalamus (1). Embryologically, these neurons originate from the olfactory area and then migrate to their adult locations (2). These GnRH-secreting neurons project axons that terminate on the portal vessels at the median eminence where GnRH is secreted for delivery to the anterior pituitary. The continual pulsatile secretion of GnRH is necessary because its short half-life is only 2–4 minutes as a result of rapid proteolytic cleavage.

GnRH stimulates the production, secretion and storage of FSH and LH from anterior hypophysis. (3). It is also an unique releasing hormone for the regulation of the simultaneous secretion of two hormones in human body (4). GnRH performs this special affect according to its pulsatile secretion. In the follicular phase, its secretion is characterized by frequent, small-amplitude pulses, however during the luteal phase, there is a progressive lengthening of the interval between pulses with higher amplitude (5).

GnRH is primarily involved in endocrine regulation of gonadotropin secretion from the pituitary. However, the regulation of GnRH secretion is various (Table 1). The pulsatile secretion of GnRH is directly affected by catecholaminergic system including the activator of

Sex Hormones and Infertility 83

complications. Most importantly, clinical pregnancy rates and live birth rates per retrieval were significantly higher using the long protocol. The benefits including higher pregnancy rates and lower OHSS rates of using the long protocol for administration of GnRH agonists greatly outweigh its disadvantages, which include daily administration, increased requirement for gonadotropins, and an overall increase in the cost of medication (10, 11).

GnRH antagonists produce a competitive blockage of GnRH receptors, preventing stimulation by endogenous GnRH and causing an immediate fall in gonadotropin and sex steroid secretion. The clinical effect is generally observed within 24 to 72 hours. Moreover, antagonists may not show flare-up affect comparing with GnRH agonists. GnRH antagonists are also used in ART for the prevention of premature ovulation and displays similar efficacy comparing GnRH agonist (long protocol) (12). However, there were significantly fewer pregnancies with the GnRH antagonist protocol. A significant reduction in the incidence of severe OHSS and the number of gonadotropin injections were observed

in the antagonist regimen compared with the long GnRH–agonist protocol.


When we searched the Cochrane Library and ACOG Commitee on Practice Bulletins.;

Usage of these agents is generally limited to 6 months because of the adverse effects as listed above. The most important side effect is osteoporosis. Many side effects can be minimized by providing add-back therapy in addition to the agents.The addition of 2.5 mg of norethindrone or 0.625 mg of conjugated estrogens with 5 mg/d of medroxyprogesterone acetate seems to relieve these side effects of GnRH analogs. The addition of 5 mg daily of norethindrone acetate alone or in conjunction with low-dose conjugated equine estrogen

The gonadotropins FSH and LH are produced by the anterior pituitary gonadotroph cells and are responsible for ovarian follicular stimulation. Structurally, there is great similarity between FSH and LH. They are both glycoproteins that share identical α subunits and differ only in the structure of their β subunits, which confer receptor specificity (14). The synthesis

seems to eliminate the loss of bone mineral density effectively as well (13).

**3.2 GnRH antagonists (cetrorelix, ganirelix)** 

Clinically usage of GnRH analogs listed below;




**4. Gonadotropins** 





Table 1. The control of GnRH secretion

norepinephrine and the inhibitor of dopamin. This system is basically regulated by endogenous opioids (6).

These opioids are three groups;


Endogenous opioids, inhibit the gonadotropin secretion to swage the GnRH secretion from hypothalamus.

Sex steroids affect GnRH by increasing the secretion of the endogenous opioids in the central nervous system (7).

Although estrogen stimulates the secretion of endogenous opioids, estrogen plus progesterone increase this effect. Clinically, increased endogenous opioids may cause hypothalamic amenorrhea.

#### **3. GnRH analogs**

#### **3.1 GnRH agonists (leuprolide, goserelin, nafarelin, buserelin)**

GnRH agonists are modifications of the native molecule to either increase receptor affinity or decrease degradation (8). The pulsatile usage of GnRH agonists that resemble endogenous GnRH leads to increase the secretion of FSH and LH. However the constant GnRH usage leads to suppression of gonadotropin secretion by the downregulation of its receptor. An initial release of gonadotropins is followed by a profound suppression of secretion. The initial release of gonadotropins represents the secretion of pituitary stores in response to receptor binding and activation. With continued activation of the gonadotroph GnRH receptor, however, there is a downregulation effect and a decrease in the concentration of GnRH receptors. As a result, gonadotropin secretion decreases and sex steroid production falls to castrate levels (9).

The most commonly used regimen for superovulation in ART is called the long, or luteal, downregulation protocol. In this protocol, GnRH agonist is started in the luteal phase (day 21) of the previous cycle, which minimizes its flare effect and prevents the follicular recruitment that is thought to begin in the luteal phase. The couple undergoing treatment is advised to abstain from intercourse during the cycle before the start of COH; however, concomitant use of GnRH agonist in the presence of an unsuspected pregnancy has not been reported to be associated with increased spontaneous abortion, congenital abnormalities, or pregnancy

norepinephrine and the inhibitor of dopamin. This system is basically regulated by

Endogenous opioids, inhibit the gonadotropin secretion to swage the GnRH secretion from

Sex steroids affect GnRH by increasing the secretion of the endogenous opioids in the

Although estrogen stimulates the secretion of endogenous opioids, estrogen plus progesterone increase this effect. Clinically, increased endogenous opioids may cause

GnRH agonists are modifications of the native molecule to either increase receptor affinity or decrease degradation (8). The pulsatile usage of GnRH agonists that resemble endogenous GnRH leads to increase the secretion of FSH and LH. However the constant GnRH usage leads to suppression of gonadotropin secretion by the downregulation of its receptor. An initial release of gonadotropins is followed by a profound suppression of secretion. The initial release of gonadotropins represents the secretion of pituitary stores in response to receptor binding and activation. With continued activation of the gonadotroph GnRH receptor, however, there is a downregulation effect and a decrease in the concentration of GnRH receptors. As a result, gonadotropin secretion decreases and sex

The most commonly used regimen for superovulation in ART is called the long, or luteal, downregulation protocol. In this protocol, GnRH agonist is started in the luteal phase (day 21) of the previous cycle, which minimizes its flare effect and prevents the follicular recruitment that is thought to begin in the luteal phase. The couple undergoing treatment is advised to abstain from intercourse during the cycle before the start of COH; however, concomitant use of GnRH agonist in the presence of an unsuspected pregnancy has not been reported to be associated with increased spontaneous abortion, congenital abnormalities, or pregnancy

**3.1 GnRH agonists (leuprolide, goserelin, nafarelin, buserelin)** 

**Inhibitors of GnRH secretion Activators of GnRH secretion** 

Dopamine Norepinephrin Gonadotrophins (negative feedback) Catecholamins Endogenous opioids Neuropeptide Y Estradiol Acetylcholine

Progesterone VIP CRH Naloxone

Table 1. The control of GnRH secretion

endogenous opioids (6).

central nervous system (7).

hypothalamic amenorrhea.

steroid production falls to castrate levels (9).

**3. GnRH analogs** 

1. Endorphins 2. Enkephalins 3. Dynorphins

hypothalamus.

These opioids are three groups;

Melatonin Serotonin GABA

complications. Most importantly, clinical pregnancy rates and live birth rates per retrieval were significantly higher using the long protocol. The benefits including higher pregnancy rates and lower OHSS rates of using the long protocol for administration of GnRH agonists greatly outweigh its disadvantages, which include daily administration, increased requirement for gonadotropins, and an overall increase in the cost of medication (10, 11).

#### **3.2 GnRH antagonists (cetrorelix, ganirelix)**

GnRH antagonists produce a competitive blockage of GnRH receptors, preventing stimulation by endogenous GnRH and causing an immediate fall in gonadotropin and sex steroid secretion. The clinical effect is generally observed within 24 to 72 hours. Moreover, antagonists may not show flare-up affect comparing with GnRH agonists. GnRH antagonists are also used in ART for the prevention of premature ovulation and displays similar efficacy comparing GnRH agonist (long protocol) (12). However, there were significantly fewer pregnancies with the GnRH antagonist protocol. A significant reduction in the incidence of severe OHSS and the number of gonadotropin injections were observed in the antagonist regimen compared with the long GnRH–agonist protocol.

When we searched the Cochrane Library and ACOG Commitee on Practice Bulletins.; Clinically usage of GnRH analogs listed below;


Side effects of GnRH analogs listed below;


Usage of these agents is generally limited to 6 months because of the adverse effects as listed above. The most important side effect is osteoporosis. Many side effects can be minimized by providing add-back therapy in addition to the agents.The addition of 2.5 mg of norethindrone or 0.625 mg of conjugated estrogens with 5 mg/d of medroxyprogesterone acetate seems to relieve these side effects of GnRH analogs. The addition of 5 mg daily of norethindrone acetate alone or in conjunction with low-dose conjugated equine estrogen seems to eliminate the loss of bone mineral density effectively as well (13).

#### **4. Gonadotropins**

The gonadotropins FSH and LH are produced by the anterior pituitary gonadotroph cells and are responsible for ovarian follicular stimulation. Structurally, there is great similarity between FSH and LH. They are both glycoproteins that share identical α subunits and differ only in the structure of their β subunits, which confer receptor specificity (14). The synthesis

activin.

**6.1 IGF** 

production

free IGF.

growth factor.

Sex Hormones and Infertility 85

affects of activin is inhibited by inhibin and follistatin. Follistatin that secretes from granulosa cells and the basophilic cells of hypophysis inhibits FSH activity by binding

IGF plays role in; the stimulation of LH activity on theca cells to increase androgen

All of IGF binds insulin like growth factor binding protein (IGF-BP) in the circulation. FSH and insulin inhibit the synthesis of IGF-BP, so efficacy of IGF may increase by increasing

Epidermal growth factor (EGF) that is an important inhibitor of FSH in the ovary is another

Sex steroid hormones are synthesized in the gonads, adrenal gland, and placenta. Cholesterol is the primary building block in steroidogenesis, and all steroid-producing tissues except the placenta are capable of synthesizing cholesterol from the 2-carbon precursor, acetate. Steroid hormone production, which involves at least 17 enzymes, primarily occurs in the abundant

Steroids are metabolized mainly in the liver and to a lesser extent in the kidney and intestinal mucosa. Accordingly, administration of certain pharmacologic steroid hormones may be contraindicated in those with active liver disease. Sex steroids are divided into three groups based on the number of carbon atoms that they contain. Each carbon in this structure is assigned a number identifier, and each ring is assigned a letter. The 21-carbon series includes progestins as well as glucocorticoids and mineralocorticoids. Androgens contain 19 carbons, whereas estrogens have 18. However the ovary is deficient in 21-hydroxylase and 11 -hydroxylase and therefore is unable to produce corticosteroids. Most important steroidogenic enzymes are listed in Table 2. Steroidogenesis is summarized in Figure 1.

IGF secretes from granulosa cells and theca cells. Its affect is similar to GH.




**7. Steroid hormones in reproduction** 

Table 2. Steroidogenic enzymes

smooth endoplasmic reticulum found in steroidogenic cells.

**Enzyme Cellular Location Reactions** 

P450scc Mitochondria Cholesterol side chain

P450c11 Mitochondria 11-Hydroxylase

P450c17 Endoplasmic reticulum 17-Hydroxylase

P450c21 Endoplasmic reticulum 21-Hydroxylase P450arom Endoplasmic reticulum Aromatase

cleavage

18-Hydroxylase 19-Methyloxidase

17, 20-Lyase

of the β subunits is the rate-regulating step in gonadotropin biosynthesis (Lalloz MRA, et al. GnRH desensitization preferentially inhibits expression of the LH β-subunit gene in vivo. Endocrinology 1988;122:1689–1694.). Thyroid-stimulating hormone and placental human chorionic gonadotropin (hCG) also share identical α subunits with the gonadotropins. The structural similarity between FSH, LH, TSH and hCG defines as the α subunits identical and the β subunits differ.

The gonadotropins were metabolized in liver and kidney then excreted by the way of urine. The half life of LH, FSH and hCG is 20 minute, 3-4 hours and 24-36 hours, respectively.

#### **4.1 FSH**

Receptors of FSH are found on granulosa cells.

FSH plays role in; granulosa cell proliferation in follicules and estrogen production


In the beginning of follicular development, there is no LH receptor on granulosa cells, however, during the 11-12nd days of cycle, FSH stimulates the production of LH receptors on granulosa cells.

#### **4.2 LH**

Receptors of LH are found on theca cells.

LH plays role in; internal thecal cell proliferation in follicules and androgen production


Although FSH plays an important role for the early maturation of follicules, FSH and LH are responsible together for the maturation of follicules before the ovulation.

#### **5. Inhibin**

Inhibin secretes from granulosa cells, sertoli cells, placenta and the basophilic cells of hypophysis. In the cycle, Inhibin selectively inhibits the secretion of FSH. There are two forms; Inhibin A and Inhibin B. The affect of Inhibin B mostly shows on follicular phase, but the affect of Inhibin A mostly shows on luteal phase of cycle. During luteofollicular transition of cycle FSH increase by decreasing of Inhibin A levels. Inhibin also stimulates LH activity and IGF secretion from granulosa and theca cells to increase androgen production.

#### **6. Activin**

Activin secretes from granulosa cells and the basophilic cells of hypophysis. In the cycle, Activin selectively activates the secretion of FSH. Therefore, it activates all affects of FSH on granulosa cells. Activin also inhibits LH activity, androgen production from theca cells and progesterone production from granulosa cells. Additionally, activin inhibits the secretion of IGF from ovary and the secretion of prolactin, ACTH, and GH from hypophysis. These affects of activin is inhibited by inhibin and follistatin. Follistatin that secretes from granulosa cells and the basophilic cells of hypophysis inhibits FSH activity by binding activin.

#### **6.1 IGF**

84 Sex Hormones

of the β subunits is the rate-regulating step in gonadotropin biosynthesis (Lalloz MRA, et al. GnRH desensitization preferentially inhibits expression of the LH β-subunit gene in vivo. Endocrinology 1988;122:1689–1694.). Thyroid-stimulating hormone and placental human chorionic gonadotropin (hCG) also share identical α subunits with the gonadotropins. The structural similarity between FSH, LH, TSH and hCG defines as the α subunits identical and

The gonadotropins were metabolized in liver and kidney then excreted by the way of urine. The half life of LH, FSH and hCG is 20 minute, 3-4 hours and 24-36 hours, respectively.

In the beginning of follicular development, there is no LH receptor on granulosa cells, however, during the 11-12nd days of cycle, FSH stimulates the production of LH receptors

LH plays role in; internal thecal cell proliferation in follicules and androgen production - luteinization and the production of progesterone when LH receptors found on

Although FSH plays an important role for the early maturation of follicules, FSH and LH are

Inhibin secretes from granulosa cells, sertoli cells, placenta and the basophilic cells of hypophysis. In the cycle, Inhibin selectively inhibits the secretion of FSH. There are two forms; Inhibin A and Inhibin B. The affect of Inhibin B mostly shows on follicular phase, but the affect of Inhibin A mostly shows on luteal phase of cycle. During luteofollicular transition of cycle FSH increase by decreasing of Inhibin A levels. Inhibin also stimulates LH activity and IGF secretion from granulosa and theca cells to increase androgen production.

Activin secretes from granulosa cells and the basophilic cells of hypophysis. In the cycle, Activin selectively activates the secretion of FSH. Therefore, it activates all affects of FSH on granulosa cells. Activin also inhibits LH activity, androgen production from theca cells and progesterone production from granulosa cells. Additionally, activin inhibits the secretion of IGF from ovary and the secretion of prolactin, ACTH, and GH from hypophysis. These

FSH plays role in; granulosa cell proliferation in follicules and estrogen production

the β subunits differ.

Receptors of FSH are found on granulosa cells.

Receptors of LH are found on theca cells.





responsible together for the maturation of follicules before the ovulation.


**4.1 FSH** 


**4.2 LH** 

on granulosa cells.



**5. Inhibin** 

**6. Activin** 

IGF secretes from granulosa cells and theca cells. Its affect is similar to GH.

IGF plays role in; the stimulation of LH activity on theca cells to increase androgen production


All of IGF binds insulin like growth factor binding protein (IGF-BP) in the circulation. FSH and insulin inhibit the synthesis of IGF-BP, so efficacy of IGF may increase by increasing free IGF.

Epidermal growth factor (EGF) that is an important inhibitor of FSH in the ovary is another growth factor.

### **7. Steroid hormones in reproduction**

Sex steroid hormones are synthesized in the gonads, adrenal gland, and placenta. Cholesterol is the primary building block in steroidogenesis, and all steroid-producing tissues except the placenta are capable of synthesizing cholesterol from the 2-carbon precursor, acetate. Steroid hormone production, which involves at least 17 enzymes, primarily occurs in the abundant smooth endoplasmic reticulum found in steroidogenic cells.

Steroids are metabolized mainly in the liver and to a lesser extent in the kidney and intestinal mucosa. Accordingly, administration of certain pharmacologic steroid hormones may be contraindicated in those with active liver disease. Sex steroids are divided into three groups based on the number of carbon atoms that they contain. Each carbon in this structure is assigned a number identifier, and each ring is assigned a letter. The 21-carbon series includes progestins as well as glucocorticoids and mineralocorticoids. Androgens contain 19 carbons, whereas estrogens have 18. However the ovary is deficient in 21-hydroxylase and 11 -hydroxylase and therefore is unable to produce corticosteroids. Most important steroidogenic enzymes are listed in Table 2. Steroidogenesis is summarized in Figure 1.


Table 2. Steroidogenic enzymes

Sex Hormones and Infertility 87

theory of ovarian steroidogenesis explains that estrogen biosynthesis requires the combined action of two gonadotropins (LH and FSH) on two cell types (theca and granulosa cells) (15). Until the late antral stage of follicular development, LH-receptor expression is limited to the thecal compartment and FSH-receptor expression is limited to the granulosa cells. Theca cells express all of the genes needed to produce androstenedione. This includes high levels of CYP17 gene expression, whose enzyme product catalyzes 17-hydroxylationation the ratelimiting step in the conversion of progesterones to androgens (16). This enzyme is absent in the granulosa cells, so they are incapable of producing the precursor needed to produce estrogens by themselves. Granulosa cells therefore rely on the theca cells as their primary source for estrogen precursors. In response to LH stimulation, theca cells synthesize the androgens, androstenedione and testosterone. These androgens are secreted into the extracellular fluid and diffuse across the basement membrane to the granulosa cells to provide precursors for estrogen production. In contrast to theca cells, granulosa cells express high levels of aromatase activity in response to FSH stimulation. Thus, these cells efficiently convert androgens to estrogens, primarily the potent estrogen, estradiol. In sum, ovarian steroidogenesis is dependent on the effects of LH and FSH acting independently on the

theca cells and granulosa cells, respectively.

Fig. 2. Estrogen biosynthesis

Estrogen is metabolized in liver and excreted to bile.

Fig. 1. The steps of the steroidogenesis pathway

Most steroids in the peripheral circulation are bound to carrier proteins, either specific proteins such as sex-hormone binding globulin (SHBG) or corticosteroid-binding globulin, or to nonspecific proteins such as albumin. Only 1 to 2 percent of androgens and estrogens are unbound or free. Tablo 3 shows the steroid transformations. Levels of SHBG are increased by hyperthyroidism, pregnancy, and estrogen administration. In contrast, androgens, progestins, GH, insulin, and corticoids decrease SHBG levels.

#### **8. Estrogens**

Steroids with 18 C classified as;

Estron (E1): Poor estrogenic affect, basically peripheric estrogen are the properties. It is dominant estrogen in prepubertal and postmenopausal periods.

Estradiol (E2): The most potent estrogen is mainly produced in the reproductive age.

Estratriol (E3): The least potent estrogen is mainly produced in pregnancy and synthesized by maternal and fetal units together. Therefore, E3 is an indicator to show the normal fetoplacental unit.

Estetrol (E4): There is not estrogenic affect. It is synthesized from fetal liver and increases in term.

Estrogens are produces by the aromatization of androstenedione and testosterone in ovary and the peripheral aromatization of androstenedione in skin, fat tissue, muscle and endometrium (Figure 2). There is a two-cell theory of ovarian steroidogenesis. The two-cell

Most steroids in the peripheral circulation are bound to carrier proteins, either specific proteins such as sex-hormone binding globulin (SHBG) or corticosteroid-binding globulin, or to nonspecific proteins such as albumin. Only 1 to 2 percent of androgens and estrogens are unbound or free. Tablo 3 shows the steroid transformations. Levels of SHBG are increased by hyperthyroidism, pregnancy, and estrogen administration. In contrast,

Estron (E1): Poor estrogenic affect, basically peripheric estrogen are the properties. It is

Estratriol (E3): The least potent estrogen is mainly produced in pregnancy and synthesized by maternal and fetal units together. Therefore, E3 is an indicator to show the normal

Estetrol (E4): There is not estrogenic affect. It is synthesized from fetal liver and increases in

Estrogens are produces by the aromatization of androstenedione and testosterone in ovary and the peripheral aromatization of androstenedione in skin, fat tissue, muscle and endometrium (Figure 2). There is a two-cell theory of ovarian steroidogenesis. The two-cell

Estradiol (E2): The most potent estrogen is mainly produced in the reproductive age.

androgens, progestins, GH, insulin, and corticoids decrease SHBG levels.

dominant estrogen in prepubertal and postmenopausal periods.

Fig. 1. The steps of the steroidogenesis pathway

**8. Estrogens** 

fetoplacental unit.

term.

Steroids with 18 C classified as;

theory of ovarian steroidogenesis explains that estrogen biosynthesis requires the combined action of two gonadotropins (LH and FSH) on two cell types (theca and granulosa cells) (15). Until the late antral stage of follicular development, LH-receptor expression is limited to the thecal compartment and FSH-receptor expression is limited to the granulosa cells. Theca cells express all of the genes needed to produce androstenedione. This includes high levels of CYP17 gene expression, whose enzyme product catalyzes 17-hydroxylationation the ratelimiting step in the conversion of progesterones to androgens (16). This enzyme is absent in the granulosa cells, so they are incapable of producing the precursor needed to produce estrogens by themselves. Granulosa cells therefore rely on the theca cells as their primary source for estrogen precursors. In response to LH stimulation, theca cells synthesize the androgens, androstenedione and testosterone. These androgens are secreted into the extracellular fluid and diffuse across the basement membrane to the granulosa cells to provide precursors for estrogen production. In contrast to theca cells, granulosa cells express high levels of aromatase activity in response to FSH stimulation. Thus, these cells efficiently convert androgens to estrogens, primarily the potent estrogen, estradiol. In sum, ovarian steroidogenesis is dependent on the effects of LH and FSH acting independently on the theca cells and granulosa cells, respectively.

Fig. 2. Estrogen biosynthesis

Estrogen is metabolized in liver and excreted to bile.

Sex Hormones and Infertility 89

include, for example, rapid activation of the adenylate cyclase, which results in cyclic adenosine monophosphate (cAMP)–dependent activation of protein kinase A (PKA). Estrogens can also stimulate the mitogen-activated protein kinase (MAPK) pathways and

The combined production of estradiol and inhibin B by the dominant follicle results in the decline of follicular phase FSH levels, and at least in part, may be responsible for the failure of other follicles to reach preovulatory status during any one cycle. This model predicts that follicles that lack adequate FSH receptor and granulosa cell number will remain primarily androgenic and will therefore become atretic. In support of this model, an increased androgen:estrogen ratio is found in the follicular fluid of atretic follicles and a number of studies have demonstrated that high estrogen levels prevent apoptosis. IGF also has apoptosis-suppressing activity, and is produced by granulosa cells. This action of IGF-I is suppressed by certain IGF-binding proteins that are present in the follicular fluid of atretic follicles. The action of FSH to prevent atresia may therefore result, in part, from its ability to

stimulate IGF-I synthesis and suppress the synthesis of the IGF-binding proteins.

*Clomiphene citrate* + \_ + + *Tamoxifen,* \_ + + + *Raloxifen* \_ \_ + +

Table 4. The effects of selective estrogen receptor modulators on some tissues

in follicle-stimulating hormone (FSH), in turn, drives ovarian follicular activity.

clomiphene citrate (CC), tamoxifen, and raloxifen (Table 4).

Clinically, there are some selective estrogen receptor modulators (SERM) such as

CC is the initial treatment for most anovulatory infertile women. Chemically similar to tamoxifen, CC is a nonsteroidal triphenylethylene derivative that demonstrates both estrogen agonist and antagonist properties. Antagonist properties predominate except at very low estrogen levels. As a result, negative feedback that is normally produced by estrogen in the hypothalamus is reduced. Gonadotropin-releasing hormone (GnRH) secretion is improved and stimulates pituitary gonadotropin release. The resulting increase

Breast Genital Kemik Lipid

Estrogen % 1 % 30 % 69 - Testosterone % 1-2 % 20-32 % 66-78 - DHEA % 4 % 88 % 8 - Androstenedione % 7 % 85 % 8 - DHT % 1 % 71 % 28 - Progesterone % 2 % 80 % 1> % 18 Cortisol % 10 % 15 - % 75

Free Albumin SHBG Transcortin

rapidly activate the Erk1/Erk2 proteins (17).

Table 3. Steroid transformations

The effects of estrogen on;

Genitourinary system:


Breast

Development of ductus

Seconder sex characters


Bone

 Increase the osteoblastic activity in bone and bone mineral density Skin

Increase vascularity and collagen

Liver


Estrogens exert a variety of effects on growth and development of different tissues. The effects of estrogens are mediated via estrogen receptors (ER), intracellular proteins that function as ligand-activated transcription factors and belong to the nuclear receptor superfamily. Two mammalian ERs have been identified, denoted ERα and ERβ. The structure of both receptors is similar and consists of six domains named A through F from the N- to C-terminus, encoded by 8 to 9 exons. Genes that are regulated by activated ERs include early gene responses such as c-myc, c-fos, and d-jun, as well as genes encoding for growth factors such as insulin growth factor (IGF-1 and IGF-2), epidermal growth factor (EGF), transforming growth factor-α, and colony-stimulating factor (CSF-1).

In addition to the described genomic effects of estrogens, there is growing evidence for nongenomic effects of estrogens on intracellular signal transduction pathways. These effects

Stimulation of vaginal epithelial proliferation and superficial cells of epithelium may

Increases the cervical mucus and elasticity and decreases the cervical viscosity (Spinn-

Increase the gap junctions, connexion proteins, and oxytocin sensitivity of smooth

muscles in uterus. Therefore, estrogen increases the uterin contractility.

Increases the crystallization of NaCl in cervical mucus and may cause ferning image.

The effects of estrogen on; Genitourinary system:

Barkeit)

Breast

Bone

Skin

Liver

Stimulation of urethral epithelial proliferation

 Proliferation of endometrial stroma and glands Production of endometrial progesterone receptor

Increase the ciliary activity and motility in fallopian tubes

Development of axillary and pubic hairs in puberty (pubarche)

Increase the osteoblastic activity in bone and bone mineral density

Increase coagulation factors as Factor II, VII, VIII, IX, and X

Increase concentration of bile acids and the development of cholelithiasis

(EGF), transforming growth factor-α, and colony-stimulating factor (CSF-1).

Estrogens exert a variety of effects on growth and development of different tissues. The effects of estrogens are mediated via estrogen receptors (ER), intracellular proteins that function as ligand-activated transcription factors and belong to the nuclear receptor superfamily. Two mammalian ERs have been identified, denoted ERα and ERβ. The structure of both receptors is similar and consists of six domains named A through F from the N- to C-terminus, encoded by 8 to 9 exons. Genes that are regulated by activated ERs include early gene responses such as c-myc, c-fos, and d-jun, as well as genes encoding for growth factors such as insulin growth factor (IGF-1 and IGF-2), epidermal growth factor

In addition to the described genomic effects of estrogens, there is growing evidence for nongenomic effects of estrogens on intracellular signal transduction pathways. These effects

Increase triglycerides, total cholesterol, and HDL

become dominant in vagina. Decreases the vaginal pH (3.8-4.5)

Increase cervical mucus pH

Facilitate follicular stimulation

Increase vascularity and collagen

Decrease antithrombin production

 Increase SHBG synthesis Increase transcortin synthesis Increase angiotensinogen

Decrease LDL

 Development of ductus Seconder sex characters

 Inhibits FSH (negative feed-back effect) Positive feed-back on LH before the ovulation Inhibits GnRH (Increase central opioids)

Development of breast in puberty (thelarche)

include, for example, rapid activation of the adenylate cyclase, which results in cyclic adenosine monophosphate (cAMP)–dependent activation of protein kinase A (PKA). Estrogens can also stimulate the mitogen-activated protein kinase (MAPK) pathways and rapidly activate the Erk1/Erk2 proteins (17).


Table 3. Steroid transformations

The combined production of estradiol and inhibin B by the dominant follicle results in the decline of follicular phase FSH levels, and at least in part, may be responsible for the failure of other follicles to reach preovulatory status during any one cycle. This model predicts that follicles that lack adequate FSH receptor and granulosa cell number will remain primarily androgenic and will therefore become atretic. In support of this model, an increased androgen:estrogen ratio is found in the follicular fluid of atretic follicles and a number of studies have demonstrated that high estrogen levels prevent apoptosis. IGF also has apoptosis-suppressing activity, and is produced by granulosa cells. This action of IGF-I is suppressed by certain IGF-binding proteins that are present in the follicular fluid of atretic follicles. The action of FSH to prevent atresia may therefore result, in part, from its ability to stimulate IGF-I synthesis and suppress the synthesis of the IGF-binding proteins.

Clinically, there are some selective estrogen receptor modulators (SERM) such as clomiphene citrate (CC), tamoxifen, and raloxifen (Table 4).


Table 4. The effects of selective estrogen receptor modulators on some tissues

CC is the initial treatment for most anovulatory infertile women. Chemically similar to tamoxifen, CC is a nonsteroidal triphenylethylene derivative that demonstrates both estrogen agonist and antagonist properties. Antagonist properties predominate except at very low estrogen levels. As a result, negative feedback that is normally produced by estrogen in the hypothalamus is reduced. Gonadotropin-releasing hormone (GnRH) secretion is improved and stimulates pituitary gonadotropin release. The resulting increase in follicle-stimulating hormone (FSH), in turn, drives ovarian follicular activity.

Bone

Liver

Decrease SHBG synthesis

**10. Androgens** 

reserve.

Sex Hormones and Infertility 91

Most progesterone actions on the female reproductive tract are mediated through nuclear hormone receptors. Progesterone enters cells by diffusion and in responsive tissues becomes associated with progesterone receptors (Conneely OM, et al: Reproductive functions of progesterone receptors. Recent Prog Horm Res 57:339, 2002). There are multiple isoforms of the human progesterone receptor. The best understood isoforms are the progesterone receptor type A (PR-A) and B (PR-B). Both arise from a single gene, are members of the steroid receptor superfamily of transcription factors, and regulate transcription of target genes. These receptors have unique actions. When PR-A and PR-B receptors are coexpressed, it appears that PR-A can inhibit PR-B gene regulation. The inhibitory effect of PR-

The ovary produces primarily androstenedione and dehydroepiandrosterone (DHEA) with small amounts of testosterone. Although the adrenal cortex primarily produces mineralocorticoids and glucocorticoids, it also contributes to approximately one-half of the daily production of androstenedione, DHEA, and essentially all of the sulfated form of DHEA (DHEAS). Twenty-five percent of circulating testosterone is secreted by the ovary, 25 percent is secreted by the adrenal gland, and the remaining 50 percent is produced by

AMH has been identified as a dimeric glycoprotein and a member of the transforming growth factor beta (TGFb) family of growth and differentiation factors. The pool of primordial follicles in the ovary is related to the number of growing antral follicles. Antral follicles are responsive to gonadotrophin stimulation and the measure of ovarian reserve can be defined as the total number of follicles, which can be stimulated to grow under maximal stimulation. Classically, age, FSH levels in the early follicular phase, antral follicle count and inhibin B have been used as markers of ovarian reserve. More recently, AMH,

AMH is initially expressed in ovarian granulosa cells of primary follicles, maximal expression occurs in pre-antral and small antral follicles. Antral follicles measuring <6 mm express the greatest amount of AMH, and that expression declines as antral follicles increase in size. AMH is not expressed by atretic follicles and during FSH dependent final stages of follicular growth. AMH inhibits initial primordial follicles recruitment and decreases the sensitivity of preantral and small antral follicles to FSH. Serum AMH concentrations decline with increasing age and constitute a sensitive marker for ovarian aging. Recently, AMH is

Basal serum levels of AMH may more accurately reflect the total developing follicular cohort and consequently potential ovarian response to FSH in cycles of ART. AMH, antral follicle count, inhibin B, FSH and ovarian volume have been demonstrated to reflect ovarian

A may extend to actions on other steroid receptors, including estrogen receptors.

peripheral conversion of androstenedione to testosterone (Figure 3).

have been used by various groups to assess the ovarian reserve (20).

used as pretreatment assessment of ovarian reserve.

**11. Anti-Müllerian hormone (AMH)** 

Antiresorptive effects on bone and increase bone mineral density

The SERM tamoxifen is an estrogen antagonist in the breast that is used in the treatment of estrogen-receptor positive breast cancer. Tamoxifen (20 mg) also has been approved for the prevention of breast cancer in high-risk women, resulting in an approximately 50% reduction in the risk of disease (18).

Raloxifene is a SERM that has been approved for both the prevention and treatment of osteoporosis. Raloxifene exercises estrogen-like actions on bone and lipids without stimulating the breast or endometrium. Raloxifene also may reduce the risk of breast cancer. Postmenopausal women receiving raloxifene as part of a large osteoporosis treatment trial experienced a 76% reduction in the risk of invasive breast cancer compared with placebotreated women (19).


Table 5. Androgen biosynthesis

#### **9. Progesterone**

Progesterone is the 21 C steroid that secretes mainly from corpus luteum and placenta. It minimally secretes from the cortex of adrenal gland. Although its level is 1 ng/mL in preovulatuary phase, it is 3-15 ng/mL in luteal phase. Also, progesterone has a thermogenic affect.

The effects of progesterone on;

Genitourinary system:


Breast

Development of alveols and lobules

Bone

90 Sex Hormones

The SERM tamoxifen is an estrogen antagonist in the breast that is used in the treatment of estrogen-receptor positive breast cancer. Tamoxifen (20 mg) also has been approved for the prevention of breast cancer in high-risk women, resulting in an approximately 50%

Raloxifene is a SERM that has been approved for both the prevention and treatment of osteoporosis. Raloxifene exercises estrogen-like actions on bone and lipids without stimulating the breast or endometrium. Raloxifene also may reduce the risk of breast cancer. Postmenopausal women receiving raloxifene as part of a large osteoporosis treatment trial experienced a 76% reduction in the risk of invasive breast cancer compared with placebo-

Androgens Potence Ovary Adrenal Peripheral DHEA \_ % 25 % 50 % 25 DHEAS 5 \_ % 100 \_ Androstenedione 10 % 50 % 50 \_ Testosterone 100 % 25 % 25 % 50 DHT 300 \_ \_ % 100

Progesterone is the 21 C steroid that secretes mainly from corpus luteum and placenta. It minimally secretes from the cortex of adrenal gland. Although its level is 1 ng/mL in preovulatuary phase, it is 3-15 ng/mL in luteal phase. Also, progesterone has a thermogenic

Decraeses the gap junctions, connexion proteins, and oxytocin sensitivity of smooth

Antiestrogenic activity according to the decrease in estrogen receptor and the increase

Intermediate cells of epithelium may become dominant in vagina.

Secretuar changes on endometrium for implantation

Decrease the ciliary activity and motility in fallopian tubes

in transformation of E2 to E1 (stimulates 17 OHSD enzyme)

Increases the cervical viscosity and decreases the cervical mucus and elasticity

muscles in uterus. Therefore, progesterone decreases the uterin contractility

Antiproliferative and antimitotic effects on endometrial stroma and glands

reduction in the risk of disease (18).

Table 5. Androgen biosynthesis

The effects of progesterone on;

Increases the vaginal pH (> 4.5)

Decreases cervical mucus pH

Inhibits LH (negative feed-back effect)

Development of alveols and lobules

 Positive feed-back on FSH before the ovulation Inhibits GnRH (Increase central opioids)

Genitourinary system:

**9. Progesterone** 

affect.

Breast

treated women (19).

Antiresorptive effects on bone and increase bone mineral density

Liver

Decrease SHBG synthesis

Most progesterone actions on the female reproductive tract are mediated through nuclear hormone receptors. Progesterone enters cells by diffusion and in responsive tissues becomes associated with progesterone receptors (Conneely OM, et al: Reproductive functions of progesterone receptors. Recent Prog Horm Res 57:339, 2002). There are multiple isoforms of the human progesterone receptor. The best understood isoforms are the progesterone receptor type A (PR-A) and B (PR-B). Both arise from a single gene, are members of the steroid receptor superfamily of transcription factors, and regulate transcription of target genes. These receptors have unique actions. When PR-A and PR-B receptors are coexpressed, it appears that PR-A can inhibit PR-B gene regulation. The inhibitory effect of PR-A may extend to actions on other steroid receptors, including estrogen receptors.

#### **10. Androgens**

The ovary produces primarily androstenedione and dehydroepiandrosterone (DHEA) with small amounts of testosterone. Although the adrenal cortex primarily produces mineralocorticoids and glucocorticoids, it also contributes to approximately one-half of the daily production of androstenedione, DHEA, and essentially all of the sulfated form of DHEA (DHEAS). Twenty-five percent of circulating testosterone is secreted by the ovary, 25 percent is secreted by the adrenal gland, and the remaining 50 percent is produced by peripheral conversion of androstenedione to testosterone (Figure 3).

#### **11. Anti-Müllerian hormone (AMH)**

AMH has been identified as a dimeric glycoprotein and a member of the transforming growth factor beta (TGFb) family of growth and differentiation factors. The pool of primordial follicles in the ovary is related to the number of growing antral follicles. Antral follicles are responsive to gonadotrophin stimulation and the measure of ovarian reserve can be defined as the total number of follicles, which can be stimulated to grow under maximal stimulation. Classically, age, FSH levels in the early follicular phase, antral follicle count and inhibin B have been used as markers of ovarian reserve. More recently, AMH, have been used by various groups to assess the ovarian reserve (20).

AMH is initially expressed in ovarian granulosa cells of primary follicles, maximal expression occurs in pre-antral and small antral follicles. Antral follicles measuring <6 mm express the greatest amount of AMH, and that expression declines as antral follicles increase in size. AMH is not expressed by atretic follicles and during FSH dependent final stages of follicular growth. AMH inhibits initial primordial follicles recruitment and decreases the sensitivity of preantral and small antral follicles to FSH. Serum AMH concentrations decline with increasing age and constitute a sensitive marker for ovarian aging. Recently, AMH is used as pretreatment assessment of ovarian reserve.

Basal serum levels of AMH may more accurately reflect the total developing follicular cohort and consequently potential ovarian response to FSH in cycles of ART. AMH, antral follicle count, inhibin B, FSH and ovarian volume have been demonstrated to reflect ovarian reserve.

Sex Hormones and Infertility 93

Test Normal Value Abnormal Value Cycle Day 3 FSH < 10-15 mIU/mL > 10-15 mIU/mL

estradiol < 80 pg/mL > 80 pg/mL Inhibin B > 45 pg/mL < 45 pg/mL

> > 2.7 ng/mL associated with improved oocyte quality as reflected in a higher implantation rate and trend toward better

challenge test FSH < 26 mIU/mL on Day 10 FSH > 26 mIU/mL

\*Note: Different infertility centers use different tests. Cut-off values may differ from center to center on

On the one hand, the results of these tests will help with designing treatment (to choose the appropriate treatment, stimulation protocol, and gonadotropin dose), and on the other hand they will be useful for counseling the couple. It is very important that a couple undergoing

In addition to these tests, it is useful to perform an ultrasound midcycle to assess the ovary and uterus and to document ovulation. Midcycle ultrasound will document follicle growth and allow us to look at the endometrial lining (eg, thickness and type). Ovulation can be documented in several ways. The easiest is to measure a midluteal phase progesterone level. Changes in the basal body temperature, urinary LH kits, luteal phase endometrial biopsy,

When the cycles are irregular, other hormonal measurements -- such as testosterone, dehydroepiandrosterone sulfate (DHEAS), 17-OH progesterone, cortisol, prolactin -- as well as thyroid function tests and dynamic evaluation of pituitary function may be necessary for the infertility work-up. If the results of any of these tests are considered abnormal,

Ovulation induction has a role in the management of patients with anovulation/oligoovulation or regular cycles. In the case of oligo-ovulation, the goal is to restore mono-

Various drugs can be used to restore ovulation. Selective estrogen receptor modulators (eg, CC, tamoxifen) are usually administered first. CC is the agent for which most experience has accumulated. It is administered from Day 3 or 5 of the cycle for 5 days. The starting dose is 50 mg, but if needed the dose can be increased by 50 mg daily during subsequent stimulation. Usually, a daily dose > 150 mg is not recommended, as higher doses compromise endometrial development, and pregnancy rates are very low. Ovulation rates are high (80%)

conducting imaging studies (eg, MRI, CT, thyroid scan) may be the appropriate step.

Estradiol level elevation and subsequent

Low levels

on Day 10

No elevation of estradiol level

clinical pregnancy rate[56]

on ultrasound > 3-4 < 3-4

ultrasound > 3 mL < 3 mL

Table 7. Cut-off Values\* for the Most Commonly Used Ovarian Reserve Tests

any form of assisted reproduction has realistic expectations (22).

and serial ultrasounds are alternatives for assessing ovulation.

**13.2 Ovulation induction, controlled ovarian hyperstimulation** 

decrease

Cycle Day 3

Anti-Müllerian hormone

Gonadotropin stimulation test

Clomiphene citrate

Antral follicle count

Ovarian volume on

ovulatory cycles.

the basis of their experience and results.

#### **12. Infertility**

Infertility is defined as 1 year of unprotected intercourse without pregnancy. This condition may be further classified as primary infertility, in which no previous pregnancies have occurred, and secondary infertility, in which a prior pregnancy, although not necessarily a live birth, has occurred. Infertility affects about 10% to 15% of reproductive age couples. (21). Various factors may be responsible for the inability to achieve a successful pregnancy. Ovulatory, anatomic, immunologic, or hormonal factors on the woman's side and abnormalities of the semen parameters on the man's side are the most common (Table 6). After a thorough work-up, treatment can be planned that aims to correct the problem identified or, in the case of unexplained infertility, tries to improve all steps of the reproductive process.


Table 6. Causes of Infertility

#### **13. Evaluation of infertility**

The most important tests for evaluation of infertility are to assess the ovarian function.

#### **13.1 Ovarian function**

Ovarian function can be evaluated by various methods. Regular menstrual cycles are a sign of ovulation in 95% of the cycles. Because the ovaries also "age," however, the regularity of the cycles alone is not enough to characterize ovarian function. The number of follicles in the ovaries decreases from birth. As a result, from the age of 30 onward a slow decline in fertility occurs. This decline parallels the reduction in the number and quality of the follicles and oocytes. The first sign of reduced ovarian activity is the shortening of the follicular phase, which reduces the length of the ovulatory cycle. The decrease in the number of follicles is followed by hormonal changes. Inhibin B is produced by the small antral follicles, and as their number declines, the ovarian output of inhibin B decreases. This is paralleled by a rise in FSH level.

For the everyday practice, there are several tests to assess ovarian reserve. Measurement of the early follicular phase FSH and estradiol levels to determine the FSH/estradiol ratio; measurement of inhibin B or anti-Müllerian hormone levels; or the early follicular phase antral follicle count are options (Table 7). Dynamic tests evaluate the ovaries during clomiphene citrate (CC) challenge or during gonadotropin-releasing hormone agonist (GnRHa) or gonadotropin stimulation.

Infertility is defined as 1 year of unprotected intercourse without pregnancy. This condition may be further classified as primary infertility, in which no previous pregnancies have occurred, and secondary infertility, in which a prior pregnancy, although not necessarily a live birth, has occurred. Infertility affects about 10% to 15% of reproductive age couples. (21). Various factors may be responsible for the inability to achieve a successful pregnancy. Ovulatory, anatomic, immunologic, or hormonal factors on the woman's side and abnormalities of the semen parameters on the man's side are the most common (Table 6). After a thorough work-up, treatment can be planned that aims to correct the problem identified or, in the case of unexplained infertility, tries to improve all steps of the

40–55

The most important tests for evaluation of infertility are to assess the ovarian function.

Ovarian function can be evaluated by various methods. Regular menstrual cycles are a sign of ovulation in 95% of the cycles. Because the ovaries also "age," however, the regularity of the cycles alone is not enough to characterize ovarian function. The number of follicles in the ovaries decreases from birth. As a result, from the age of 30 onward a slow decline in fertility occurs. This decline parallels the reduction in the number and quality of the follicles and oocytes. The first sign of reduced ovarian activity is the shortening of the follicular phase, which reduces the length of the ovulatory cycle. The decrease in the number of follicles is followed by hormonal changes. Inhibin B is produced by the small antral follicles, and as their number declines, the ovarian output of inhibin B decreases. This is paralleled by

For the everyday practice, there are several tests to assess ovarian reserve. Measurement of the early follicular phase FSH and estradiol levels to determine the FSH/estradiol ratio; measurement of inhibin B or anti-Müllerian hormone levels; or the early follicular phase antral follicle count are options (Table 7). Dynamic tests evaluate the ovaries during clomiphene citrate (CC) challenge or during gonadotropin-releasing hormone agonist

Prevalence of the etiologies of infertility (%)

30–40 30–40 10–15 10–15

**12. Infertility** 

reproductive process.

Female factor


Table 6. Causes of Infertility

**13.1 Ovarian function** 

a rise in FSH level.

(GnRHa) or gonadotropin stimulation.

**13. Evaluation of infertility** 

Male factor 25–40

Both male and female factors 10 Unexplained infertility 10


\*Note: Different infertility centers use different tests. Cut-off values may differ from center to center on the basis of their experience and results.

Table 7. Cut-off Values\* for the Most Commonly Used Ovarian Reserve Tests

On the one hand, the results of these tests will help with designing treatment (to choose the appropriate treatment, stimulation protocol, and gonadotropin dose), and on the other hand they will be useful for counseling the couple. It is very important that a couple undergoing any form of assisted reproduction has realistic expectations (22).

In addition to these tests, it is useful to perform an ultrasound midcycle to assess the ovary and uterus and to document ovulation. Midcycle ultrasound will document follicle growth and allow us to look at the endometrial lining (eg, thickness and type). Ovulation can be documented in several ways. The easiest is to measure a midluteal phase progesterone level. Changes in the basal body temperature, urinary LH kits, luteal phase endometrial biopsy, and serial ultrasounds are alternatives for assessing ovulation.

When the cycles are irregular, other hormonal measurements -- such as testosterone, dehydroepiandrosterone sulfate (DHEAS), 17-OH progesterone, cortisol, prolactin -- as well as thyroid function tests and dynamic evaluation of pituitary function may be necessary for the infertility work-up. If the results of any of these tests are considered abnormal, conducting imaging studies (eg, MRI, CT, thyroid scan) may be the appropriate step.

#### **13.2 Ovulation induction, controlled ovarian hyperstimulation**

Ovulation induction has a role in the management of patients with anovulation/oligoovulation or regular cycles. In the case of oligo-ovulation, the goal is to restore monoovulatory cycles.

Various drugs can be used to restore ovulation. Selective estrogen receptor modulators (eg, CC, tamoxifen) are usually administered first. CC is the agent for which most experience has accumulated. It is administered from Day 3 or 5 of the cycle for 5 days. The starting dose is 50 mg, but if needed the dose can be increased by 50 mg daily during subsequent stimulation. Usually, a daily dose > 150 mg is not recommended, as higher doses compromise endometrial development, and pregnancy rates are very low. Ovulation rates are high (80%)

Sex Hormones and Infertility 95

mild male factor or cervical factor infertility is diagnosed. A wide range of pregnancy rates have been reported after insemination (5% to 20% per cycle). Pregnancy rates are higher when gonadotropin stimulation is used in conjunction with IUI. Pregnancy rates are affected by the age of the female partner, semen parameters, tubal status, the presence of endometriosis, and the order of the treatment cycle. The pregnancy and multiple gestation rates are highest with the first treatment cycle. Some even recommend performing the first IUI in a natural unstimulated cycle to avoid multiple gestations and only to proceed with stimulation if the first attempt fails. Usually IUI should not be repeated more than 3 or 4 times. Two exceptions are when donor sperm is used and when the patient has oligoovulation; in these cases, a significant number of further pregnancies have been reported in a 5th or 6th cycle. The decision should be made individually, and the availability of IVF

The first baby conceived after IVF treatment was born in 1978. Since then, the field has undergone enormous development, and IVF is now routinely used in the management of various forms of infertility. Initially, it was used for the treatment of tubal factor infertility, but today it is used to help patients with male factor infertility, unexplained infertility,

Early on, IVF was carried out during the patients' natural cycle. Later, CC was added to the protocol to increase efficacy. These cycles were characterized by relatively high cancellation rates as a result of premature ovulation and low pregnancy rates. With the advent of GnRH agonists, antagonists, and different types of gonadotropins, new stimulation protocols have been developed. Cycles with these protocols, by contrast, are characterized by very low cancellation rates, a higher number of oocytes, better-quality embryos, and significantly

A typical IVF cycle is made up of 3 parts: stimulation, egg retrieval, and embryo transfer. Stimulation usually consists of pretreatment and stimulation. Pretreatment with oral contraceptive pills or a GnRH agonist allows flexible cycle scheduling and a more simultaneous follicle growth. In the various stimulation protocols, GnRH agonist or antagonist can be given to prevent premature LH surges. The GnRH agonist can be initiated in the luteal phase of the preceding cycle (long protocol) or with the onset of menstruation together with gonadotropins (short, ultrashort protocols). The GnRH agonist initially depletes the pituitary gonadotropin stores ("flare up" effect) before it prevents further FSH and LH release (usually

A GnRH antagonist has a different mechanism of action. It competes with GnRH for its pituitary receptors. Upon administration it immediately prevents FSH and LH release. In GnRH antagonist cycles, the antagonist is administered either on Day 6 of stimulation (fixed

There are 5 or 6 different stimulation protocols in use by IVF centers. Subtle differences in the management of the pretreatment phase or in the type and dose of gonadotropins do exist between IVF clinics. Several patient characteristics are considered before one decides about the protocol to be used. Typically, age, results of the ovarian reserve testing, and response to previous stimulation help with the decision about the appropriate stimulation protocol (35). Cycle monitoring (ultrasound and estradiol measurements) usually starts after 5 days of stimulation. When at least 2 follicles reach 17 to 18 mm in diameter, the final steps of oocyte maturation are induced by 5000 to 10,000 IU hCG. In those cycles during which GnRH

obviously influences the decision (33, 34).

higher implantation and pregnancy rates.

**15. In vitro fertilization, intracytoplasmic sperm injection** 

after 7 to 12 days). This initial flare effect is used with the short protocols.

protocol) or when the lead follicle reaches 14 mm in diameter (flexible protocol).

genetic problems, and those who fail in vivo treatments.

with CC, but cumulative pregnancy rates are only around 40%. The difference between the high ovulation rates and relatively low pregnancy rates is most likely due to the antiestrogenic effects of CC on the periphery, most prominently at the level of the endometrium. If pregnancy does not occur after a maximum of 6 cycles, other options need to be explored (23).

CC stimulation can be combined with ovulation induction with human chorionic gonadotropin (hCG), especially when a spontaneous LH surge cannot be documented. The multifetal gestation rate is about 10% with CC use. CC has relatively few side effects, with gastrointestinal symptoms, visual changes, and hot flashes being more common.

Aromatase inhibitors (eg, letrozole, anastrozole) have been explored recently. Aromatase is an enzyme that regulates the androgen-estrogen conversion. Aromatase inhibitors work by reducing estradiol level and therefore increasing pituitary gonadotropin output (resulting in decreased estradiol negative feedback). Their use is seldom associated with multifollicular development. Pregnancy rates are about 15% to 20 % per cycle. No adverse perinatal outcome following aromatase inhibitor use has yet been reported in the published, peer-reviewed literature, although the authors of a study presented during the American Society for Reproductive Medicine meeting in 2005 reported a higher rate of congenital anomalies with 5 mg anastrozole (24, 25, 26). Notably, letrozole has warned clinicians against prescribing drugs for ovulation induction on the basis of reports of birth defects and spontaneous miscarriages in its safety database (27). Letrozole is not approved for ovulation induction.

Insulin-sensitizing agents have been successfully used to treat infertile patients with PCOS. Metformin (1500 to 2000 mg daily) has been used most widely. With metformin, ovulation can be documented in about 50% to 60% of cases. Metformin can also be combined with CC in CCresistant cases. Lower miscarriage rates and fewer cases of gestational diabetes have been reported with metformin use. Metformin is a category B drug; no serious adverse effects have been reported with use during pregnancy. Gastrointestinal side effects are often reported upon initiation of treatment. It is a good approach to start with a lower daily dose and slowly increase it to the therapeutic range. Metformin should not be used in women with liver or renal disease. It takes at least 2 to 3 months for insulin sensitizers to take full effect (28, 29, 30, 31, 32).

Gonadotropins can be administered when oral preparations are ineffective or do not lead to pregnancy after repeated attempts. Gonadotropins can be used alone or in combination with oral preparations and are usually started on Day 3 of the cycle at an initial dose of 75 to 150 IU. Cycle monitoring (ultrasound ± estradiol measurement) begins after 5 days of stimulation. When gonadotropins are combined with oral preparations, the pill is initiated first (usually on Day 3) and the injections are administered starting 2 days later. Injections are usually administered on every other day. These protocols can be adjusted depending on the response. Although pregnancy rates are higher following gonadotropin stimulation, the risks for multiple gestations and ovarian hyperstimulation syndrome (OHSS) are increased as well.

Ovulation induction cycles can be completed in different ways. Urinary LH kits can be used to predict ovulation and to time intercourse or insemination. Alternatively, when the lead follicle is around 18 to 20 mm in diameter, human chorionic gonadotropin (hCG) can be administered to induce ovulation. When hCG is used, intercourse or insemination is scheduled 36 to 40 hrs after the injection.

#### **14. Intrauterine Insemination**

Intrauterine insemination (IUI) further improves the chances of pregnancy. IUI is more effective than intracervical or intravaginal insemination. Its use is especially indicated when

with CC, but cumulative pregnancy rates are only around 40%. The difference between the high ovulation rates and relatively low pregnancy rates is most likely due to the antiestrogenic effects of CC on the periphery, most prominently at the level of the endometrium. If pregnancy does not occur after a maximum of 6 cycles, other options need to be explored (23). CC stimulation can be combined with ovulation induction with human chorionic gonadotropin (hCG), especially when a spontaneous LH surge cannot be documented. The multifetal gestation rate is about 10% with CC use. CC has relatively few side effects, with

Aromatase inhibitors (eg, letrozole, anastrozole) have been explored recently. Aromatase is an enzyme that regulates the androgen-estrogen conversion. Aromatase inhibitors work by reducing estradiol level and therefore increasing pituitary gonadotropin output (resulting in decreased estradiol negative feedback). Their use is seldom associated with multifollicular development. Pregnancy rates are about 15% to 20 % per cycle. No adverse perinatal outcome following aromatase inhibitor use has yet been reported in the published, peer-reviewed literature, although the authors of a study presented during the American Society for Reproductive Medicine meeting in 2005 reported a higher rate of congenital anomalies with 5 mg anastrozole (24, 25, 26). Notably, letrozole has warned clinicians against prescribing drugs for ovulation induction on the basis of reports of birth defects and spontaneous miscarriages in

Insulin-sensitizing agents have been successfully used to treat infertile patients with PCOS. Metformin (1500 to 2000 mg daily) has been used most widely. With metformin, ovulation can be documented in about 50% to 60% of cases. Metformin can also be combined with CC in CCresistant cases. Lower miscarriage rates and fewer cases of gestational diabetes have been reported with metformin use. Metformin is a category B drug; no serious adverse effects have been reported with use during pregnancy. Gastrointestinal side effects are often reported upon initiation of treatment. It is a good approach to start with a lower daily dose and slowly increase it to the therapeutic range. Metformin should not be used in women with liver or renal disease.

Gonadotropins can be administered when oral preparations are ineffective or do not lead to pregnancy after repeated attempts. Gonadotropins can be used alone or in combination with oral preparations and are usually started on Day 3 of the cycle at an initial dose of 75 to 150 IU. Cycle monitoring (ultrasound ± estradiol measurement) begins after 5 days of stimulation. When gonadotropins are combined with oral preparations, the pill is initiated first (usually on Day 3) and the injections are administered starting 2 days later. Injections are usually administered on every other day. These protocols can be adjusted depending on the response. Although pregnancy rates are higher following gonadotropin stimulation, the risks for multiple gestations and ovarian hyperstimulation syndrome (OHSS) are increased as well. Ovulation induction cycles can be completed in different ways. Urinary LH kits can be used to predict ovulation and to time intercourse or insemination. Alternatively, when the lead follicle is around 18 to 20 mm in diameter, human chorionic gonadotropin (hCG) can be administered to induce ovulation. When hCG is used, intercourse or insemination is

Intrauterine insemination (IUI) further improves the chances of pregnancy. IUI is more effective than intracervical or intravaginal insemination. Its use is especially indicated when

It takes at least 2 to 3 months for insulin sensitizers to take full effect (28, 29, 30, 31, 32).

gastrointestinal symptoms, visual changes, and hot flashes being more common.

its safety database (27). Letrozole is not approved for ovulation induction.

scheduled 36 to 40 hrs after the injection.

**14. Intrauterine Insemination** 

mild male factor or cervical factor infertility is diagnosed. A wide range of pregnancy rates have been reported after insemination (5% to 20% per cycle). Pregnancy rates are higher when gonadotropin stimulation is used in conjunction with IUI. Pregnancy rates are affected by the age of the female partner, semen parameters, tubal status, the presence of endometriosis, and the order of the treatment cycle. The pregnancy and multiple gestation rates are highest with the first treatment cycle. Some even recommend performing the first IUI in a natural unstimulated cycle to avoid multiple gestations and only to proceed with stimulation if the first attempt fails. Usually IUI should not be repeated more than 3 or 4 times. Two exceptions are when donor sperm is used and when the patient has oligoovulation; in these cases, a significant number of further pregnancies have been reported in a 5th or 6th cycle. The decision should be made individually, and the availability of IVF obviously influences the decision (33, 34).

#### **15. In vitro fertilization, intracytoplasmic sperm injection**

The first baby conceived after IVF treatment was born in 1978. Since then, the field has undergone enormous development, and IVF is now routinely used in the management of various forms of infertility. Initially, it was used for the treatment of tubal factor infertility, but today it is used to help patients with male factor infertility, unexplained infertility, genetic problems, and those who fail in vivo treatments.

Early on, IVF was carried out during the patients' natural cycle. Later, CC was added to the protocol to increase efficacy. These cycles were characterized by relatively high cancellation rates as a result of premature ovulation and low pregnancy rates. With the advent of GnRH agonists, antagonists, and different types of gonadotropins, new stimulation protocols have been developed. Cycles with these protocols, by contrast, are characterized by very low cancellation rates, a higher number of oocytes, better-quality embryos, and significantly higher implantation and pregnancy rates.

A typical IVF cycle is made up of 3 parts: stimulation, egg retrieval, and embryo transfer. Stimulation usually consists of pretreatment and stimulation. Pretreatment with oral contraceptive pills or a GnRH agonist allows flexible cycle scheduling and a more simultaneous follicle growth. In the various stimulation protocols, GnRH agonist or antagonist can be given to prevent premature LH surges. The GnRH agonist can be initiated in the luteal phase of the preceding cycle (long protocol) or with the onset of menstruation together with gonadotropins (short, ultrashort protocols). The GnRH agonist initially depletes the pituitary gonadotropin stores ("flare up" effect) before it prevents further FSH and LH release (usually after 7 to 12 days). This initial flare effect is used with the short protocols.

A GnRH antagonist has a different mechanism of action. It competes with GnRH for its pituitary receptors. Upon administration it immediately prevents FSH and LH release. In GnRH antagonist cycles, the antagonist is administered either on Day 6 of stimulation (fixed protocol) or when the lead follicle reaches 14 mm in diameter (flexible protocol).

There are 5 or 6 different stimulation protocols in use by IVF centers. Subtle differences in the management of the pretreatment phase or in the type and dose of gonadotropins do exist between IVF clinics. Several patient characteristics are considered before one decides about the protocol to be used. Typically, age, results of the ovarian reserve testing, and response to previous stimulation help with the decision about the appropriate stimulation protocol (35).

Cycle monitoring (ultrasound and estradiol measurements) usually starts after 5 days of stimulation. When at least 2 follicles reach 17 to 18 mm in diameter, the final steps of oocyte maturation are induced by 5000 to 10,000 IU hCG. In those cycles during which GnRH

Sex Hormones and Infertility 97

gestational diabetes have been reported with metformin use. Metformin is a category B drug; no adverse effects have been reported with use during pregnancy. Once follicle growth is achieved, adding hCG can help the timing (intercourse or insemination). Without

The patient's husband has a low sperm count. Therefore, IUI could improve this couple's chances for conception. IVF/ICSI would be recommended if IUI was not successful after 3 to

[1] Plant TM, et al. The arcuate nucleus and the control of the gonadotropin and prolactin secretion in the female rhesus monkey. Endocrinology 1978;102:52–62. [2] Schwanzel-Fukuda M, et al. Origin of luteinizing hormone releasing hormone neurons.

[3] Blackwell RE. Concomitant release of FSH and LH induced by native and synthetic LRF.

[4] Knobil E. Neuroendocrine control of the menstrual cycle. Recent Prog Horm Res

[5] Filicori M, et al. Characterization of the physiological pattern of episodic gonadotropin

[6] Howlett TA, et al. Endogenous opioid peptide and hypothalamo-pituitary function.

[7] Reid Rl, et al. Effects of exogenous β-endorphin on pituitary hormone secretion and its

[8] Karten MJ, et al. Gonadotropin-releasing hormone analog design. Structure function

[9] Conn PM, Crowley WF Jr. Gonadotropin-releasing hormone and its analogs. Annu Rev

[10] Ron–El R, et al. Gonadotropins and combined gonadotropin– releasing hormone

[11] San Roman GA, et al. A prospective randomized comparison of luteal phase versus

[12] Al–Inany H, et al. Gonadotrophin–releasing hormone antagonists for assisted

[13] Surrey ES, et al. Prolonged GnRH agonist and add-back therapy for symptomatic

[14] Vaitukaitis JL, et al. Gonadotropins and their subunits: basic and clinical studies.

[15] Peters H, Joint A: The Ovary: A Correlation of Structure and Function in Mammals.

[16] Sasano H, et al. Immunolocalization of aromatase, 17 alpha-hydroxylase and side-

[17] Speroff L, et al: Neuroendocrinology. In Clinical Gynecologic Endocrinology and

chain-cleavage cytochromes P-450 in the human ovary. J Reprod Fertil 85:163, 1989.

endometriosis: Long term follow-up. Obstet Gynecol 2002; 99: 709.

Infertility, 7th ed. Baltimore, Lippincott Williams & Wilkins, 2005.

secretion throughout the human menstrual cycle. J Clin Endocrinol Metab 1986;62:

disappearance rate in normal human subjects. J Clin Endocrinol Metab

studies towards the development of agonists and antagonists: rationale and

agonist gonadotropins protocols in a randomized prospective study. Fertil Steril

concurrent follicular phase initiation of gonadotropin–releasing hormone agonist

a mature follicle, however, hCG alone does not work.

Nature 1989;338:161–164.

1980;36:53–88.

1981;52:1179–1184.

Med 1994;45:391–405.

1991;55:574–578.

1136–1144.

Am J Physiol 1973;224:170–175.

Annu Rev Physiol 1986;48:527–536.

perspective. Endocr Rev 1986;7:44–66.

Recent Prog Horm Res 1976;32:289–331.

Berkeley, University of California Press, 1980.

for in vitro fertilization. Fertil Steril 1992;58:744–749.

conception. Cochrane Database Syst Rev 2001;CD001750.

6 attempts.

**16. References** 

agonist downregulation is not applied, the final maturation of the oocytes can be induced with GnRH agonist as well. This method is associated with a lower incidence of OHSS. Oocyte retrieval is scheduled 35 to 36 hours after the final injection.

Oocyte retrieval is an ultrasound-guided vaginal procedure that is performed under intravenous sedation. Oocytes are collected in culture medium and are processed for fertilization. Human tubular fluid was used as an example to design culture medium. Currently, several companies produce culture medium. Use of sequential media tries to satisfy the changing needs of the developing embryo.

Fertilization may occur spontaneously when the sperm number, motility and morphology are within the normal range or can be done using intracytoplasmic sperm injection (ICSI). ICSI is used when the sperm parameters are suboptimal or when fertilization was poor in a previous cycle. During ICSI, the immobilized sperm is transferred through the zona pellucida with a fine glass needle to allow fertilization to take place (36).

The day after the retrieval, the oocytes are checked for signs of fertilization (presence of 2 pronuclei) and are cultured for an additional 2 to 4 days. Transfer usually takes place on Day 3 or 5 after the retrieval. Embryos are assessed on the basis of blastomere number and morphology. Usually 2 or 3 good-quality embryos are transferred. The decision is influenced by the order of the cycle, the patient's age, the number and quality of the embryos, the couple's wishes, and by regulations in those countries where the number of embryos to be transferred is limited. Surplus good-quality embryos can be frozen and stored for later use. To reduce the number of multiple gestations, there is tendency toward transferring fewer embryos. In some countries, the transfer of only a single embryo is allowed. Although pregnancy rates per transfer are lower, following the transfer of 1 fresh and 1 cryopreserved embryo, the cumulative pregnancy rates are comparable to rates following the transfer of 2 embryos. Multiple pregnancies occur significantly less often. An efficient cryopreservation program needs to be in place, however, before one can comfortably offer elective single embryo transfer (37).

This patient has oligo-anovulation; therefore, the assessment of her hormonal status is important. Most commonly, irregular ovarian activity has an endocrine etiology including thyroid disease, hyperprolactinemia, androgen excess, PCOS, premature ovarian failure. Transvaginal ultrasound will assess the morphology of the ovaries (ie, whether they are polycystic or not), myometrium, and endometrium. Serial ultrasound will document follicle growth and allows us to look at the changes in the endometrial lining (eg, thickness and type). Once the etiology of the irregular cycles is known, the appropriate treatment can be planned.

Women with PCOS are at increased for impaired glucose tolerance (and diabetes), dylipidemia, and hypertension. Therefore, the baseline evaluation of these metabolic markers should be part of the work-up for this patient.

Weight loss (life-style modification), CC, or insulin sensitizers could be recommended. At least half of women with PCOS are obese. Obesity is associated with insulin resistance that will further compromise ovarian activity. Weight loss and regular exercise are integral parts of their treatment. Weight loss is associated not only with improved ovarian function but also with lower risk for metabolic complications. CC and insulin sensitizers have both been shown to be effective for ovulation induction among women with oligo-ovulation.

Adding an insulin sensitizer such as metformin would be the next step. A daily dose > 150 mg of CC is not recommended, as higher doses compromise endometrial development, and pregnancy rates are very low. Insulin-sensitizing agents have been successfully used to treat infertile patients with PCOS. Metformin (1500-2000 mg daily) has been used most widely. With metformin, ovulation can be documented in about 50% to 60% of the cases. Metformin can be combined with CC in CC-resistant cases. Lower miscarriage rates and fewer cases of

agonist downregulation is not applied, the final maturation of the oocytes can be induced with GnRH agonist as well. This method is associated with a lower incidence of OHSS.

Oocyte retrieval is an ultrasound-guided vaginal procedure that is performed under intravenous sedation. Oocytes are collected in culture medium and are processed for fertilization. Human tubular fluid was used as an example to design culture medium. Currently, several companies produce culture medium. Use of sequential media tries to

Fertilization may occur spontaneously when the sperm number, motility and morphology are within the normal range or can be done using intracytoplasmic sperm injection (ICSI). ICSI is used when the sperm parameters are suboptimal or when fertilization was poor in a previous cycle. During ICSI, the immobilized sperm is transferred through the zona

The day after the retrieval, the oocytes are checked for signs of fertilization (presence of 2 pronuclei) and are cultured for an additional 2 to 4 days. Transfer usually takes place on Day 3 or 5 after the retrieval. Embryos are assessed on the basis of blastomere number and morphology. Usually 2 or 3 good-quality embryos are transferred. The decision is influenced by the order of the cycle, the patient's age, the number and quality of the embryos, the couple's wishes, and by regulations in those countries where the number of embryos to be transferred is limited. Surplus good-quality embryos can be frozen and stored for later use. To reduce the number of multiple gestations, there is tendency toward transferring fewer embryos. In some countries, the transfer of only a single embryo is allowed. Although pregnancy rates per transfer are lower, following the transfer of 1 fresh and 1 cryopreserved embryo, the cumulative pregnancy rates are comparable to rates following the transfer of 2 embryos. Multiple pregnancies occur significantly less often. An efficient cryopreservation program needs to be in

Oocyte retrieval is scheduled 35 to 36 hours after the final injection.

pellucida with a fine glass needle to allow fertilization to take place (36).

place, however, before one can comfortably offer elective single embryo transfer (37).

This patient has oligo-anovulation; therefore, the assessment of her hormonal status is important. Most commonly, irregular ovarian activity has an endocrine etiology including thyroid disease, hyperprolactinemia, androgen excess, PCOS, premature ovarian failure. Transvaginal ultrasound will assess the morphology of the ovaries (ie, whether they are polycystic or not), myometrium, and endometrium. Serial ultrasound will document follicle growth and allows us to look at the changes in the endometrial lining (eg, thickness and type). Once the etiology of the irregular cycles is known, the appropriate treatment can be planned. Women with PCOS are at increased for impaired glucose tolerance (and diabetes), dylipidemia, and hypertension. Therefore, the baseline evaluation of these metabolic

Weight loss (life-style modification), CC, or insulin sensitizers could be recommended. At least half of women with PCOS are obese. Obesity is associated with insulin resistance that will further compromise ovarian activity. Weight loss and regular exercise are integral parts of their treatment. Weight loss is associated not only with improved ovarian function but also with lower risk for metabolic complications. CC and insulin sensitizers have both been

Adding an insulin sensitizer such as metformin would be the next step. A daily dose > 150 mg of CC is not recommended, as higher doses compromise endometrial development, and pregnancy rates are very low. Insulin-sensitizing agents have been successfully used to treat infertile patients with PCOS. Metformin (1500-2000 mg daily) has been used most widely. With metformin, ovulation can be documented in about 50% to 60% of the cases. Metformin can be combined with CC in CC-resistant cases. Lower miscarriage rates and fewer cases of

shown to be effective for ovulation induction among women with oligo-ovulation.

satisfy the changing needs of the developing embryo.

markers should be part of the work-up for this patient.

gestational diabetes have been reported with metformin use. Metformin is a category B drug; no adverse effects have been reported with use during pregnancy. Once follicle growth is achieved, adding hCG can help the timing (intercourse or insemination). Without a mature follicle, however, hCG alone does not work.

The patient's husband has a low sperm count. Therefore, IUI could improve this couple's chances for conception. IVF/ICSI would be recommended if IUI was not successful after 3 to 6 attempts.

#### **16. References**


**1. Introduction** 

Source: Wikipedia.

**1.1 Progesterone synthesis** 

**5** 

*Sweden* 

**Progesterone in Human** 

*Department of Woman and Child Health,* 

Ylva Vladic Stjernholm

**Pregnancy and Parturition** 

*Karolinska University Hospital and Institute, Stockholm,* 

Progesterone was independently discovered by different research groups (Butenandt & Westphal 1934, Allen 1935). Allen and collaborators discovered progesterone in 1933, and were the first to determine the molecular weight and partial molecular structure. The name

Fig. 1. Steroid hormone synthesis. The precursor cholesterol from the maternal circulation is

converted to 21 carbon (C21) progestagens. Progestagens can be converted to C21 glucocorticoids, or to C19 androgens. Androgens serve as precursors for C18 estrogens.

*progesterone* derives from progestational steroidal ketone *(Allen 1935).* 


### **Progesterone in Human Pregnancy and Parturition**

Ylva Vladic Stjernholm *Department of Woman and Child Health, Karolinska University Hospital and Institute, Stockholm, Sweden* 

#### **1. Introduction**

98 Sex Hormones

[18] Fisher B, et al. Tamoxifen for prevention of breast cancer: report of the national surgical adjuvant breast and bowel project P-1 study. J Natl Cancer Inst 1998;90:1371–1388. [19] Cummings S, et al. The effect of raloxifene on risk of breast cancer in postmenopausal women: results from the MORE randomized trial. JAMA 1999;281:2189–2197. [20] Jayaprakasan K, et al. A prospective, comparative analysis of anti-Mullerian hormone,

[22] Hillier SG Gonadotropic control of ovarian follicular growth and development Mol

[23] Hopps CV, et al. The diagnosis and treatment of the azoospermic patient in the age of intracytoplasmic sperm injection. Urol Clin North Am. 2002; 29: 895-911. [24] Adashi EY. Clomiphene citrate-initiated ovulation. A clinical update. Semin Reprod

[25] Mitwally MF, et al. Use of an aromatase inhibitor for induction of ovulation in patients with an inadequate response to clomiphene citrate. Fertil Steril. 2001; 75: 305-309. [26] Healy S, et al. Effects of letrozole on superovulation with gonadotropins in women undergoing intrauterine insemination. Fertil Steril. 2003; 80: 1325-1329. [27] Biljan MM, et al. The outcome of 150 babies following treatment with letrozole or letrozole and gonadotropins. ASRM/CFAS Annual Meeting; October 15-19, 2005. [28] Novartis warns doctors on off-label Femara use. Reuters Health. November 30, 2005. Available at: http://www.medscape.com/viewarticle/518136. 2005. [29] The Rotterdam ESHRE/ASRM-sponsored PCOS consensus workshop group Revised

2003 consensus on diagnostic criteria and long-term health risks related to

gestational diabetes in women with polycystic ovary syndrome. Fertil Steril. 2002;

polycystic ovary syndrome appears to safely reduce first-trimester abortion: a pilot

follicles on the outcome of multiple cycles of clomiphene-citrate intrauterine

[32] Glueck CJ, et al. Continuing metformin throughout pregnancy in women with

[33] Nestler JE, et al. Strategies for the use of insulin-sensitizing drugs to treat infertility in women with polycystic ovary syndrome. Fertil Steril. 2002; 77: 209-215. [34] Hughes EG The effectiveness of ovulation induction and intrauterine insemination in the treatment of persistent infertility: a meta-analysis. Hum Reprod. 1997; 12: 1865-1872. [35] Dickey R, et al. Effect of diagnosis, age, sperm quality, and number of preovulatory

[36] Arslan M, et al. Controlled ovarian hyperstimulation protocols for in vitro fertilization: two decades of experience after the birth of Elizabeth Carr. Fertil Steril. 2005; 84: 555-569. [37] Palermo GD, et al. Pregnancies after intracytoplasmic injection of single spermatozoon

polycystic ovarian syndrome (PCOS). Hum Reprod. 2003; 19: 41-47. [30] Costello MF, et al. A systematic review of the reproductive system effects of metformin in patients with polycystic ovary syndrome. Fertil Steril. 2003; 79: 1-13. [31] Glueck CJ, et al Metformin therapy throughout pregnancy reduces the development of

[21] National Center for Health Statistics. Infertility. Available at: http://www.cdc.gov/nchs/fastats/fertile.htm. 2005.

Cell Endocrinol. 2001; 179: 39-46.

Endocrinol. 1986; 4: 255-275.

855-864.

77: 520-525.

study. Fertil Steril. 2001; 75: 46-52.

insemination. Fertil Steril. 2002; 78: 1088-1095.

into an oocyte. Lancet. 1992; 340: 17-18.

inhibin-B, and three-dimensional ultrasound determinants of ovarian reserve in the prediction of poor response to controlled ovarian stimulation. Fertil Steril 2010; 93:

#### **1.1 Progesterone synthesis**

Progesterone was independently discovered by different research groups (Butenandt & Westphal 1934, Allen 1935). Allen and collaborators discovered progesterone in 1933, and were the first to determine the molecular weight and partial molecular structure. The name *progesterone* derives from progestational steroidal ketone *(Allen 1935).* 

Fig. 1. Steroid hormone synthesis. The precursor cholesterol from the maternal circulation is converted to 21 carbon (C21) progestagens. Progestagens can be converted to C21 glucocorticoids, or to C19 androgens. Androgens serve as precursors for C18 estrogens. Source: Wikipedia.

Progesterone in Human Pregnancy and Parturition 101

Fig. 2. The steroid hormone receptor family. A. Phylogenetic tree of the steroid hormone receptors showing the evolutionary interrelationships between the receptors . B. Sequence homologies of intracellular steroid hormone receptor proteins showing the N-terminal domain (A/B), the DNA-binding domain (DBD, C), the hinge region (D) and the C-terminal ligand

Steroid hormones have been shown to initiate rapid actions, which cannot be explained by the slow genomic mechanisms. Such rapid actions occur within seconds through the activation of intracellular signaling pathways resulting in alterations in ion fluxes and intracellular free calcium concentrations *(Blackmore et al 1991)*, and within minutes through the activation of other second messengers, such as cyclic nucleotides and extracellular-

binding domain (LBD, E). The human estrogen receptor subtypes (ERand ER), glucocorticoid receptor (GR), progesterone receptor isoforms (PRA and PRB), androgen receptor (AR), and mineralocorticoid receptor (MR) are described. The estrogen receptor is unique in that it contains an additional C-terminal F domain. Numbers represent the amino acid sequency of the receptors. In Griekspoor A, et al. Nuclear Receptor Signaling (2007) 5, e003.

**2.2 Non-genomic effects** 

Progesterone belongs to the C21 group of progestagens and is the evolutionary most conserved of the reproductive steroid hormones. The synthesis of progesterone from its precursor cholesterol in the maternal circulation requires only two enzymatic steps to form pregnenolone, which is readily transformed to progesterone. The main source of progesterone in humans is the corpus luteum in the ovary. After conception, the corpus luteum is supported by the secretion of human chorionic gonadotropin (hCG) from the conceptus, and produces progesterone until approximately the 10th gestational week. After a transition period by 7-10 gestational weeks the placenta becomes the major progesterone source, using circulating cholesterol as a substrate, after which maternal serum levels of progesterone increase markedly. Progesterone in serum is to 95-99% bound to corticosteroid binding globulin (CBG) almost as tightly as glucocorticoids *(Speroff et al 1994).*

Progesterone accompanies and modulates estrogen action. Whereas progesterone is synthesized in the placenta, neither the placenta nor the fetal adrenal glands are capable of producing sufficient quantities of precursors for estrogen synthesis. This observation led to the coining of the unique endocrine system "the maternal-fetal-placental unit" (*Diczfalucy 1969*). In early pregnancy, the maternal circulation provides androgen precursors for estrogen synthesis. By 20 gestational weeks the majority of androgen precursors, predominantly dehydroepiandrosterone sulphate (DHEAS), are derived from the fetal adrenals. The fetal compartment is extremely efficient in sulphate conjugation of steroid hormones, protecting the fetus from high steroid concentrations*.* About 30% of circulating estrogens are loosely bound to albumin, whereas the major amount is tightly bound to sex hormone binding globulin (SHBG) *(Speroff et al 1994).* 

#### **2. Mechanisms of action**

#### **2.1 Genomic effects**

The nuclear progesterone receptor (nPR) belongs to the steroid supergroup of transcription factor proteins (*O´Malley et al 1990*). All steroid receptor proteins are composed of a variable N-terminal domain which activates gene transcription and protein-protein interactions, determining the biological response of the steroid, an evolutionary highly conserved DNAbinding domain, a flexible hinge region and a C-terminal ligand-binding domain. The classic genomic mechanism of steroid action involving mRNA and protein synthesis is slow, occurring over hours to days.

The nPR binds to progesterone, and with a much less affinity to cortisol *(Sanborn et al 1976).* The biological response to progesterone is dependent on the levels and ratios of the nPR isoforms. The nPR isoforms A (nPR-A) (94 kDa) and nPR-B (116 kDa) are transcribed from the same gene, being activated by different promoters. The nPR-B isoform contains an additional 164 amino acids at the N-terminal and activates progesterone responsive genes. The nPR-A isoform is a weaker activator of transcription than nPR-B and can act as an inhibitor of nPR-B and other steroid receptors such as the nuclear estrogen (nER) and nuclear glucocorticoid (nGR) receptors *(Vegeto et al 1993, Pieber et al 2001).* A third nPR-C isoform has been identified in human myometrium *(Condon et al 2006).* nPR-C lacks a large segment of the N-terminal and a major part of the DNA-binding domain, and therefore cannot bind to DNA.

Progesterone belongs to the C21 group of progestagens and is the evolutionary most conserved of the reproductive steroid hormones. The synthesis of progesterone from its precursor cholesterol in the maternal circulation requires only two enzymatic steps to form pregnenolone, which is readily transformed to progesterone. The main source of progesterone in humans is the corpus luteum in the ovary. After conception, the corpus luteum is supported by the secretion of human chorionic gonadotropin (hCG) from the conceptus, and produces progesterone until approximately the 10th gestational week. After a transition period by 7-10 gestational weeks the placenta becomes the major progesterone source, using circulating cholesterol as a substrate, after which maternal serum levels of progesterone increase markedly. Progesterone in serum is to 95-99% bound to corticosteroid binding globulin (CBG) almost as tightly as glucocorticoids

Progesterone accompanies and modulates estrogen action. Whereas progesterone is synthesized in the placenta, neither the placenta nor the fetal adrenal glands are capable of producing sufficient quantities of precursors for estrogen synthesis. This observation led to the coining of the unique endocrine system "the maternal-fetal-placental unit" (*Diczfalucy 1969*). In early pregnancy, the maternal circulation provides androgen precursors for estrogen synthesis. By 20 gestational weeks the majority of androgen precursors, predominantly dehydroepiandrosterone sulphate (DHEAS), are derived from the fetal adrenals. The fetal compartment is extremely efficient in sulphate conjugation of steroid hormones, protecting the fetus from high steroid concentrations*.* About 30% of circulating estrogens are loosely bound to albumin, whereas the major amount is tightly bound to sex

The nuclear progesterone receptor (nPR) belongs to the steroid supergroup of transcription factor proteins (*O´Malley et al 1990*). All steroid receptor proteins are composed of a variable N-terminal domain which activates gene transcription and protein-protein interactions, determining the biological response of the steroid, an evolutionary highly conserved DNAbinding domain, a flexible hinge region and a C-terminal ligand-binding domain. The classic genomic mechanism of steroid action involving mRNA and protein synthesis is slow,

The nPR binds to progesterone, and with a much less affinity to cortisol *(Sanborn et al 1976).* The biological response to progesterone is dependent on the levels and ratios of the nPR isoforms. The nPR isoforms A (nPR-A) (94 kDa) and nPR-B (116 kDa) are transcribed from the same gene, being activated by different promoters. The nPR-B isoform contains an additional 164 amino acids at the N-terminal and activates progesterone responsive genes. The nPR-A isoform is a weaker activator of transcription than nPR-B and can act as an inhibitor of nPR-B and other steroid receptors such as the nuclear estrogen (nER) and nuclear glucocorticoid (nGR) receptors *(Vegeto et al 1993, Pieber et al 2001).* A third nPR-C isoform has been identified in human myometrium *(Condon et al 2006).* nPR-C lacks a large segment of the N-terminal and a major part of the DNA-binding domain, and therefore

*(Speroff et al 1994).*

hormone binding globulin (SHBG) *(Speroff et al 1994).* 

**2. Mechanisms of action** 

occurring over hours to days.

cannot bind to DNA.

**2.1 Genomic effects** 

Fig. 2. The steroid hormone receptor family. A. Phylogenetic tree of the steroid hormone receptors showing the evolutionary interrelationships between the receptors . B. Sequence homologies of intracellular steroid hormone receptor proteins showing the N-terminal domain (A/B), the DNA-binding domain (DBD, C), the hinge region (D) and the C-terminal ligand binding domain (LBD, E). The human estrogen receptor subtypes (ERand ER), glucocorticoid receptor (GR), progesterone receptor isoforms (PRA and PRB), androgen receptor (AR), and mineralocorticoid receptor (MR) are described. The estrogen receptor is unique in that it contains an additional C-terminal F domain. Numbers represent the amino acid sequency of the receptors. In Griekspoor A, et al. Nuclear Receptor Signaling (2007) 5, e003.

#### **2.2 Non-genomic effects**

Steroid hormones have been shown to initiate rapid actions, which cannot be explained by the slow genomic mechanisms. Such rapid actions occur within seconds through the activation of intracellular signaling pathways resulting in alterations in ion fluxes and intracellular free calcium concentrations *(Blackmore et al 1991)*, and within minutes through the activation of other second messengers, such as cyclic nucleotides and extracellular-

Progesterone in Human Pregnancy and Parturition 103

Serum levels of progesterone increase progressively during human pregnancy and remain high until delivery of the placenta *(Csapo et al 1973, Speroff et al 1994, Stjernholm et al 1997).* In other species, such as rodents and rabbits, which depend on an active corpus luteum for progesterone synthesis throughout pregnancy, labor is initiated by prostaglandin F2PGF2 from the endometrium, activating prostaglandin F (FP)-receptors in the corpus luteum leading to luteolysis *(Sugimoto et al 1997).* These observations led to the concept of a "functional progesterone withdrawal" at parturition in humans *(Hertelendy & Zakar 2004).*

Placental concentrations of progesterone reach 1-10 M (*Stites & Siiteri 1983, Miyaura & Iwata 2002),* whereas serum concentrations reach 100-500 nM until term pregnancy before labor

Increased levels of prostacyclin (PGI2) are considered to be a factor behind the physiological angiotensin resistance observed in normal pregnancy *(Friedman 1988).* A progesterone induced mechanism behind this refractoriness to angiotensin has been suggested *(Everett et al 1978, Rupnow et al 2002).* Nitric oxide (NO) and protein kinase C (PKC) pathways are involved in the regulation of vascular tone during pregnancy *(Kublickiene et al 1997, Chang et* 

The pulmonary function is not impaired by pregnancy, but the tidal volume, minute ventilator volume and minute oxygen uptake increase with advancing gestation. This pregnancy-induced respiratory alkalosis is partially compensated for by increased renal excretion of bicarbonate. As a consequence, maternal arterial pH is increased to 7.46. The increased respiratory effort and decrease in PCO2 has been related to progesterone and to a

Successful maintenance of pregnancy depends on maternal tolerance of the fetal semiallograft *(Szekeres-Bartho 2002).* Progesterone, cortisol and prolactin have strong immunomodulatory effects leading to immunotolerance during pregnancy (*Stites & Siiteri 1983, Speroff et al 1994)*. The human decidua is adjacent to the myometrium, the fetal trophoblasts of the placenta and to the fetal membranes. Natural killer (NK) cells is the predominant immune cell in the decidua before implantation and in early pregnancy, constituting 70% of decidual immune cells, followed by macrophages constituting about 10%of total decidual cells, dendritic cells (DC) and T lymphocytes. The local endocrine environment regulates the recruitment of monocytes into the uterus, and the subsequent differentiation of monocytes into macrophages with specific phenotypes promoting immunotolerance or inflammation (*Stout et al 2004*). Colony-stimulating factor (CSF)-1, macrophage migration inhibitory factor (MIF), monocyte chemoattractant protein (MCP)-1 and regulated on activation, normal T cell expressed, and secreted (RANTES) have been

**3. Systemic effects** 

**3.1 The placenta** 

*al 2008).* 

**4. The uterus** 

**3.2 The vascular system** 

**3.3 The respiratory system**

**4.1 The decidua at implantation** 

*(Stjernholm et al 1997; Miyaura & Iwata 2002).* 

lesser degree to estrogen *(Wolfe et al 1998, Jensen et al 2005).*

regulated kinase (ERK) 1 and 2 (*Filardo et al 2000)*. Recently, three new putative membrane progesterone receptors (mPRs), mPRα, mPRβ, and mPRγ were identified in humans (*Zhu et al 2003*).

Fig. 3. G (guanine nucleotide-binding) protein-coupled transmembrane (TM) receptors communicate signals from hormones and other signaling factors to intracellular messengers. They consist of the Gand the tightly associated G subunits. Here guanosinetriphosphate (GTP) is hydrolyzed by Gsubunit to guanosine-diphosphate (GDP). Source: CellMosaic, Worcester, MA, US.

Fig. 4. Nuclear and transmembrane-bound progesterone receptors mediating genomic and non-genomic effects. Progesterone (P) activates A. Genomic pathways through nuclear receptor proteins (PR-A, PR-B) resulting in gene activation (slow process) and/or B. Nongenomic pathways via membrane-bound receptors, which activate secondary messengers (fast process).

#### **3. Systemic effects**

102 Sex Hormones

regulated kinase (ERK) 1 and 2 (*Filardo et al 2000)*. Recently, three new putative membrane progesterone receptors (mPRs), mPRα, mPRβ, and mPRγ were identified in humans (*Zhu et* 

Fig. 3. G (guanine nucleotide-binding) protein-coupled transmembrane (TM) receptors communicate signals from hormones and other signaling factors to intracellular messengers.

triphosphate (GTP) is hydrolyzed by Gsubunit to guanosine-diphosphate (GDP). Source:

Fig. 4. Nuclear and transmembrane-bound progesterone receptors mediating genomic and non-genomic effects. Progesterone (P) activates A. Genomic pathways through nuclear receptor proteins (PR-A, PR-B) resulting in gene activation (slow process) and/or B. Nongenomic pathways via membrane-bound receptors, which activate secondary messengers

They consist of the Gand the tightly associated G subunits. Here guanosine-

*al 2003*).

CellMosaic, Worcester, MA, US.

(fast process).

Serum levels of progesterone increase progressively during human pregnancy and remain high until delivery of the placenta *(Csapo et al 1973, Speroff et al 1994, Stjernholm et al 1997).* In other species, such as rodents and rabbits, which depend on an active corpus luteum for progesterone synthesis throughout pregnancy, labor is initiated by prostaglandin F2PGF2 from the endometrium, activating prostaglandin F (FP)-receptors in the corpus luteum leading to luteolysis *(Sugimoto et al 1997).* These observations led to the concept of a "functional progesterone withdrawal" at parturition in humans *(Hertelendy & Zakar 2004).*

#### **3.1 The placenta**

Placental concentrations of progesterone reach 1-10 M (*Stites & Siiteri 1983, Miyaura & Iwata 2002),* whereas serum concentrations reach 100-500 nM until term pregnancy before labor *(Stjernholm et al 1997; Miyaura & Iwata 2002).* 

#### **3.2 The vascular system**

Increased levels of prostacyclin (PGI2) are considered to be a factor behind the physiological angiotensin resistance observed in normal pregnancy *(Friedman 1988).* A progesterone induced mechanism behind this refractoriness to angiotensin has been suggested *(Everett et al 1978, Rupnow et al 2002).* Nitric oxide (NO) and protein kinase C (PKC) pathways are involved in the regulation of vascular tone during pregnancy *(Kublickiene et al 1997, Chang et al 2008).* 

#### **3.3 The respiratory system**

The pulmonary function is not impaired by pregnancy, but the tidal volume, minute ventilator volume and minute oxygen uptake increase with advancing gestation. This pregnancy-induced respiratory alkalosis is partially compensated for by increased renal excretion of bicarbonate. As a consequence, maternal arterial pH is increased to 7.46. The increased respiratory effort and decrease in PCO2 has been related to progesterone and to a lesser degree to estrogen *(Wolfe et al 1998, Jensen et al 2005).*

#### **4. The uterus**

#### **4.1 The decidua at implantation**

Successful maintenance of pregnancy depends on maternal tolerance of the fetal semiallograft *(Szekeres-Bartho 2002).* Progesterone, cortisol and prolactin have strong immunomodulatory effects leading to immunotolerance during pregnancy (*Stites & Siiteri 1983, Speroff et al 1994)*. The human decidua is adjacent to the myometrium, the fetal trophoblasts of the placenta and to the fetal membranes. Natural killer (NK) cells is the predominant immune cell in the decidua before implantation and in early pregnancy, constituting 70% of decidual immune cells, followed by macrophages constituting about 10%of total decidual cells, dendritic cells (DC) and T lymphocytes. The local endocrine environment regulates the recruitment of monocytes into the uterus, and the subsequent differentiation of monocytes into macrophages with specific phenotypes promoting immunotolerance or inflammation (*Stout et al 2004*). Colony-stimulating factor (CSF)-1, macrophage migration inhibitory factor (MIF), monocyte chemoattractant protein (MCP)-1 and regulated on activation, normal T cell expressed, and secreted (RANTES) have been

Progesterone in Human Pregnancy and Parturition 105

The progesterone induced protective immune environment in the decidua during early pregnancy includes production of the immunomodulatory progesterone-induced blocking factor (PIBF) protein by decidual cells *(Szekeres-Bartho et al 1985, Piccinni et al 1995).* The presence of nPRs in immune cells has been debated. nPRs in the thymus are necessary for progesterone induced involution of the thymus during pregnancy (*Tibbetts et al 1999).* Most studies have showed an absence of nPRs in lymphocytes from nonpregnant women *(Szekeres-Bartho et al 1990, Mansour et al 1994, Bamberger et al 1999).* Recently, transcripts for mPRand mPRbut not mPR, were detected in human peripheral blood leukocytes and T lymphocytes. Progesterone activated an inhibitory G-protein (Gi), suggesting that mPRs are coupled to Gi. These results suggest a potential novel mechanism for progesterone's

The establishment of human pregnancy is associated with an adequate synthesis of leukemia inhibitory factor (LIF), and macrophage colony-stimulating factor (M-CSF) producing T-cells. Progesterone at concentrations comparable to those in the maternal-fetal

The corpus uteri is a muscular organ with about 70% smooth muscle cells surrounded by extracellular matrix (Danforth 1954). Progesterone is holding the uterine myometrium in a quiescent state, "a progesterone block", during pregnancy by suppressing the propagation of electrical activity between the excitable myocyte membranes (*Csapo 1956, Csapo et al 1973*). The genomic and nongenomic pathways co-operate to maintain myometrial relaxation. At parturition, a functional progesterone withdrawal occurs by increased expression of the nPR-A and/or nPR-C to nPR-B ratios and changes in nPR co-regulator levels which result in repression of the nPR-B transcriptional activity. The diminished progesterone influence leads to an estrogen dominance *(Mesiano et al 2002).* Prostaglandins have been shown to induce an increased nPR-A/nPR-B ratio through the protein kinase C (PKC) pathway in human myometrial cells *(Madsen et al 2004).* Proinflammatory IL-1 up-regulates nPR-C in human myometrial cells, leading to diminished activation of nPR-B *(Condon et al 2006).* The increased expression of specific membrane-associated PRs (mPRs) at parturition augments contractility by decreasing intracellular cyclic adenosine monophosphate (cAMP) and altering intracellular Ca2+ levels. *(Pieber et al 2001, Mesiano et al 2002, Madsen et al 2004,* 

The cervix uteri is up to 85% composed by connective tissue, which is dominated by collagen fibers. Fibroblasts, smooth muscle cells, T and B lymphocytes, leukocytes and Langerhans cells are scattered within the tissue (Danforth & Evanston 1954, Schwalm & Dubrauszky 1966, White et al 1997). Cervical remodeling is a prerequisite for cervical effacement and dilatation prior to labor and is characterized by increased levels of vascular adhesion molecules (VCAM), diapedesis and activation of neutrophils, monocytes/macrophages, T lymphocytes, mast cells, eosinophils, the release of proinflammatory cytokines such as IL-1and the strong chemotactor IL-8, and increased tissue concentrations of metalloproteinase enzymes (MMPs) *(Junquiera et al 1980, Liggins 1981, Uldbjerg et al 1983, Bokström et al 1997, Sennström et al 2000, Stygar et al 2002, Winkler et al 2003*). At parturition, a functional progesterone withdrawal occurs in the cervix uteri with decreased total nPR and an increased nPR-A/nPR-B ratio

immunoregulatory function through activation of mPRs (*Dosiou et al 2008*).

interface induces LIF and M-CSF (*Piccinni 2010*).

**4.2 The myometrium** 

*Mesiano 2007).* 

**5. The cervix uteri** 

suggested as factors involved in the recruitment and modulation of decidual macrophages, and are synthesized by decidual stromal cells, NK-cells and trophoblasts at the maternalfetal interface (*Wood et al, 1997; Lockwood et al 2006*). Resident decidual macrophages appear to express immunosuppressive actions that favor the maintenance of pregnancy. In contrast, monocytes/macrophages migrating into the lower uterine segment prior to parturition are involved in the inflammatory process associated with cervical ripening and labor initiation *(Nagamatsu et al 2010).* A switch in decidual type 1 (Th1) to type 2 (Th2) T cell dominance in the fetal-placental interface has been suggested to play a crucial role in the establishment of pregnancy (*Wegmann et al 1993*). Human Th1 T-cells are the main effectors of host defence and Th1-type cytokines produce proinflammtory responses. The Th-1 response involves interferon (IFN)-, interleukin-2 (IL-2), tumor necrosis factor (TNF)-, and the generation of cell-mediated immunity. On the contrary, human Th2 T cells inhibit macrophage functions. A Th2 response involves IL-4, IL-5, anti-inflammatory IL-10, IL-10, IL-13, and the stimulation of humoral immunity *(Abbas et al 1996, Weiner et al 2001).* 

Fig. 5. Endocrine and immune cross-talk in the fetal-maternal interface at implantation. CL= corpus luteum, HCG= human chorionic gonadotropin. In Fujiwara H. Molecular Human Reproduction (2009) 15, 335–343.

Progesterone at concentrations higher than in serum but comparable to those in the maternal-fetal interface induces differentiation of T cells along the Th2 pathway (*Stites & Siiteri 1983; Piccinni et al 1995, Miyaura & Iwata 2002*). Glucocorticoids and 1,25-dihydroxy Vitamin D increase IL-4 (*Rook et al 1994*), whereas dihydrotestosterone decreases IL-4 and IL-5 production (*Vacca et al 1990*).

The progesterone induced protective immune environment in the decidua during early pregnancy includes production of the immunomodulatory progesterone-induced blocking factor (PIBF) protein by decidual cells *(Szekeres-Bartho et al 1985, Piccinni et al 1995).* The presence of nPRs in immune cells has been debated. nPRs in the thymus are necessary for progesterone induced involution of the thymus during pregnancy (*Tibbetts et al 1999).* Most studies have showed an absence of nPRs in lymphocytes from nonpregnant women *(Szekeres-Bartho et al 1990, Mansour et al 1994, Bamberger et al 1999).* Recently, transcripts for mPRand mPRbut not mPR, were detected in human peripheral blood leukocytes and T lymphocytes. Progesterone activated an inhibitory G-protein (Gi), suggesting that mPRs are coupled to Gi. These results suggest a potential novel mechanism for progesterone's immunoregulatory function through activation of mPRs (*Dosiou et al 2008*).

The establishment of human pregnancy is associated with an adequate synthesis of leukemia inhibitory factor (LIF), and macrophage colony-stimulating factor (M-CSF) producing T-cells. Progesterone at concentrations comparable to those in the maternal-fetal interface induces LIF and M-CSF (*Piccinni 2010*).

#### **4.2 The myometrium**

104 Sex Hormones

suggested as factors involved in the recruitment and modulation of decidual macrophages, and are synthesized by decidual stromal cells, NK-cells and trophoblasts at the maternalfetal interface (*Wood et al, 1997; Lockwood et al 2006*). Resident decidual macrophages appear to express immunosuppressive actions that favor the maintenance of pregnancy. In contrast, monocytes/macrophages migrating into the lower uterine segment prior to parturition are involved in the inflammatory process associated with cervical ripening and labor initiation *(Nagamatsu et al 2010).* A switch in decidual type 1 (Th1) to type 2 (Th2) T cell dominance in the fetal-placental interface has been suggested to play a crucial role in the establishment of pregnancy (*Wegmann et al 1993*). Human Th1 T-cells are the main effectors of host defence and Th1-type cytokines produce proinflammtory responses. The Th-1 response involves interferon (IFN)-, interleukin-2 (IL-2), tumor necrosis factor (TNF)-, and the generation of cell-mediated immunity. On the contrary, human Th2 T cells inhibit macrophage functions. A Th2 response involves IL-4, IL-5, anti-inflammatory IL-10, IL-10, IL-13, and the

Fig. 5. Endocrine and immune cross-talk in the fetal-maternal interface at implantation. CL= corpus luteum, HCG= human chorionic gonadotropin. In Fujiwara H. Molecular Human

Progesterone at concentrations higher than in serum but comparable to those in the maternal-fetal interface induces differentiation of T cells along the Th2 pathway (*Stites & Siiteri 1983; Piccinni et al 1995, Miyaura & Iwata 2002*). Glucocorticoids and 1,25-dihydroxy Vitamin D increase IL-4 (*Rook et al 1994*), whereas dihydrotestosterone decreases IL-4 and

Reproduction (2009) 15, 335–343.

IL-5 production (*Vacca et al 1990*).

stimulation of humoral immunity *(Abbas et al 1996, Weiner et al 2001).* 

The corpus uteri is a muscular organ with about 70% smooth muscle cells surrounded by extracellular matrix (Danforth 1954). Progesterone is holding the uterine myometrium in a quiescent state, "a progesterone block", during pregnancy by suppressing the propagation of electrical activity between the excitable myocyte membranes (*Csapo 1956, Csapo et al 1973*). The genomic and nongenomic pathways co-operate to maintain myometrial relaxation. At parturition, a functional progesterone withdrawal occurs by increased expression of the nPR-A and/or nPR-C to nPR-B ratios and changes in nPR co-regulator levels which result in repression of the nPR-B transcriptional activity. The diminished progesterone influence leads to an estrogen dominance *(Mesiano et al 2002).* Prostaglandins have been shown to induce an increased nPR-A/nPR-B ratio through the protein kinase C (PKC) pathway in human myometrial cells *(Madsen et al 2004).* Proinflammatory IL-1 up-regulates nPR-C in human myometrial cells, leading to diminished activation of nPR-B *(Condon et al 2006).* The increased expression of specific membrane-associated PRs (mPRs) at parturition augments contractility by decreasing intracellular cyclic adenosine monophosphate (cAMP) and altering intracellular Ca2+ levels. *(Pieber et al 2001, Mesiano et al 2002, Madsen et al 2004, Mesiano 2007).* 

#### **5. The cervix uteri**

The cervix uteri is up to 85% composed by connective tissue, which is dominated by collagen fibers. Fibroblasts, smooth muscle cells, T and B lymphocytes, leukocytes and Langerhans cells are scattered within the tissue (Danforth & Evanston 1954, Schwalm & Dubrauszky 1966, White et al 1997). Cervical remodeling is a prerequisite for cervical effacement and dilatation prior to labor and is characterized by increased levels of vascular adhesion molecules (VCAM), diapedesis and activation of neutrophils, monocytes/macrophages, T lymphocytes, mast cells, eosinophils, the release of proinflammatory cytokines such as IL-1and the strong chemotactor IL-8, and increased tissue concentrations of metalloproteinase enzymes (MMPs) *(Junquiera et al 1980, Liggins 1981, Uldbjerg et al 1983, Bokström et al 1997, Sennström et al 2000, Stygar et al 2002, Winkler et al 2003*). At parturition, a functional progesterone withdrawal occurs in the cervix uteri with decreased total nPR and an increased nPR-A/nPR-B ratio

Progesterone in Human Pregnancy and Parturition 107

suggested as the primary functions of PGs in human parturition *(Hertelendy & Zakar 2004)*. Human decidual macrophages synthetize PGs (*Norwitz 1991*). Mechanical stretch of the lower uterine segment, proinflammatory cytokines such as IL-1 and the peptide hormone oxytocin

Successful treatment with PG-E2 for cervical priming before labor induction, allowing for resulting in cervical effacement and dilatation allowing for parturition was associated with diminished cervical progesterone and androgen receptor concentrations *(Vladic Stjernholm,* 

After delivery of the placenta, serum concentrations of estrogen and progesterone decrease within hours, and the puerperuim (*puer*: infant, *pario*: give birth) is a hypoestrogenic and hypoprogestagenic state. The high progesterone level during pregnancy inhibits lactation. The fall in progesterone levels after delivery is one factor that stimulates milk production

Natural progesterone and synthetic progestins do both exert a progestogenic effect, defined as the decidualizing effect on estrogen-primed rabbit endometium *(Elton 1966, Schindler et al* 

In clinical practice, progestin treatment was practised since the 1950s as luteal phase support to prevent miscarriage during the first trimester of pregnancy. The amount of data from wellcontrolled clinical trials is limited. Further studies are required to establish the optimal treatment situation as well as type and dose of progestin *(LeVine et al 1964, Daya & Gunby 2004).*

Since the 1960s studies on treatment with synthetic progestins for preventing premature childbirth have reported beneficial effects. Human pregnancy lasts 40 gestational weeks and birth between 22 and 37 weeks is defined as premature *(WHO 1977*). The highly active progesterone ester 17-hydroxyprogestrone caproate has a long duration allowing for

induce PG synthesis *(Molnar et al 1999, Allport et al 2001, Leguizamon et al 2001)*.

*2009).*

**7. The puerperium** 

**8. Progestin and progesterone treatment** 

**8.1 Progestins and progesterone for preventing miscarriage** 

**8.2 Progestins and progesterone for preventing premature birth** 

Fig. 7. The synthetic progestin 17-hydroxyprogesterone caproate.

*(Tucker 1979).*

*2003).*

*(Stjernholm et al 1997, Vladic Stjernholm et al 2004).* These endocrine and inflammatory events are followed by an up to 30-50% decreased collagen concentration, and an altered proteoglycan composition with a decreased density of the small proteoglycan decorin and an increased density of the large proteoglycan Versican. These events result in dispersed collagen fibrils clinically recognized as cervical effacement and dilatation (*Uldbjerg et al 1983, Ekman et al 1986, Norman et al 1993, Stjernholm et al 1997).* Evidence suggest that progesterone effects on the cervix uteri are even more pronounced than its effects on the myometrium (*Romero 2007*).

Fig. 6. Cervical effacement and dilatation before labor onset.

#### **6. Parturition**

#### **6.1 Animal studies**

Classical experiments in sheep demonstrated that parturition in this species is initiated by activation of the fetal hypothalamic pituitary adrenal (HPA) axis leading to increased fetal cortisol secretion and induction of placental P450 enzymes (17-hydroxylase and 17-20-lyase activities), which favor the conversion of C21 to C18 steroids *(Liggins 1974, Anderson et al 1975).*

#### **6.2 Human parturition**

Progesterone is the main progestational hormone in humans, whereas the HPA axis has a modulatory function *(Hertelendy & Zakar 2004).* Prostaglandins (PGs) from the E and F series are considered to be the main promoters of cervical ripening and myometrial contractility, and the influence of PG-E2 in promotion of cervical maturation and uterine vasodilatation has been suggested as the primary functions of PGs in human parturition *(Hertelendy & Zakar 2004)*. Human decidual macrophages synthetize PGs (*Norwitz 1991*). Mechanical stretch of the lower uterine segment, proinflammatory cytokines such as IL-1 and the peptide hormone oxytocin induce PG synthesis *(Molnar et al 1999, Allport et al 2001, Leguizamon et al 2001)*.

Successful treatment with PG-E2 for cervical priming before labor induction, allowing for resulting in cervical effacement and dilatation allowing for parturition was associated with diminished cervical progesterone and androgen receptor concentrations *(Vladic Stjernholm, 2009).*

### **7. The puerperium**

106 Sex Hormones

*(Stjernholm et al 1997, Vladic Stjernholm et al 2004).* These endocrine and inflammatory events are followed by an up to 30-50% decreased collagen concentration, and an altered proteoglycan composition with a decreased density of the small proteoglycan decorin and an increased density of the large proteoglycan Versican. These events result in dispersed collagen fibrils clinically recognized as cervical effacement and dilatation (*Uldbjerg et al 1983, Ekman et al 1986, Norman et al 1993, Stjernholm et al 1997).* Evidence suggest that progesterone effects on the cervix uteri are even more pronounced than its effects on the myometrium (*Romero 2007*).

Fig. 6. Cervical effacement and dilatation before labor onset.

Classical experiments in sheep demonstrated that parturition in this species is initiated by activation of the fetal hypothalamic pituitary adrenal (HPA) axis leading to increased fetal cortisol secretion and induction of placental P450 enzymes (17-hydroxylase and 17-20-lyase activities), which favor the conversion of C21 to C18 steroids *(Liggins 1974, Anderson et al 1975).*

Progesterone is the main progestational hormone in humans, whereas the HPA axis has a modulatory function *(Hertelendy & Zakar 2004).* Prostaglandins (PGs) from the E and F series are considered to be the main promoters of cervical ripening and myometrial contractility, and the influence of PG-E2 in promotion of cervical maturation and uterine vasodilatation has been

**6. Parturition** 

**6.1 Animal studies** 

**6.2 Human parturition** 

After delivery of the placenta, serum concentrations of estrogen and progesterone decrease within hours, and the puerperuim (*puer*: infant, *pario*: give birth) is a hypoestrogenic and hypoprogestagenic state. The high progesterone level during pregnancy inhibits lactation. The fall in progesterone levels after delivery is one factor that stimulates milk production *(Tucker 1979).*

#### **8. Progestin and progesterone treatment**

Natural progesterone and synthetic progestins do both exert a progestogenic effect, defined as the decidualizing effect on estrogen-primed rabbit endometium *(Elton 1966, Schindler et al 2003).*

#### **8.1 Progestins and progesterone for preventing miscarriage**

In clinical practice, progestin treatment was practised since the 1950s as luteal phase support to prevent miscarriage during the first trimester of pregnancy. The amount of data from wellcontrolled clinical trials is limited. Further studies are required to establish the optimal treatment situation as well as type and dose of progestin *(LeVine et al 1964, Daya & Gunby 2004).*

#### **8.2 Progestins and progesterone for preventing premature birth**

Since the 1960s studies on treatment with synthetic progestins for preventing premature childbirth have reported beneficial effects. Human pregnancy lasts 40 gestational weeks and birth between 22 and 37 weeks is defined as premature *(WHO 1977*). The highly active progesterone ester 17-hydroxyprogestrone caproate has a long duration allowing for

Fig. 7. The synthetic progestin 17-hydroxyprogesterone caproate.

Progesterone in Human Pregnancy and Parturition 109

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intramuscular administration 1-3 times weekly. It has been administered to risk groups with previous recurrent abortions or previous premature births and to patients with premature contractions and short cervices *(Johnsson et al 1976, Meis et al 2003, Dodd et al 2006*). Natural progesterone has been administered as of vaginal gel to such risk groups and in situations with premature contractions and short cervices *(daFonseca et al 2003, deFranco et al 2007, O´Brien et al 2007).* Reduced incidence of premature birth before 32, 34 and 37 gestational weeks and improved neonatal outcome were reported (*Brent 2005).* Further studies are required to establish the optimal dose and type of agent as well as long term effects on the newborn.

#### **9. Summary**

Progesterone is the evolutionary most conserved of the reproductive steroid hormones. It is the main progestational hormone in humans, and its strong immunomodulatory effects are important for the physiological immunotolerance at implantation. After a transition period by 7-10 gestational weeks the placenta becomes the major progesterone source. Placental concentrations of progesterone reach 1-10 M and serum concentrations 100-500 nM until term pregnancy. Progesterone exerts its effects through genomic nuclear receptor mediated and non-genomic transmembrane receptor mediated processes, keeping the myometrium in a quiescent state and stabilizing the cervix uteri during pregnancy. A functional progesterone withdrawal occurs at human parturition with a diminished total receptor density and altered isoform ratios. In clinical practice, progestin treatment has been given as luteal phase support to prevent miscarriage during the first trimester of pregnancy. Treatment with synthetic progestins and natural progesterone has been shown to reduce the incidence of premature birth. Further studies are required to establish the optimal dose and type of agent as well as long term effects on the newborn.

#### **10. Acknowledgements**

I thank the editor for inviting me to present this work and Dr Tomislav Vladic for critical comments.

#### **11. References**

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Allen WM (1935) The isolation of crystalline progestin. Science 82 (2118): 89–93.


intramuscular administration 1-3 times weekly. It has been administered to risk groups with previous recurrent abortions or previous premature births and to patients with premature contractions and short cervices *(Johnsson et al 1976, Meis et al 2003, Dodd et al 2006*). Natural progesterone has been administered as of vaginal gel to such risk groups and in situations with premature contractions and short cervices *(daFonseca et al 2003, deFranco et al 2007, O´Brien et al 2007).* Reduced incidence of premature birth before 32, 34 and 37 gestational weeks and improved neonatal outcome were reported (*Brent 2005).* Further studies are required to establish the optimal dose and type of agent as well as long term effects on the

Progesterone is the evolutionary most conserved of the reproductive steroid hormones. It is the main progestational hormone in humans, and its strong immunomodulatory effects are important for the physiological immunotolerance at implantation. After a transition period by 7-10 gestational weeks the placenta becomes the major progesterone source. Placental concentrations of progesterone reach 1-10 M and serum concentrations 100-500 nM until term pregnancy. Progesterone exerts its effects through genomic nuclear receptor mediated and non-genomic transmembrane receptor mediated processes, keeping the myometrium in a quiescent state and stabilizing the cervix uteri during pregnancy. A functional progesterone withdrawal occurs at human parturition with a diminished total receptor density and altered isoform ratios. In clinical practice, progestin treatment has been given as luteal phase support to prevent miscarriage during the first trimester of pregnancy. Treatment with synthetic progestins and natural progesterone has been shown to reduce the incidence of premature birth. Further studies are required to establish the optimal dose and

I thank the editor for inviting me to present this work and Dr Tomislav Vladic for critical

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**6** 

*Poland* 

Andrzej Gomuła\*

*The Andropause Institute, Warsaw,* 

**Late - Onset Hypogonadism - New Point of View** 

Long-term testosterone deficiency related to age may adversely affect health, anatomy and physiology of man. The implementation of testosterone boost therapy only at the time, when for many years, as the result of testosterone deficiency, irreversible anatomical changes have occurred, is clearly too late. Age-related progressive decrease in testosterone serum concentration levels causes anatomical and functional abnormalities. It is the cause of lipid disorders; it excarberates type-2 diabetes, it is also the common cause of cardiovascular diseases. It contributes to other health problems such as atherosclerosis, hypertension, osteoporosis and obesity and it manifests itself by decreased libido and potency. There is also a strong relationship between age-related decrease in testosterone and Parkinson's disease and Alzheimer's disease. Benign prostatic hyperplasia (BPH) and carcinoma of the prostate are closely associated with testosterone deficiency and comedo-carcinoma — the most malignant form of prostate cancer — is directly proportional to the decrease in serum testosterone. A good therapy for increasing testosterone serum levels can reverse the problems associated with aging such as type-2 diabetes, sexual dysfunction, osteoporosis, hyper-lipidemia and ischemic heart disease. It can even reverse symptoms of Parkinson's disease. Using synthetic testosterone is often recommended in the treatment of testosterone deficiency. Unfortunately, synthetic testosterone can cause side effects such as infertility and a long-term use of testosterone may also lead to irreversible testicular atrophy. Therefore, patients receiving long-term testosterone therapy are all dependent on adequate doses of synthetic testosterone until the end of their lives. Meanwhile, intramuscular administration of hCG to stimulate the endogenous testosterone synthesis, has been known since the 1950s. The induction of endogenous testosterone production by hCG has been effective in all age groups while being safe at the same time. In this paper, the author presents problems caused by testosterone deficiency and outlines the possibility of the treatment, which increases the induction of testosterone endosynthesis by hCG. It has not yet been determined how to diagnose testosterone deficiency. The age-related serum testosterone concentration reference range has not been established yet either. The paper presents the first attempt to establish international standards for testosterone serum concentration levels in different age groups.

Late-onset hypogonadism (LOH) is a testosterone deficiency syndrome resulting in the aging process. LOH leads to metabolic disorders and functional or anatomical abnormalities. It is

**1. Introduction** 

**2. Late-onset hypogonadism** 

Corresponding Author

 \*


### **Late - Onset Hypogonadism - New Point of View**

### Andrzej Gomuła\*

*The Andropause Institute, Warsaw, Poland* 

#### **1. Introduction**

114 Sex Hormones

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Dev 46:62-70.

RANTES in macrophage recruitment during successful pregnancy. Mol Reprod

of genes in humans and other vertebrates homologous to a fish membrane

Long-term testosterone deficiency related to age may adversely affect health, anatomy and physiology of man. The implementation of testosterone boost therapy only at the time, when for many years, as the result of testosterone deficiency, irreversible anatomical changes have occurred, is clearly too late. Age-related progressive decrease in testosterone serum concentration levels causes anatomical and functional abnormalities. It is the cause of lipid disorders; it excarberates type-2 diabetes, it is also the common cause of cardiovascular diseases. It contributes to other health problems such as atherosclerosis, hypertension, osteoporosis and obesity and it manifests itself by decreased libido and potency. There is also a strong relationship between age-related decrease in testosterone and Parkinson's disease and Alzheimer's disease. Benign prostatic hyperplasia (BPH) and carcinoma of the prostate are closely associated with testosterone deficiency and comedo-carcinoma — the most malignant form of prostate cancer — is directly proportional to the decrease in serum testosterone. A good therapy for increasing testosterone serum levels can reverse the problems associated with aging such as type-2 diabetes, sexual dysfunction, osteoporosis, hyper-lipidemia and ischemic heart disease. It can even reverse symptoms of Parkinson's disease. Using synthetic testosterone is often recommended in the treatment of testosterone deficiency. Unfortunately, synthetic testosterone can cause side effects such as infertility and a long-term use of testosterone may also lead to irreversible testicular atrophy. Therefore, patients receiving long-term testosterone therapy are all dependent on adequate doses of synthetic testosterone until the end of their lives. Meanwhile, intramuscular administration of hCG to stimulate the endogenous testosterone synthesis, has been known since the 1950s. The induction of endogenous testosterone production by hCG has been effective in all age groups while being safe at the same time. In this paper, the author presents problems caused by testosterone deficiency and outlines the possibility of the treatment, which increases the induction of testosterone endosynthesis by hCG. It has not yet been determined how to diagnose testosterone deficiency. The age-related serum testosterone concentration reference range has not been established yet either. The paper presents the first attempt to establish international standards for testosterone serum concentration levels in different age groups.

#### **2. Late-onset hypogonadism**

Late-onset hypogonadism (LOH) is a testosterone deficiency syndrome resulting in the aging process. LOH leads to metabolic disorders and functional or anatomical abnormalities. It is

<sup>\*</sup> Corresponding Author

Late - Onset Hypogonadism - New Point of View 117

this is associated with a simultaneous increase of SHBG levels, bioavailable testosterone may decline more significantly than apparent total testosterone (Snyder, 2001, Vermeulen, 2001). Only the testosterone circulating in the body which is not bound to SHBG is biologically active in the target organs. Therefore the measurement of bioavailable testosterone levels more accurately reflects a patient's clinical status than the measurement of total testosterone levels. The study of 810 men aged 24-90 years showed a strong correlation between age and a decrease in bioavailable testosterone level and in estradiol level (r =- 0.52). It was still strong when other variables were taken into account such as alcohol intake, BMI, smoking, caffeine intake, and diabetes (Ferrini & Barrett-Connor, 1998). Also significant but weaker (r=- 0.13) correlations were found between age and total testosterone and estradiol levels. Both the decreased testosterone production and its increased conversion can explain a reduction in total testosterone levels, while age-related increase in sex hormone binding capacity can cause reduced biologically available testosterone production. The correlation between age and the level of bioavailable and of total estradiol in men has not been studied previously. Bioavailable estradiol levels might decrease due to a decline in levels of testosterone, which is the main substrate for male estradiol production. It also contributes to an increase of SHBG with age. In one of the largest studies, which involved 2,623 men aged 65 and over, enjoying good health, levels of free and bioavailable testosterone and estradiol levels correlated with each other (Orwoll, et al. 2006). Higher age, higher BMI, and worse health status were associated with slightly lower total testosterone levels. The concentration of SHBG rose with age. The decrease in testosterone levels fell approximately 10% for 10 years. Many old men still had testosterone levels established for young men. However, it is unknown which level of testosterone is sufficient for an elderly person and for that person's specific tissues (i.e. muscles, bones, and nervous system). The number of androgen binding sites in the hippocampus and the number of the layers of the skin tissues that cover the penis increase with age (Hijazi & Cunningham, 2005). Testosterone production peaks at age 20 and then begins a gradual decline, usually giving rise to the first symptoms after the age of 50. In a group of men aged 80, the level of free

testosterone is half of what they had when they were younger (Vermeulen, et al., 1996).

Classic categorization of hypogonadism differentiates its primary and secondary types. In the case of primary hypogonadism, testicles are unable to synthesize estosterone, while in case of secondary hypogonadism low testosterone is caused by pituitary insufficiency. Until present from the pathophysiology point of view, LOH has been regarded as a mixed type of hypogonadism conditioned by the changes undergoing both in gonads (component of primary hypogonadism) and on the level of central controllers of testicular functions; hypothalamus and pituitary (components of secondary hypogonadism). Hypothalamus and pituitary regulate, on the basis of feedback, functions of Leydig cells in testicles. Gonadotrophin-releasing hormone is a trophin hypothalamus hormone relative to the Leydig cells. Its effect on pituitary results in the synthesis of luteinizing hormone (LH) and folicule stimulating hormone (FSH) in gonadotrophin cells. Gonadotrophin LH stimulates the synthesis of testosterone through the Leydig cells in testicle. With age, pulsating secretion of LH becomes disturbed. Pulses become more scarce (decreased frequency), of lower amplitude and their duration extends. The result is an increase of LH concentration with age. If LH concentration increases with age, thus pituitary insufficiency, i.e. secondary hypogonadism, does not exist and testicles retain their capability of high endosynthesis of testosterone, hence in case of LOH features of primary or of secondary hypogonadism are

**2.1 Hypogonadism – Primary, secondary, LOH** 

now beyond dispute that, as testosterone deficit increases with age, it adversely affects the function of multiple organ systems. Therefore the implementation of appropriate strategies to increase testosterone levels only when as a result of testosterone deficiency irreversible anatomical changes have occurred, is clearly too late, which operates to the detriment of men.

Recent studies suggest that there are large numbers of men in the community whose testosterone deficiency is neither being diagnosed nor treated (Trinick et al., 2011).

Testosterone exerts influence on multiple life processes such as blood cell production, bone formation, lipid metabolism, protein metabolism, carbohydrate metabolism, liver function, and spermiogenesis (Gooren, 2000). This can affect the structure and function of many organs in human body. They include skin, hair, muscle, brain and bones. It also has a significant effect on fertility and sexual behaviour. Testosterone plays a very important role in a man's life. At the mitochondrial level of each single cell it is the catalyst for protein synthesis. It has an effect on the brain's function, and thus determines the physical and sexual condition. It increases libido and improves sexual potency. It stimulates the immune system and affects multiple metabolic processes. It reduces body fat accumulation. It can affect muscle mass and strength. It increases bone mass and accelerates wound healing. Testosterone is responsible for our memory processes. Computer memory circuits are manufactured using metal arranged on silicon. In the process of human memory, proteins are involved. The testosterone that catalyzes the synthesis of various proteins is also a catalyst for storage protein synthesis. Protein synthesis becomes less efficient with age, proteins formed are unstable. Their decay means that an old man remembers what was years ago and cannot remember what just happened. In the literature, there have been isolated reports about the role of testosterone in the aetiology of Alzheimer's disease (Hogervorst, 2004).

Testosterone also aids in immune system protein synthesis, which protects us against infection. Hormone intake in pregnant women during the first trimester of pregnancy significantly affects hormone balance in young men. Although hormone therapy allows the woman to keep her pregnancy from failing, it is also essential for organogenesis, and in particular for the reproductive organs of male foetuses. According to the definition in Standards of Endocrinology, "Testosterone deficit disorder manifests itself in a decrease in libido and potency, in constant fatigue, in deterioration of mood and in sleep quality, in nervousness, in hot flushes, in low testosterone, and in elevated serum gonadotropin concentrations" (Zgliczyński & Zgliczyński, 2002). However, according to the definition provided by ISSAM, the International Society of Andrology (ISA) and the European Association of Urology (EAU), it is "a clinical and biochemical syndrome associated with advancing age, characterized by typical symptoms and a deficiency in serum testosterone levels. It can cause a significant deterioration in over-all quality of life and adversely affect the function of many different organs and systems" (Nieschlag, et al., 2005). It is now beyond dispute that, as testosterone deficit increases with age, it adversely affects the function of multiple organ systems. Previously adopted minimum standard testosterone levels, at which there has been best evidence for treatment, are far too low. The implementation of the therapy to boost testosterone levels at the time when for many years, as a result of testosterone deficiency, irreversible anatomical changes have occurred, is clearly too late, which operates to the detriment of men (Gomuła & Rabijewski, 2010). According to Tenover, if we consider the norm based on the level of total testosterone, in a group of 55-year-old men, 20% of them will be hypogonadal; and in the same group of men, when we adopt a standard based on the level of bio-available testosterone, hypogonadism will be diagnosed in half of them (Tenover, 1997). Cross-sectional studies have shown that, since

now beyond dispute that, as testosterone deficit increases with age, it adversely affects the function of multiple organ systems. Therefore the implementation of appropriate strategies to increase testosterone levels only when as a result of testosterone deficiency irreversible anatomical changes have occurred, is clearly too late, which operates to the detriment of men. Recent studies suggest that there are large numbers of men in the community whose

Testosterone exerts influence on multiple life processes such as blood cell production, bone formation, lipid metabolism, protein metabolism, carbohydrate metabolism, liver function, and spermiogenesis (Gooren, 2000). This can affect the structure and function of many organs in human body. They include skin, hair, muscle, brain and bones. It also has a significant effect on fertility and sexual behaviour. Testosterone plays a very important role in a man's life. At the mitochondrial level of each single cell it is the catalyst for protein synthesis. It has an effect on the brain's function, and thus determines the physical and sexual condition. It increases libido and improves sexual potency. It stimulates the immune system and affects multiple metabolic processes. It reduces body fat accumulation. It can affect muscle mass and strength. It increases bone mass and accelerates wound healing. Testosterone is responsible for our memory processes. Computer memory circuits are manufactured using metal arranged on silicon. In the process of human memory, proteins are involved. The testosterone that catalyzes the synthesis of various proteins is also a catalyst for storage protein synthesis. Protein synthesis becomes less efficient with age, proteins formed are unstable. Their decay means that an old man remembers what was years ago and cannot remember what just happened. In the literature, there have been isolated reports about the role of testosterone in the aetiology of

Testosterone also aids in immune system protein synthesis, which protects us against infection. Hormone intake in pregnant women during the first trimester of pregnancy significantly affects hormone balance in young men. Although hormone therapy allows the woman to keep her pregnancy from failing, it is also essential for organogenesis, and in particular for the reproductive organs of male foetuses. According to the definition in Standards of Endocrinology, "Testosterone deficit disorder manifests itself in a decrease in libido and potency, in constant fatigue, in deterioration of mood and in sleep quality, in nervousness, in hot flushes, in low testosterone, and in elevated serum gonadotropin concentrations" (Zgliczyński & Zgliczyński, 2002). However, according to the definition provided by ISSAM, the International Society of Andrology (ISA) and the European Association of Urology (EAU), it is "a clinical and biochemical syndrome associated with advancing age, characterized by typical symptoms and a deficiency in serum testosterone levels. It can cause a significant deterioration in over-all quality of life and adversely affect the function of many different organs and systems" (Nieschlag, et al., 2005). It is now beyond dispute that, as testosterone deficit increases with age, it adversely affects the function of multiple organ systems. Previously adopted minimum standard testosterone levels, at which there has been best evidence for treatment, are far too low. The implementation of the therapy to boost testosterone levels at the time when for many years, as a result of testosterone deficiency, irreversible anatomical changes have occurred, is clearly too late, which operates to the detriment of men (Gomuła & Rabijewski, 2010). According to Tenover, if we consider the norm based on the level of total testosterone, in a group of 55-year-old men, 20% of them will be hypogonadal; and in the same group of men, when we adopt a standard based on the level of bio-available testosterone, hypogonadism will be diagnosed in half of them (Tenover, 1997). Cross-sectional studies have shown that, since

testosterone deficiency is neither being diagnosed nor treated (Trinick et al., 2011).

Alzheimer's disease (Hogervorst, 2004).

this is associated with a simultaneous increase of SHBG levels, bioavailable testosterone may decline more significantly than apparent total testosterone (Snyder, 2001, Vermeulen, 2001). Only the testosterone circulating in the body which is not bound to SHBG is biologically active in the target organs. Therefore the measurement of bioavailable testosterone levels more accurately reflects a patient's clinical status than the measurement of total testosterone levels. The study of 810 men aged 24-90 years showed a strong correlation between age and a decrease in bioavailable testosterone level and in estradiol level (r =- 0.52). It was still strong when other variables were taken into account such as alcohol intake, BMI, smoking, caffeine intake, and diabetes (Ferrini & Barrett-Connor, 1998). Also significant but weaker (r=- 0.13) correlations were found between age and total testosterone and estradiol levels. Both the decreased testosterone production and its increased conversion can explain a reduction in total testosterone levels, while age-related increase in sex hormone binding capacity can cause reduced biologically available testosterone production. The correlation between age and the level of bioavailable and of total estradiol in men has not been studied previously. Bioavailable estradiol levels might decrease due to a decline in levels of testosterone, which is the main substrate for male estradiol production. It also contributes to an increase of SHBG with age. In one of the largest studies, which involved 2,623 men aged 65 and over, enjoying good health, levels of free and bioavailable testosterone and estradiol levels correlated with each other (Orwoll, et al. 2006). Higher age, higher BMI, and worse health status were associated with slightly lower total testosterone levels. The concentration of SHBG rose with age. The decrease in testosterone levels fell approximately 10% for 10 years. Many old men still had testosterone levels established for young men. However, it is unknown which level of testosterone is sufficient for an elderly person and for that person's specific tissues (i.e. muscles, bones, and nervous system). The number of androgen binding sites in the hippocampus and the number of the layers of the skin tissues that cover the penis increase with age (Hijazi & Cunningham, 2005). Testosterone production peaks at age 20 and then begins a gradual decline, usually giving rise to the first symptoms after the age of 50. In a group of men aged 80, the level of free testosterone is half of what they had when they were younger (Vermeulen, et al., 1996).

#### **2.1 Hypogonadism – Primary, secondary, LOH**

Classic categorization of hypogonadism differentiates its primary and secondary types. In the case of primary hypogonadism, testicles are unable to synthesize estosterone, while in case of secondary hypogonadism low testosterone is caused by pituitary insufficiency. Until present from the pathophysiology point of view, LOH has been regarded as a mixed type of hypogonadism conditioned by the changes undergoing both in gonads (component of primary hypogonadism) and on the level of central controllers of testicular functions; hypothalamus and pituitary (components of secondary hypogonadism). Hypothalamus and pituitary regulate, on the basis of feedback, functions of Leydig cells in testicles. Gonadotrophin-releasing hormone is a trophin hypothalamus hormone relative to the Leydig cells. Its effect on pituitary results in the synthesis of luteinizing hormone (LH) and folicule stimulating hormone (FSH) in gonadotrophin cells. Gonadotrophin LH stimulates the synthesis of testosterone through the Leydig cells in testicle. With age, pulsating secretion of LH becomes disturbed. Pulses become more scarce (decreased frequency), of lower amplitude and their duration extends. The result is an increase of LH concentration with age. If LH concentration increases with age, thus pituitary insufficiency, i.e. secondary hypogonadism, does not exist and testicles retain their capability of high endosynthesis of testosterone, hence in case of LOH features of primary or of secondary hypogonadism are

Late - Onset Hypogonadism - New Point of View 119

followed by andropause, (Morley & Perry, 2003) which stayed in medicine for good. Many learned societies with the term of andropause in the centre were established around the world. However the term of andropause is erroneous by definition. Such a phenomenon as pause, break in production of testosterone with men does not exist. Since a pause is a stage of an activity as discontinuation and a pause (a break) is followed by return to the activity from before the pause. At the end of previous century notions of *Androgen Deficiency in Aging Male* or *Androgen Deficiency in Adult Male* /ADAM/ (Carruthers, 2004) came to being. It must be emphasized that an adult man is aged between 20-25 years, while an aging man according to *International Society for the Study of the Aging Male* (ISSAM) is a gentleman above 60-65 (Carruthers, 2004). This is where the dilemma arises, does the testosterone shortfall issue concern exclusively elder men or younger, adult men? Testosterone shortfall has earned yet another name— PADAM (*partial androgen deficiency in aging male*), which defines the testosterone shortfall in a not sufficiently explicit manner. The concept lacks specification of boarder testosterone concentration and thus the value according to which a shortfall can be recognized. Therefore the term PADAM should not be used at all. What does partial shortfall mean? No accounting system recognizes partial deficit, it either exists or does not. Can a woman be partially pregnant? Another term was established; secondary or late onset hypogonadism but it still did not represent the core of the problem (Morales, et al., 2006). As clinical symptoms of androgen shortfall manifest themselves already at the break of forth and fifth decade (Mosby, 1998) (35–45 years), i.e. in the first half of life, can it be said that hypogonadism appears late? The latest name of the disease, which so far has not been explicitly defined, is *testosterone deficiency syndrome /TDS /* (Morales, et al., 2006) currently a fashionable term. Yet science and fashion follow totally different criteria. What counts in fashion is a one-season success, while in science exact, indisputable facts forming a solid basis for many years should be essecial. In the meantime, testosterone shortfall, an unfavourable phenomenon leading to various metabolic disorders or issue pathologies, has

not been named a disease until now.

**2.3 Late - onset hypogonadism and age related testosterone levels** 

year-old man, whose level now, at 70, is more than 35 nmol/L?

nmol/L – 350% difference (Lazarou, et al., 2006)

Many studies have now demonstrated that as men age, their serum testosterone concentrations fall at an average rate of 0.8%–1% per year (Feldman, et al., 2002, Kaufmann & Vermeulen, 2005). Concurrently, free and bioavailable testosterone levels fall by 2% per year (Kaufmann & Vermeulen, 2005). Obesity, alcohol abuse, diabetes, hypertension, heart

Therefore, and also due to an increase in the concentrations of carrier proteins, significantly decreased testosterone levels characterise approximately 8% of men of 40-60 and 20% men of 60-80 (Kaufmann & Vermeulen, 2005). The question arises: how to measure the decrease in testosterone levels during the life of a man? By the hormonal status of young people today? The hormonal status of their fathers? What was the hormonal status of their grandparents in their youth, since testosterone levels in men over age 70 often reach the upper range of the standard? And another question: what was the testosterone level of a 30-

Standard testosterone levels vary in different analytical laboratories. In American research laboratories alone, the lower limit of normal testosterone level ranges from 4.5 to 15.6

The effects of an objectively measurable drop in testosterone levels may also vary. Researchers wonder if it would be most appropriate to establish separate ranges of normal testosterone

disease, cancer and ulcers have intensified the negative impact (Harman, et al., 2001).

non-existent. It has been acknowledged that testicle stimulation with hCG does not cause any substantial, sufficient increase of endosynthesis of testosterone. And here lies the fundamental error – the statement that hCG does not result in any substantial increase of testosterone endosynthesis is untrue!!! Therapy based on hCG allows doubling the concentration of testosterone, which in over 90% of those treated enables restoring a normal hormone condition. I base my statement on over a 10-year long observation of 1200 patients I treated with hCG to induce endosynthesis of testosterone.

Secretion of GnRH is distorted as well. Hypothalamus response to change of LH pulse nature and to lowered secretion of androgens is incommensurate. The phenomenon is referred to as dysregulation of hypothalamus pulse generator. The reasons for alternations of pulsating secretion of GnRH and LH are unclear, however of importance are genetic factors and backward changes in gonadotrophin cells and in reflective microcirculation on hypothalamus and pituitary axis (Snyder, et al., 2000). Ferrini, Wang, Hakim et al. have established that changes in functioning of hypothalamus and pituitary may not be the major cause of gonads dysfunctions in men (Ferrini, et al., 2001). They have found evidence for a substantial increase of hypothalamus and pituitary apoptosis, two changes clarifying development of hypogonadism at old age. Until now it was believed that together with hypothalamus and pituitary dysfunction gonadal backward changes played an important role in etiopathogenesis of gonads. The process of decreasing testosterone synthesis starts approximately at the age of 35-40 years and is of extreme individual variability. Changes within testicles are noted for progressive decrease of the number of Leydig cells and deterioration of Sertoli cells functions and as a result decrease of secretion of inhibin, impaired micro-circulation and blood-supply of testicle cells and backward changes within perivascular parenchyma (Deslypere & Vermulen, 1984, Snyder, 2001). From the analysis of own material it is considered that to-date views on TDS aetiology are unfounded. As it has been demonstrated in the material consisting of 908 men treated with induction of endosynthesis, it is possible with every age group, but it decreas in terms of physiology with age. Nevertheless, the capability of efficient endosynthesis is retained until advanced years, which contradicts views of atrophy of Leydig cells functions. From a clinical point of view, LOH resembles pathology of incorrect LH bioactivity described in men of 47XY karyotype who after adolescence undergo hypogonadism with decrease of testosterone and increase of LH concentration. These patients demonstrate correct gonads response to LH stimulation. It has been acknowledged that in these cases LH bioactivity is decreased, while immunoreactivity is normal. It results from glutamine changing into arginine in position 54 of beta chains.

Currently, it is not possible to define explicitly the aetiologic factor of LOH leading to testosterone shortfall. Testosterone shortfall results in metabolic syndrome causing disorders of many bodily functions such as memory disorders, difficulties with falling asleep, excessive nervousness, deterioration of ability to associate and concentrate, heat waves, depression tendencies, vertigo and headache, fall of strength and muscle bulk, as well as numerous cardiovascular system complaints. Testosterone concentration drop leads to lipid disorders and diabetes type 2.

#### **2.2 Andropause, ADAM, PADAM, LOH, TDS**

The concept of "male menopause" has been known since 1960s (Wang, et al., 1996) yet until today it has not been unambiguously defined. The names of the clinical state caused by testosterone shortfall have changed many times over decades which explicitly proves lack of firm facts based on indisputable ground. Names of andropause, sarcopenia, viropause were

non-existent. It has been acknowledged that testicle stimulation with hCG does not cause any substantial, sufficient increase of endosynthesis of testosterone. And here lies the fundamental error – the statement that hCG does not result in any substantial increase of testosterone endosynthesis is untrue!!! Therapy based on hCG allows doubling the concentration of testosterone, which in over 90% of those treated enables restoring a normal hormone condition. I base my statement on over a 10-year long observation of 1200 patients

Secretion of GnRH is distorted as well. Hypothalamus response to change of LH pulse nature and to lowered secretion of androgens is incommensurate. The phenomenon is referred to as dysregulation of hypothalamus pulse generator. The reasons for alternations of pulsating secretion of GnRH and LH are unclear, however of importance are genetic factors and backward changes in gonadotrophin cells and in reflective microcirculation on hypothalamus and pituitary axis (Snyder, et al., 2000). Ferrini, Wang, Hakim et al. have established that changes in functioning of hypothalamus and pituitary may not be the major cause of gonads dysfunctions in men (Ferrini, et al., 2001). They have found evidence for a substantial increase of hypothalamus and pituitary apoptosis, two changes clarifying development of hypogonadism at old age. Until now it was believed that together with hypothalamus and pituitary dysfunction gonadal backward changes played an important role in etiopathogenesis of gonads. The process of decreasing testosterone synthesis starts approximately at the age of 35-40 years and is of extreme individual variability. Changes within testicles are noted for progressive decrease of the number of Leydig cells and deterioration of Sertoli cells functions and as a result decrease of secretion of inhibin, impaired micro-circulation and blood-supply of testicle cells and backward changes within perivascular parenchyma (Deslypere & Vermulen, 1984, Snyder, 2001). From the analysis of own material it is considered that to-date views on TDS aetiology are unfounded. As it has been demonstrated in the material consisting of 908 men treated with induction of endosynthesis, it is possible with every age group, but it decreas in terms of physiology with age. Nevertheless, the capability of efficient endosynthesis is retained until advanced years, which contradicts views of atrophy of Leydig cells functions. From a clinical point of view, LOH resembles pathology of incorrect LH bioactivity described in men of 47XY karyotype who after adolescence undergo hypogonadism with decrease of testosterone and increase of LH concentration. These patients demonstrate correct gonads response to LH stimulation. It has been acknowledged that in these cases LH bioactivity is decreased, while immunoreactivity is normal. It results

I treated with hCG to induce endosynthesis of testosterone.

from glutamine changing into arginine in position 54 of beta chains.

to lipid disorders and diabetes type 2.

**2.2 Andropause, ADAM, PADAM, LOH, TDS** 

Currently, it is not possible to define explicitly the aetiologic factor of LOH leading to testosterone shortfall. Testosterone shortfall results in metabolic syndrome causing disorders of many bodily functions such as memory disorders, difficulties with falling asleep, excessive nervousness, deterioration of ability to associate and concentrate, heat waves, depression tendencies, vertigo and headache, fall of strength and muscle bulk, as well as numerous cardiovascular system complaints. Testosterone concentration drop leads

The concept of "male menopause" has been known since 1960s (Wang, et al., 1996) yet until today it has not been unambiguously defined. The names of the clinical state caused by testosterone shortfall have changed many times over decades which explicitly proves lack of firm facts based on indisputable ground. Names of andropause, sarcopenia, viropause were followed by andropause, (Morley & Perry, 2003) which stayed in medicine for good. Many learned societies with the term of andropause in the centre were established around the world. However the term of andropause is erroneous by definition. Such a phenomenon as pause, break in production of testosterone with men does not exist. Since a pause is a stage of an activity as discontinuation and a pause (a break) is followed by return to the activity from before the pause. At the end of previous century notions of *Androgen Deficiency in Aging Male* or *Androgen Deficiency in Adult Male* /ADAM/ (Carruthers, 2004) came to being. It must be emphasized that an adult man is aged between 20-25 years, while an aging man according to *International Society for the Study of the Aging Male* (ISSAM) is a gentleman above 60-65 (Carruthers, 2004). This is where the dilemma arises, does the testosterone shortfall issue concern exclusively elder men or younger, adult men? Testosterone shortfall has earned yet another name— PADAM (*partial androgen deficiency in aging male*), which defines the testosterone shortfall in a not sufficiently explicit manner. The concept lacks specification of boarder testosterone concentration and thus the value according to which a shortfall can be recognized. Therefore the term PADAM should not be used at all. What does partial shortfall mean? No accounting system recognizes partial deficit, it either exists or does not. Can a woman be partially pregnant? Another term was established; secondary or late onset hypogonadism but it still did not represent the core of the problem (Morales, et al., 2006). As clinical symptoms of androgen shortfall manifest themselves already at the break of forth and fifth decade (Mosby, 1998) (35–45 years), i.e. in the first half of life, can it be said that hypogonadism appears late? The latest name of the disease, which so far has not been explicitly defined, is *testosterone deficiency syndrome /TDS /* (Morales, et al., 2006) currently a fashionable term. Yet science and fashion follow totally different criteria. What counts in fashion is a one-season success, while in science exact, indisputable facts forming a solid basis for many years should be essecial. In the meantime, testosterone shortfall, an unfavourable phenomenon leading to various metabolic disorders or issue pathologies, has not been named a disease until now.

#### **2.3 Late - onset hypogonadism and age related testosterone levels**

Many studies have now demonstrated that as men age, their serum testosterone concentrations fall at an average rate of 0.8%–1% per year (Feldman, et al., 2002, Kaufmann & Vermeulen, 2005). Concurrently, free and bioavailable testosterone levels fall by 2% per year (Kaufmann & Vermeulen, 2005). Obesity, alcohol abuse, diabetes, hypertension, heart disease, cancer and ulcers have intensified the negative impact (Harman, et al., 2001).

Therefore, and also due to an increase in the concentrations of carrier proteins, significantly decreased testosterone levels characterise approximately 8% of men of 40-60 and 20% men of 60-80 (Kaufmann & Vermeulen, 2005). The question arises: how to measure the decrease in testosterone levels during the life of a man? By the hormonal status of young people today? The hormonal status of their fathers? What was the hormonal status of their grandparents in their youth, since testosterone levels in men over age 70 often reach the upper range of the standard? And another question: what was the testosterone level of a 30 year-old man, whose level now, at 70, is more than 35 nmol/L?

Standard testosterone levels vary in different analytical laboratories. In American research laboratories alone, the lower limit of normal testosterone level ranges from 4.5 to 15.6 nmol/L – 350% difference (Lazarou, et al., 2006)

The effects of an objectively measurable drop in testosterone levels may also vary. Researchers wonder if it would be most appropriate to establish separate ranges of normal testosterone

Late - Onset Hypogonadism - New Point of View 121

The evaluation of 1267 men aged 20-89 years has found that the average testosterone level in

Fig. 1. The average testosterone levels of 1267 men in different age groups. The horizontal line on the graph represents an accepted lower limit of normal testosterone level 12 nmol/L /according to the International Society for the Study of the Aging Male/ (Lunenfeld, B. Et al., 2005, Nieschlag, et al., 2005). If we assume that the standard testosterone level is higher than 12 nmol/L, in the age group 20-29 years testosterone deficit has been found in 11.5% of those examined. In the age group 30-39 years the deficit has been found in 5.9% of those examined. In the age group 40-49 years – in 21% of those examined; in the age group 50-59 years – in 24% of those examined; in the age group 60-69 years – in 27.8%; in the age group 70-79 years – in 20.6%; and in the age group 80-89 years – in 24% of those examined. Testosterone deficiency (serum testosterone concentrations below 12 nmol/L) in different

> **Age N pts %**  20–29 9/78 11.5% 30–39 6/102 5.9% 40–49 37/176 21% 50–59 88/367 24% 60–69 78/281 27.8% 70–79 44/213 20.6% 80–89 12/50 24%

On the basis of the TDI ratio, testosterone deficiency has been found in 49.55% of those in the study group. The smaller number of patients who had TDI determined is due to the absence of the ratio of LH for 48 out of 1267 patients. Summary of parameters to provide a

basic index of the Andropause status for 1219 patients is shown in Table 3.

all age groups is higher than 12 nmol/L – what is shown in Fig. 1.

age groups is shown in Table 2.

Table 2. Testosterone deficiency in different age groups.

levels for the young and for the old (Lunenfeld & Gooren, 2002). It is also unknown whether the plasma testosterone threshold values vary with age. There is preliminary evidence that the threshold value necessary for the proper functioning and for the proper effects of testosterone also increases with age (Schiavi, et al., 1993). Bancroft hypothesizes that the threshold required for the behavioral effects of testosterone increases with age (Bancroft, 1989). Although many older men have testosterone levels within the normal range, their levels may not be sufficient for normal sexual functioning. Probably organ sensitivity to androgens is not the same for the young and for the old. Schiavi reported that nocturnal erections are androgen-dependent and are disrupted in healthy elderly men who do not meet the criteria for hypogonadism, which seems to confirm Bancroft's hypothesis (Bancroft, 1989, Schiavi, et al. (1993). Based on the analysis of the material provided by 1267 men of 20-89, together with the material provided by 908 men under treatment, standard levels of testosterone have been age-determined (Gomuła & Rabijewski, 2010). Among the respondents between 20-29, the average level of testosterone was 21.99 nmol/L; among the respondents between 30-39 – 21.91 nmol/L among the respondents between 40-49 – 19.18 nmol / L; among the respondents between 40-49 - 19.18 nmol/L; among the respondents between 50-59 – 17.74 nmol/L; among the respondents between 60-69 – 15.94 nmol/L among the respondents between 70-79 – 17.66 nmol/:L and among the respondents between 80-89 – 16.65 nmol/l. The standard testosterone levels in individual age groups (minimum and maximum, average level, standard deviations and medians) are shown in Table 1.


n - number of patients in the age group , SD - standard deviation

Table 1. Testosterone levels of 1267 men in different age groups

levels for the young and for the old (Lunenfeld & Gooren, 2002). It is also unknown whether the plasma testosterone threshold values vary with age. There is preliminary evidence that the threshold value necessary for the proper functioning and for the proper effects of testosterone also increases with age (Schiavi, et al., 1993). Bancroft hypothesizes that the threshold required for the behavioral effects of testosterone increases with age (Bancroft, 1989). Although many older men have testosterone levels within the normal range, their levels may not be sufficient for normal sexual functioning. Probably organ sensitivity to androgens is not the same for the young and for the old. Schiavi reported that nocturnal erections are androgen-dependent and are disrupted in healthy elderly men who do not meet the criteria for hypogonadism, which seems to confirm Bancroft's hypothesis (Bancroft, 1989, Schiavi, et al. (1993). Based on the analysis of the material provided by 1267 men of 20-89, together with the material provided by 908 men under treatment, standard levels of testosterone have been age-determined (Gomuła & Rabijewski, 2010). Among the respondents between 20-29, the average level of testosterone was 21.99 nmol/L; among the respondents between 30-39 – 21.91 nmol/L among the respondents between 40-49 – 19.18 nmol / L; among the respondents between 40-49 - 19.18 nmol/L; among the respondents between 50-59 – 17.74 nmol/L; among the respondents between 60-69 – 15.94 nmol/L among the respondents between 70-79 – 17.66 nmol/:L and among the respondents between 80-89 – 16.65 nmol/l. The standard testosterone levels in individual age groups (minimum and maximum, average level, standard deviations and

medians) are shown in Table 1.

**50–59 years** n = 367

Median (25%, 75%) 16.20 (12.10; 21.70)

n - number of patients in the age group , SD - standard deviation Table 1. Testosterone levels of 1267 men in different age groups

Min., max. 3.10; 53.50 Average (SD) 17.74 (8.01)

**n, Testosterone in nmol/L** 

**Age/min-max, median,average,SD** 

**20–29 years** n = 78 **60–69 years** n = 281

Median (25%, 75%) 21.70 (15.80; 25.20) Median (25%, 75%) 16.10 (12.70; 21.30) **40–49 years** n = 176 **80–89 years** n = 50

Min., max. 4.64; 49.10 Min., max. 2; 40.40 Average (SD) 21.99 (9.33) Average (SD) 15.94 (6.68) Median (25%, 75%) 19.75 (15.40; 26.20) Median (25%, 75%) 15 (11.30; 19.70) **30–39 years** n = 102 **70–79 years** n = 213

Min., max. 8.90; 46.80 Min., max. 2.71; 45.60 Average (SD) 21.91 (8.18) Average (SD) 17.66 (7.87)

Min., max. 3.40; 47.20 Min., max. 3.70; 38.80 Average (SD) 19.18 (8.92) Average (SD) 16.65 (7.55) Median (25%, 75%) 17 (12.6; 24.30) Median (25%, 75%) 15.75 (12; 20.60)

**n, Testosterone in nmol/L** 

**Age/min-max, median,average,SD**  The evaluation of 1267 men aged 20-89 years has found that the average testosterone level in all age groups is higher than 12 nmol/L – what is shown in Fig. 1.

Fig. 1. The average testosterone levels of 1267 men in different age groups. The horizontal line on the graph represents an accepted lower limit of normal testosterone level 12 nmol/L /according to the International Society for the Study of the Aging Male/ (Lunenfeld, B. Et al., 2005, Nieschlag, et al., 2005). If we assume that the standard testosterone level is higher than 12 nmol/L, in the age group 20-29 years testosterone deficit has been found in 11.5% of those examined. In the age group 30-39 years the deficit has been found in 5.9% of those examined. In the age group 40-49 years – in 21% of those examined; in the age group 50-59 years – in 24% of those examined; in the age group 60-69 years – in 27.8%; in the age group 70-79 years – in 20.6%; and in the age group 80-89 years – in 24% of those examined. Testosterone deficiency (serum testosterone concentrations below 12 nmol/L) in different age groups is shown in Table 2.


Table 2. Testosterone deficiency in different age groups.

On the basis of the TDI ratio, testosterone deficiency has been found in 49.55% of those in the study group. The smaller number of patients who had TDI determined is due to the absence of the ratio of LH for 48 out of 1267 patients. Summary of parameters to provide a basic index of the Andropause status for 1219 patients is shown in Table 3.

Late - Onset Hypogonadism - New Point of View 123

Fig. 2. Testosterone levels below 12 nmol/L in relation to age.

Fig. 3. TDI (Testosterone Deficit Index) in relation to age.

deficiency, while testosterone deficiency levels have clearly been lower in the age group 70- 79 years and in the age group 80-89 years. An assessment in accordance with the testosterone-concentrations-below-12-nmol/L parameter exhibits significant deviations. It shows that, as men age, testosterone deficiency does not increase in a linear fashion. However, Figure 3 shows that the analysis of the same material on the basis of the testosterone deficiency index (TDI) demonstrates that, as men age, testosterone deficiency increases in a linear fashion. The study proves the effectiveness of the testosterone deficiency index (TDI) in assessing the degree of testosterone deficiency. On the basis of the TDI results and/or on the basis of a patient's clinical status, 903 of those examined were involved in testosterone endosynthesis induction treatment with human chorionic gonadotropin /hCG/ (Gould, 1951, Gomula, 2001, Gomula, 2002). Testosterone levels in response to hCG treatment are shown in Table 5, and graphically in Figure 4.


T — testosterone; LH — luteinizing hormone; SD — standard deviation; TDI —testosterone deficit index.

Table 3. Average serum testosterone concentrations , LH and TDI

The findings in Table 4 show, on the basis of the TDI ratio, an increased incidence of testosterone deficiency with age.


Table 4. Testosterone deficiency in different age groups for 1219 male respondents, calculated on the basis of TDI.

Two methods of assessing testosterone deficiency have been compared, based on global standards and on TDI. Figures 2 and 3 show the material discussed above under analysis. In the group of 1267 patients, there were only 274 males with serum testosterone concentrations levels below 12 nmol/L, which amounts to 21.62% of those examined, whereas, in accordance with TDI standards, 604 out of 1219 men had testosterone deficiency, which amounts to 49.55% of those examined. The observed difference between the groups is statistically significant.

If we put on the two graphs (Figure 2 and Figure 3) the lines connecting testosterone level in young men when they have the highest testosterone level (i.e., 30-39 year olds), it turns out that the testosterone deficit estimation results in accordance with the standardised testosterone-below-12-nmol/l approach are different from those in accordance with TDI standards. As shown in Figure 2, in accordance with the most rigorous standards of serum testosterone concentration, men aged 60-70 years have been diagnosed with testosterone

**Parameter n = 1219 pts T (ng/mL)** 

**LH (IU/L)** 

**T/LH** 

T — testosterone; LH — luteinizing hormone; SD — standard deviation; TDI —testosterone deficit index.

The findings in Table 4 show, on the basis of the TDI ratio, an increased incidence of

**Age TDI < 1**  20–29 21/78 (28%) 30–39 29/101 (28.7%) 40–49 56/165 (33.9%) 50–59 160/355 (45.1%) 60–69 174/271 (64.2%) 70–79 124/197 (62.9%) 80–89 38/47 (80.85%)

Table 4. Testosterone deficiency in different age groups for 1219 male respondents,

Two methods of assessing testosterone deficiency have been compared, based on global standards and on TDI. Figures 2 and 3 show the material discussed above under analysis. In the group of 1267 patients, there were only 274 males with serum testosterone concentrations levels below 12 nmol/L, which amounts to 21.62% of those examined, whereas, in accordance with TDI standards, 604 out of 1219 men had testosterone deficiency, which amounts to 49.55% of those examined. The observed difference between

If we put on the two graphs (Figure 2 and Figure 3) the lines connecting testosterone level in young men when they have the highest testosterone level (i.e., 30-39 year olds), it turns out that the testosterone deficit estimation results in accordance with the standardised testosterone-below-12-nmol/l approach are different from those in accordance with TDI standards. As shown in Figure 2, in accordance with the most rigorous standards of serum testosterone concentration, men aged 60-70 years have been diagnosed with testosterone

Median (25%, 75%) = 4.78 (3.59; 6.48)

Median (25%, 75%) = 4.8 (3.3; 6.7)

Median (25%, 75%) = 1 (0.63; 1.61) **TDI < 1** = 604/1219 (49.55%)

Min., max. = 0.58; 15.41 Average (SD)= 5.22 (2.33)

Min., max. = 0.2; 47 Average (SD) = 5.8 (4.6)

Min., max. = 0.02; 41.76 Average (SD) = 1.36 (1.67)

Table 3. Average serum testosterone concentrations , LH and TDI

testosterone deficiency with age.

calculated on the basis of TDI.

the groups is statistically significant.

Fig. 2. Testosterone levels below 12 nmol/L in relation to age.

Fig. 3. TDI (Testosterone Deficit Index) in relation to age.

deficiency, while testosterone deficiency levels have clearly been lower in the age group 70- 79 years and in the age group 80-89 years. An assessment in accordance with the testosterone-concentrations-below-12-nmol/L parameter exhibits significant deviations. It shows that, as men age, testosterone deficiency does not increase in a linear fashion. However, Figure 3 shows that the analysis of the same material on the basis of the testosterone deficiency index (TDI) demonstrates that, as men age, testosterone deficiency increases in a linear fashion. The study proves the effectiveness of the testosterone deficiency index (TDI) in assessing the degree of testosterone deficiency. On the basis of the TDI results and/or on the basis of a patient's clinical status, 903 of those examined were involved in testosterone endosynthesis induction treatment with human chorionic gonadotropin /hCG/ (Gould, 1951, Gomula, 2001, Gomula, 2002). Testosterone levels in response to hCG treatment are shown in Table 5, and graphically in Figure 4.

Late - Onset Hypogonadism - New Point of View 125

Fig. 4. Testosterone levels in different age groups before (the lower, red line) and during

Fig. 5. The prospects of testosterone endosynthesis in different age groups.

Testosterone concentrations in different age groups have risen for 908 patients under hormone therapy (testosterone endosynthesis induction treatment through the administration of hCG) by an average of between 88 percent and 128 percent. At the same time, it has been found that under hCG stimulation the prospects of endosynthesis decrease linearly with age in the age group 30-89 years. This is consistent with physiology (Figure 5). The prospects of testosterone endosynthesis decrease with age. The age group 30-39 years had an average 128% increase in testosterone levels; the age group 40-49 years – an average 126% increase in testosterone levels; the age group 50-59 years – an average 129% increase in testosterone levels; the age group 60- 69 years – an average 121% increase in testosterone levels; the age group 70-79 years – an average 99% increase in testosterone levels; the age group 80-99 years – an average 88% increase in testosterone levels. The full range of values of parameters determining the prospects of testosterone endosynthesis in different age groups has been shown in Table 5.

hCG treatment (the upper, green line).


Table 5. Testosterone concentration before (T-0) and during hCG treatment (T-1) in different age groups.

Using Student's t-test or the Wilcoxon rank-sum test, it has been examined whether the testosterone concentration change is significantly different from 0. Statistically significant results have been achieved both for the whole group and when broken down by age group.

20.30, 89.10 44.03 (13.45) 44.05 (34.70, 51.15)

20.50; 96.20 49.62 (16.12) 46.80 (37.60; 60.40)

11.40; 78.20 41.72 (14.36) 39.60 (31.60; 50.50)

10.30; 78.30 39.23 (12.63) 38.50 (29.80; 46.20)

7.50; 82.30 34.48 (13.32) 32.30 (24.70; 41.90)

6.80; 89 34.08 (14.83) 32 (23.50; 42.70)

11,50; 48,50 27.10 (10.36) 24.50 (18.80; 35.40)

Table 5. Testosterone concentration before (T-0) and during hCG treatment (T-1) in different

Using Student's t-test or the Wilcoxon rank-sum test, it has been examined whether the testosterone concentration change is significantly different from 0. Statistically significant results have been achieved both for the whole group and when broken down by age group.

1.5, 42.2 22.9 (10.9) 23.55 (14.35, 30.9)

3.9; 63.6 27.8 (13.5) 28.9 (16.6; 35.7)

2.1; 58.5 23.2 (12.09)

4.9; 62.6 22.08 (11.7)

4.3, 69.2 18.9 (11,.9)

7.2; 59 16.99 (13.32)

4.9; 29.6 12.72 (9.86)

23.13 (13.8; 32.6) < 0.001

20.1 (13.7; 29.6) < 0.001

16.8 (10.6; 25) < 0.001

15.6 (6.3; 24.2) < 0.001

12.4 (5.4; 19) < 0.001

<0.001

**Age T-0 nmol/L T-1 nmol/L Change p** 

20-29 n=44 n=44 n=44

30–39 n = 63 n = 63 n = 63

40–49 n = 131 n = 131 n = 131

50–59 n = 277 n = 277 n = 277

60–69 n = 205 n = 205 n = 205

70–79 n = 154 n = 154 n = 154

80–89 n = 29 n = 29 n = 29

6.2, 49.1 21.13 (9.7) 19.7 (14.05, 24.55)

8.9; 46,8 21.78 (8.31) 21.7 (15.9; 24)

6.3; 46.6 18.5 (8.47) 16.5 (12.3; 23.6)

3.1; 46.4 17.14 (7.72) 15.7 (11.9; 20.5)

2.71; 40.4 15.59 (6.45) 14.4 (10.8; 19.1)

2.71; 45.6 17.09 (7.5) 15.15 (12.5; 19.9)

3.7; 27.8 14.39 (5.43) 14.8 (12; 17.6)

Min., max. Average (SD) Median (25%,

Min., max. Average (SD) Median (25%,

Min., max. Average (SD) Median (25%,

Min., max. Average (SD) Median (25%,

Min., max. Average (SD) Median (25%,

Min., max. Average (SD) Median (25%,

Min., max. Average (SD) Median (25%,

75%)

75%)

75%)

75%)

75%)

75%)

75%)

age groups.

Fig. 4. Testosterone levels in different age groups before (the lower, red line) and during hCG treatment (the upper, green line).

Testosterone concentrations in different age groups have risen for 908 patients under hormone therapy (testosterone endosynthesis induction treatment through the administration of hCG) by an average of between 88 percent and 128 percent. At the same time, it has been found that under hCG stimulation the prospects of endosynthesis decrease linearly with age in the age group 30-89 years. This is consistent with physiology (Figure 5). The prospects of testosterone endosynthesis decrease with age. The age group 30-39 years had an average 128% increase in testosterone levels; the age group 40-49 years – an average 126% increase in testosterone levels; the age group 50-59 years – an average 129% increase in testosterone levels; the age group 60- 69 years – an average 121% increase in testosterone levels; the age group 70-79 years – an average 99% increase in testosterone levels; the age group 80-99 years – an average 88% increase in testosterone levels. The full range of values of parameters determining the prospects of testosterone endosynthesis in different age groups has been shown in Table 5.

Fig. 5. The prospects of testosterone endosynthesis in different age groups.

Late - Onset Hypogonadism - New Point of View 127

A very important question arises: if testosterone levels are so high, as the authors propose, are they not harmful? However, since these concentrations have been obtained by endosynthesis, it is obvious that they are in accordance with the body's physiological ability to synthesize testosterone. In addition, it has been known for more than 20 years that longterm high-dose testosterone therapy does not give significant metabolic side effects (Matsumoto, 1990). The correct threshold for proper testosterone functioning and for proper testosterone effects also increases with age (Schiavi, et al., 1993). Therefore the proposed age-related standards for testosterone must be much higher than those observed previously. A retrospective analysis of material possessed has suggested that the hormonal balance in today's young men is significantly worse than the hormonal balance for their fathers when they were in their youth. It has been shown in fact that the total testosterone level in today's young men is roughly two-thirds of their fathers' testosterone level. If we draw a line in parallel with the decline in serum testosterone levels with age for the age group of 35 to 70 years, and then draw it backwards, from the age of 90 to the earlier years, it will appear that the fathers, aged 30-35, exhibited significantly higher testosterone levels than their

Fig. 7. Concentrations of testosterone in men aged 20-90 years and the line aptly showing

Figure 7 shows that the concentration of testosterone in today's young men aged 30-35 is, on average, 23 nmol/L Concentrations of testosterone of their ancestors aged 85-89 averaged 17.2 nmol/L Therefore, standard values in the testosterone concentration range – the dispersion of values previously ranging to 31 nmol/L– have now narrowed to such an extent that the difference in testosterone levels between younger and older men is only 5 nmol/L i.e. 16% of normal range 10-42 nmol/L It practically means that young men now have testosterone levels about 35% lower than those of their ancestors. Therefore, it can be concluded that, on the one hand, the whole world population is aging. On the other hand, the biological condition of today's youth suggests that in such a metabolic-hormonal state the youth of today will not increase human life expectancy. It is highly likely that they will die at a much younger age than their ancestors did in the past. The reasons for these changes are more complex. Genetic factors, lifestyle habits, medications, toxins, free radicals, body weight, psychological and social aspects, chronic stress, mental health, and social position

descendants at comparable ages (Fig. 7).

their fathers' testosterone levels in the past.

play a significant role.

On the basis of the findings of the analysis of testosterone concentrations that occur physiologically, without treatment, during testosterone endosynthesis stimulation, it was decided to measure standard physiological testosterone levels correlated with age. During testosterone endosynthesis stimulation, testosterone concentrations obtained ade it possible for the patients to exhibit significantly better mood ratings, to develop improved physical fitness, and to increase libido and potency. The testosterone concentrations obtained also had the following positive impacts on blood pressure and biochemical parameters: normalization of the lipid profile and the reduction in HbA1c levels. Therefore, the testosterone concentrations obtained were adopted as normative in different age groups. Standard deviations of testosterone level increase while inducing testosterone endosynthesis by administering hCG varies by age group. This situation has been taken into account in measuring standard testosterone levels in different age groups. It has been assumed that normal testosterone levels in a particular age group is a number between the highest average value of testosterone while inducing testosterone endosynthesis by administering hCG and one standard deviation in the age group. These standards are illustrated graphically in Figure 6, and entered in Table 6.

Fig. 6. The proposed standard testosterone levels for men of various age groups, based on the physiological capabilities caused by testosterone endosynthesis. The bottom line – the serum testosterone concentration in different age groups, prior to treatment; top line testosterone concentrations in each age group during hCG treatment; box – proposed agerelated standard testosterone levels.


Table 6. Standard testosterone levels for men of various age groups, based on the analysis of potential testosterone endosynthesis capabilities among 908 patients treated by inducing testosterone endosynthesis by administration of hCG.

On the basis of the findings of the analysis of testosterone concentrations that occur physiologically, without treatment, during testosterone endosynthesis stimulation, it was decided to measure standard physiological testosterone levels correlated with age. During testosterone endosynthesis stimulation, testosterone concentrations obtained ade it possible for the patients to exhibit significantly better mood ratings, to develop improved physical fitness, and to increase libido and potency. The testosterone concentrations obtained also had the following positive impacts on blood pressure and biochemical parameters: normalization of the lipid profile and the reduction in HbA1c levels. Therefore, the testosterone concentrations obtained were adopted as normative in different age groups. Standard deviations of testosterone level increase while inducing testosterone endosynthesis by administering hCG varies by age group. This situation has been taken into account in measuring standard testosterone levels in different age groups. It has been assumed that normal testosterone levels in a particular age group is a number between the highest average value of testosterone while inducing testosterone endosynthesis by administering hCG and one standard deviation in the age group. These standards are illustrated

Fig. 6. The proposed standard testosterone levels for men of various age groups, based on the physiological capabilities caused by testosterone endosynthesis. The bottom line – the serum testosterone concentration in different age groups, prior to treatment; top line testosterone concentrations in each age group during hCG treatment; box – proposed age-

Age 30–39 y 40–49 y 50–59 y 60–69 y 70–79 y 80–89 y

T (nmol/lL) 33.5 ÷ 49.6 27.3 ÷ 41.7 26.6÷39.2 21 ÷ 34.4 19.2 ÷ 34.0 16.7 ÷ 27.1

Table 6. Standard testosterone levels for men of various age groups, based on the analysis of potential testosterone endosynthesis capabilities among 908 patients treated by inducing

graphically in Figure 6, and entered in Table 6.

related standard testosterone levels.

testosterone endosynthesis by administration of hCG.

A very important question arises: if testosterone levels are so high, as the authors propose, are they not harmful? However, since these concentrations have been obtained by endosynthesis, it is obvious that they are in accordance with the body's physiological ability to synthesize testosterone. In addition, it has been known for more than 20 years that longterm high-dose testosterone therapy does not give significant metabolic side effects (Matsumoto, 1990). The correct threshold for proper testosterone functioning and for proper testosterone effects also increases with age (Schiavi, et al., 1993). Therefore the proposed age-related standards for testosterone must be much higher than those observed previously. A retrospective analysis of material possessed has suggested that the hormonal balance in today's young men is significantly worse than the hormonal balance for their fathers when they were in their youth. It has been shown in fact that the total testosterone level in today's young men is roughly two-thirds of their fathers' testosterone level. If we draw a line in parallel with the decline in serum testosterone levels with age for the age group of 35 to 70 years, and then draw it backwards, from the age of 90 to the earlier years, it will appear that the fathers, aged 30-35, exhibited significantly higher testosterone levels than their descendants at comparable ages (Fig. 7).

Fig. 7. Concentrations of testosterone in men aged 20-90 years and the line aptly showing their fathers' testosterone levels in the past.

Figure 7 shows that the concentration of testosterone in today's young men aged 30-35 is, on average, 23 nmol/L Concentrations of testosterone of their ancestors aged 85-89 averaged 17.2 nmol/L Therefore, standard values in the testosterone concentration range – the dispersion of values previously ranging to 31 nmol/L– have now narrowed to such an extent that the difference in testosterone levels between younger and older men is only 5 nmol/L i.e. 16% of normal range 10-42 nmol/L It practically means that young men now have testosterone levels about 35% lower than those of their ancestors. Therefore, it can be concluded that, on the one hand, the whole world population is aging. On the other hand, the biological condition of today's youth suggests that in such a metabolic-hormonal state the youth of today will not increase human life expectancy. It is highly likely that they will die at a much younger age than their ancestors did in the past. The reasons for these changes are more complex. Genetic factors, lifestyle habits, medications, toxins, free radicals, body weight, psychological and social aspects, chronic stress, mental health, and social position play a significant role.

Late - Onset Hypogonadism - New Point of View 129

disease. Dopamine plays a crucial role in our mental health. Also, male sexual fitness is closely related to hormonal balance nd especially to dopamine and 4-hydroxyestradiol. Outlined below are some of the entities associated with testosterone deficit or with the

Dopamine 4-hydroxyestradiol

Clinical trial results have shown that men with type 2 diabetes have a significantly greater testosterone deficiency (Zitzmann & Nieschlag, 2006). In a large population study of subjects aged over 20, men with total testosterone levels in the lowest quartile and men with free testosterone in the lowest quartile had a 4-fold higher prevalence of diabetes compared with men with testosterone levels in the first quartile /p = 0.04/ (Muller et al., 2005). Metabolic syndrome, defined as a combination of lipid disorders and cardiovascular risk factors: abdominal obesity, insulin resistance, and arterial hypertension, also increases the risk for late onset hypogonadism ((Kalyani & Dobs, 2007). In my own research, out of 1200 men with late onset hypogonadism treated with hCG, more than 10 percent had diabetes. Raising the serum testosterone levels in these patients as a result of hCG therapy significantly improved glucose tolerance. Increasing testosterone levels, the average level of output being 18.3 nmol/L-38.6 nmol/L, resulted in a reduction of HbA1c by an average of 1.75%

Normalization of HbA1c followed in the not-previously-treated diabetic group, as well as in the group of patients taking their medication without changing the dose. In some patients previously treated for diabetes, at the time of hCG administration, it was necessary to lower the dose or even to discontinue antidiabetic therapy. Among patients without known diabetes (HbA1c<6.0%), as a result of hCG therapy, HbA1c reductions were observed by an average of 0.5%. In patients with diabetes, insulin level was also determined. Average output level was 16.55 mU/L, and with intensified hCG therapy the average calculated

I believe that the term "insulin resistance" is overused. Insulin resistance does not cause diabetes. The inability to metabolize glucose (burn sugar), fully and continually is the cause of type 2 diabetes. An increase in the concentration of the glucose in the extracellular space

Fig. 8. The dopamine structure and the 4-hydroxyestradiol structure.

**3.2 Late - onset hypogonadism and type 2 diabetes** 

(p<0.001). This effect is shown graphically in Figure 9.

insulin level was 7.8 mU/L.

deficit of testosterone derivatives.

#### **3. Late - onset hypogonadism and other diseases**

Testosterone levels in maturing man peak around age of 30 and then start to decline slowly. This decline in serum testosterone of aging men can lead to the development of many diseases (e.g. hypertension, ischemic heart disease etc.). The metabolic syndrome (defined as a combination of lipid abnormalities and cardiovascular risk factors), abdominal obesity, insulin resistance, arterial hypertension or raised blood pressure, are also associated with testosterone deficiency (Kalyani & Dobs, 2007). All men are at risk for erectile dysfunction and prostate diseases including Benign Prostatic Hyperplasia (BPH) and Prostate Cancer (PC), which occur in later years of life of men. Additionally, Alzheimer's disease and Parkinson's disease are both common in the elderly men, especially in those over 85. Low testosterone levels − a risk factor for development of visceral obesity − are associated with an acute decrease in circulating HDL cholesterol and increase of triglycerides. There may be a link between low testosterone levels in males and type 2 diabetes with elevated insulin levels. Some of these problems are due to the concentrations of total and free testosterone but there are also those that are associated with the derivatives of persistent testosterone deficiency. Late onset hypogonadism may result in the metabolic syndrome frequently leading to diabetes and/or to accelerated heart disease. When testosterone hormones in our body are balanced, the symptoms disappear*.* An increase in serum testosterone, results in normalization of lipid profile, improves glucose tolerance, decreases HbA1c, and produces the increase in bone mass observed in densitometry. Pharmacological correction of hormones results in marked improvements in the sexual health (increasing both libido and potency), removes depression symptoms, and reduces or completely removes all symptoms of BPH and of Parkinson's disease.

#### **3.1 Late - onset hypogonadism is not just testosterone deficiency**

So far the aging process and clinical and biochemical changes which cause aging have been linked to reduction in testosterone concentration. This approach is, however, too simplistic. Testosterone is the direct precursor to estradiol, which is the most potent endogenous estrogen, and to many other hormone derivatives which have a significant impact on the normal structure and on the function of the human body. Therefore therapy to increase testosterone is inextricably linked with estradiol supplementation. Here special attention should be paid to the fact that not all preparations of testosterone can be converted to estradiol and to other derivatives, which makes such supplementation seem not to improve, but, sometimes, to make the situation worse. Estrogen affects the skeletal bone and cardiovascular systems in many radical ways. The decline in estrogen is associated with osteoporosis, premature atherosclerosis, marked risk of myocardial infarction, and with loss of bone mass (Gooren & Bunck, 2004).

While testosterone can act directly on cells, it can also be converted to dihydroxytestosterone (DHT) by 5α-reductase. The same chemical reaction occurs which converts estradiol to 4 hydroxyestradiol. In addition to hormonal effects, this compound has the peculiarity of saturation binding for dopamine receptors widely distributed in the brain. If one compares the chemical structure of dopamine with that of 4-hydroxyestradiol, it is possible to find the key to the dopamine receptor − a benzene ring with two hydroxyl groups attached to the ring − which can be seen in Figure 8.

Dopamine receptors saturation in human brain has some very important implications of many physiological and pathological states. Dopamine deficiency causes Parkinson's

Testosterone levels in maturing man peak around age of 30 and then start to decline slowly. This decline in serum testosterone of aging men can lead to the development of many diseases (e.g. hypertension, ischemic heart disease etc.). The metabolic syndrome (defined as a combination of lipid abnormalities and cardiovascular risk factors), abdominal obesity, insulin resistance, arterial hypertension or raised blood pressure, are also associated with testosterone deficiency (Kalyani & Dobs, 2007). All men are at risk for erectile dysfunction and prostate diseases including Benign Prostatic Hyperplasia (BPH) and Prostate Cancer (PC), which occur in later years of life of men. Additionally, Alzheimer's disease and Parkinson's disease are both common in the elderly men, especially in those over 85. Low testosterone levels − a risk factor for development of visceral obesity − are associated with an acute decrease in circulating HDL cholesterol and increase of triglycerides. There may be a link between low testosterone levels in males and type 2 diabetes with elevated insulin levels. Some of these problems are due to the concentrations of total and free testosterone but there are also those that are associated with the derivatives of persistent testosterone deficiency. Late onset hypogonadism may result in the metabolic syndrome frequently leading to diabetes and/or to accelerated heart disease. When testosterone hormones in our body are balanced, the symptoms disappear*.* An increase in serum testosterone, results in normalization of lipid profile, improves glucose tolerance, decreases HbA1c, and produces the increase in bone mass observed in densitometry. Pharmacological correction of hormones results in marked improvements in the sexual health (increasing both libido and potency), removes depression symptoms, and reduces or completely removes all symptoms

**3. Late - onset hypogonadism and other diseases** 

of BPH and of Parkinson's disease.

of bone mass (Gooren & Bunck, 2004).

which can be seen in Figure 8.

**3.1 Late - onset hypogonadism is not just testosterone deficiency** 

So far the aging process and clinical and biochemical changes which cause aging have been linked to reduction in testosterone concentration. This approach is, however, too simplistic. Testosterone is the direct precursor to estradiol, which is the most potent endogenous estrogen, and to many other hormone derivatives which have a significant impact on the normal structure and on the function of the human body. Therefore therapy to increase testosterone is inextricably linked with estradiol supplementation. Here special attention should be paid to the fact that not all preparations of testosterone can be converted to estradiol and to other derivatives, which makes such supplementation seem not to improve, but, sometimes, to make the situation worse. Estrogen affects the skeletal bone and cardiovascular systems in many radical ways. The decline in estrogen is associated with osteoporosis, premature atherosclerosis, marked risk of myocardial infarction, and with loss

While testosterone can act directly on cells, it can also be converted to dihydroxytestosterone (DHT) by 5α-reductase. The same chemical reaction occurs which converts estradiol to 4 hydroxyestradiol. In addition to hormonal effects, this compound has the peculiarity of saturation binding for dopamine receptors widely distributed in the brain. If one compares the chemical structure of dopamine with that of 4-hydroxyestradiol, it is possible to find the key to the dopamine receptor − a benzene ring with two hydroxyl groups attached to the ring −

Dopamine receptors saturation in human brain has some very important implications of many physiological and pathological states. Dopamine deficiency causes Parkinson's disease. Dopamine plays a crucial role in our mental health. Also, male sexual fitness is closely related to hormonal balance nd especially to dopamine and 4-hydroxyestradiol. Outlined below are some of the entities associated with testosterone deficit or with the deficit of testosterone derivatives.

Fig. 8. The dopamine structure and the 4-hydroxyestradiol structure.

#### **3.2 Late - onset hypogonadism and type 2 diabetes**

Clinical trial results have shown that men with type 2 diabetes have a significantly greater testosterone deficiency (Zitzmann & Nieschlag, 2006). In a large population study of subjects aged over 20, men with total testosterone levels in the lowest quartile and men with free testosterone in the lowest quartile had a 4-fold higher prevalence of diabetes compared with men with testosterone levels in the first quartile /p = 0.04/ (Muller et al., 2005). Metabolic syndrome, defined as a combination of lipid disorders and cardiovascular risk factors: abdominal obesity, insulin resistance, and arterial hypertension, also increases the risk for late onset hypogonadism ((Kalyani & Dobs, 2007). In my own research, out of 1200 men with late onset hypogonadism treated with hCG, more than 10 percent had diabetes. Raising the serum testosterone levels in these patients as a result of hCG therapy significantly improved glucose tolerance. Increasing testosterone levels, the average level of output being 18.3 nmol/L-38.6 nmol/L, resulted in a reduction of HbA1c by an average of 1.75% (p<0.001). This effect is shown graphically in Figure 9.

Normalization of HbA1c followed in the not-previously-treated diabetic group, as well as in the group of patients taking their medication without changing the dose. In some patients previously treated for diabetes, at the time of hCG administration, it was necessary to lower the dose or even to discontinue antidiabetic therapy. Among patients without known diabetes (HbA1c<6.0%), as a result of hCG therapy, HbA1c reductions were observed by an average of 0.5%. In patients with diabetes, insulin level was also determined. Average output level was 16.55 mU/L, and with intensified hCG therapy the average calculated insulin level was 7.8 mU/L.

I believe that the term "insulin resistance" is overused. Insulin resistance does not cause diabetes. The inability to metabolize glucose (burn sugar), fully and continually is the cause of type 2 diabetes. An increase in the concentration of the glucose in the extracellular space

Late - Onset Hypogonadism - New Point of View 131

It is beyond discussion that women have osteoporosis as a direct result of ovarian hormone imbalance. In bone densitometry studies, 90% of those examined are females and only 10% are males; hence the great retardation of male endocrinology and of densitometric diagnosis of osteoporosis in men in relation to the same areas of medicine with regard to women. According to the recommendations of World Health Organisation, a woman should take hormone therapy early enough to avoid dangerous irreversible anatomical changes. What about a man? At puberty male estrogen can affect skeletal growth and bone mineralization. The study of men in the andropause also indicates a relation of estrogen level to bone mineral density (Riggs, et al. 2002). Studies have shown that two-thirds of the effects of testosterone replacement therapy (particularly with regard to increase in bone mineral) are due to excessive estrogen levels in aromatization of testosterone (Khosla, et al., 1998, Leder, et al., 2003). There is a clear relationship between the amount of androgens (bioavailable testosterone) and estrogens (bioavailable estradiol E2). In a large-scale study (data from 2623 men over 65, enjoying good health) levels of free and bioavailable testosterone and bioavailable estradiol levels correlated. Low levels of bioavailable estradiol were associated with age and with osteoporosis (Orwoll, et al. 2006). In our own panel of 1200 patients, it was found that raising man's testosterone levels to the values recommended for a given age was simultaneously accompanied by the increase of estradiol level. This caused the incorporation of calcium into bones, without any additional classical treatment of osteoporosis. The hormonal therapy used was to induce endogenous testosterone synthesis by human chorionic gonadotrophin (hCG). Please find below a depiction of the effects. A two year hCG therapy in a patient aged 60 years increased the bone density by 6.7% ( Figure 11 and Figure 12); and a long-term therapy could help restore bone density to that of a fine young man. Figure 13 shows densitometric data of the chapter's author (age 62y). Bone density of a fine young man − Young Ref (%) = 92.7; bone density with reference to age −

Fig. 10. Improved lipids during HCG-treatment hormonal treatment

**3.4 Late - onset hypogonadism and osteoporosis** 

Age Match (%) = 118.1 Z-Score = 1.26)

induces the increase in insulin levels. But insulin does not move sugar into cells when the cell-glucose level is equally high. Acceleration of tissue metabolism after injection of testosterone causes excessively fast burning of glucose, which decreases cell-glucose levels. Then, according to the concentration gradient, glucose enters the cell and this reduces extracellular cell concentrations of glucose. With the reduction of glucose concentration in the extracellular space, blood insulin levels fall below a certain level. Therefore "insulin resistance" does not apply in this case.

Fig. 9. HbA1c normalization as a result of hCG therapy.

#### **3.3 Late - onset hypogonadism and hyperlipidemia**

As men age, there is a dramatic decrease in testosterone production, with a corresponding decrease in the production of estradiol. Excess lipids in the blood lead to atherosclerosis. Cholesterol, which can be very annoying, is in fact absolutely necessary. Too high or too low a concentration thereof has a detrimental effect. LDL cholesterol serves as precursor for the synthesis of steroid hormones. Moreover, every cell in the body requires LDL cholesterol to maintain cell wall integrity. With age, cell turnover is reduced. What is more, there are lower cholesterol production rates to create new cells. An excessive reduction of cholesterol levels in the body results in the deficit of raw materials for cellular renewal and in the deficit in the synthesis of steroid hormones. Various formulations are present in the treatment for hypercholesterolemia. However, hypercholesterolemia can also be normalized only by accelerating the metabolic processes. Anabolic testosterone boost effectively corrects the moderate lipid metabolic disorders. The hCG hormone therapy, which results in a significant increase in testosterone concentration and in E2 concentration (in testosterone level − from 18.3 nmol/L to 38.6 nmol/L; in E2 level − from 138.6 pmol/L to 280.9 pmol/L), improves lipid profile. There is a 15 to 20% decrease in total cholesterol and its fractions. This effect is shown graphically in Figure 10.

induces the increase in insulin levels. But insulin does not move sugar into cells when the cell-glucose level is equally high. Acceleration of tissue metabolism after injection of testosterone causes excessively fast burning of glucose, which decreases cell-glucose levels. Then, according to the concentration gradient, glucose enters the cell and this reduces extracellular cell concentrations of glucose. With the reduction of glucose concentration in the extracellular space, blood insulin levels fall below a certain level. Therefore "insulin

As men age, there is a dramatic decrease in testosterone production, with a corresponding decrease in the production of estradiol. Excess lipids in the blood lead to atherosclerosis. Cholesterol, which can be very annoying, is in fact absolutely necessary. Too high or too low a concentration thereof has a detrimental effect. LDL cholesterol serves as precursor for the synthesis of steroid hormones. Moreover, every cell in the body requires LDL cholesterol to maintain cell wall integrity. With age, cell turnover is reduced. What is more, there are lower cholesterol production rates to create new cells. An excessive reduction of cholesterol levels in the body results in the deficit of raw materials for cellular renewal and in the deficit in the synthesis of steroid hormones. Various formulations are present in the treatment for hypercholesterolemia. However, hypercholesterolemia can also be normalized only by accelerating the metabolic processes. Anabolic testosterone boost effectively corrects the moderate lipid metabolic disorders. The hCG hormone therapy, which results in a significant increase in testosterone concentration and in E2 concentration (in testosterone level − from 18.3 nmol/L to 38.6 nmol/L; in E2 level − from 138.6 pmol/L to 280.9 pmol/L), improves lipid profile. There is a 15 to 20% decrease in total cholesterol and its fractions.

resistance" does not apply in this case.

Fig. 9. HbA1c normalization as a result of hCG therapy.

**3.3 Late - onset hypogonadism and hyperlipidemia** 

This effect is shown graphically in Figure 10.

Fig. 10. Improved lipids during HCG-treatment hormonal treatment

#### **3.4 Late - onset hypogonadism and osteoporosis**

It is beyond discussion that women have osteoporosis as a direct result of ovarian hormone imbalance. In bone densitometry studies, 90% of those examined are females and only 10% are males; hence the great retardation of male endocrinology and of densitometric diagnosis of osteoporosis in men in relation to the same areas of medicine with regard to women. According to the recommendations of World Health Organisation, a woman should take hormone therapy early enough to avoid dangerous irreversible anatomical changes. What about a man? At puberty male estrogen can affect skeletal growth and bone mineralization. The study of men in the andropause also indicates a relation of estrogen level to bone mineral density (Riggs, et al. 2002). Studies have shown that two-thirds of the effects of testosterone replacement therapy (particularly with regard to increase in bone mineral) are due to excessive estrogen levels in aromatization of testosterone (Khosla, et al., 1998, Leder, et al., 2003). There is a clear relationship between the amount of androgens (bioavailable testosterone) and estrogens (bioavailable estradiol E2). In a large-scale study (data from 2623 men over 65, enjoying good health) levels of free and bioavailable testosterone and bioavailable estradiol levels correlated. Low levels of bioavailable estradiol were associated with age and with osteoporosis (Orwoll, et al. 2006). In our own panel of 1200 patients, it was found that raising man's testosterone levels to the values recommended for a given age was simultaneously accompanied by the increase of estradiol level. This caused the incorporation of calcium into bones, without any additional classical treatment of osteoporosis. The hormonal therapy used was to induce endogenous testosterone synthesis by human chorionic gonadotrophin (hCG). Please find below a depiction of the effects. A two year hCG therapy in a patient aged 60 years increased the bone density by 6.7% ( Figure 11 and Figure 12); and a long-term therapy could help restore bone density to that of a fine young man. Figure 13 shows densitometric data of the chapter's author (age 62y). Bone density of a fine young man − Young Ref (%) = 92.7; bone density with reference to age − Age Match (%) = 118.1 Z-Score = 1.26)

Late - Onset Hypogonadism - New Point of View 133

It should come as no surprise to anyone that chronic prostatitis, benign prostatic hyperplasia (BPH), and prostate cancer (PC) are consequences of hormonal deficits. Prostate-specific antigen (PSA), a marker that has been used for many years in early prostate cancer detection, turned out to be less than 100% accurate to diagnose prostate malignancy. PSA was originally supposed to be a unique marker for prostate cancer. It was supposed to be just what its name implies – a specific marker. Over the past few decades an enormous amount of material has been written about the role of PSA in diagnosis of prostate cancer. Doctors started to use other PSA values - such as PSA velocity, PSA density, and percentfree PSA - to get a more accurate idea of what was happening within the prostate. When the PSA reaches or exceeds the level of 0.5 ng/mL, it is strongly recommended to take all-out diagnostic measures, in order to ascertain the absence of PC (Gould, et al., 2006). Practically in such cases, the biopsy of the prostate should be done. After a prostate biopsy, the PSA continues to rise, so the biopsy is repeated. This method often cannot give definite confirmation of prostate cancer, which has been the first dilemma. The other dilemma: what if the blood tests detect much lower levels of PSA? What if the reduction amounts to 2-3 fold? Long-term clinical experience shows that a sudden significant increase in PSA level is possible with any surgery, after virus infection regardless of its location, or with regard to a severe exacerbation of chronic inflammatory diseases. A sudden increase in one's PSA level should not escape the attention, but it is not equivalent to the occurrence or the development of prostate cancer. Changes in the PSA levels may be the consequence of the high levels of prostaglandins, which induce non-specific inflammations of the prostate

Harbitz stated over 30 years ago that male gonadal dysfunction (endocrine testicular failure) leads to adenomas and to the prostate cancer (Harbitz & Haugen, 1974). It is an undeniable fact that testosterone levels peak in a man at approximately age 30, but this does not yet cause BPH or prostate cancer. And the older the man, and the greater the chances of testosterone deficiency, the higher the prevalence of benign prostatic hyperplasia and of PC. It can no longer be doubted, questioned, whether BPH and PC are closely related to the

There are publications, which say that there was never any association between testosterone treatment, when subjects were all healthy men, and the increase of prostate volume and serum PSA level. They also say that in the treatment of men with hypogonadism, the therapy led to moderate prostate enlargement and to a 15% increase in PSA (Algarte-Genin, et al., 2004, Behre, et al., 1994, Gould & Kirby, 2006). Among my own 1200 patients under treatment for hypogonadism, more than two-fold increase in endogenous testosterone concentrations after 37 months of hCG therapy resulted in a 40% decrease in their PSA levels. The aforementioned decrease is closely related not only to concentration of testosterone, but to the level of estradiol, which stimulates LHRH mRNA synthesis and increases pituitary LH synthesis. Testosterone deficiency does not cause prostate pathology. However, the losses of testosterone and long-term E2 deficiency result in the situation in which the hypothalamus and pituitary gland can become prostate's greatest enemies. Therefore, modern hormonal medications used to treat BPH have selfstimulation points in the lateral hypothalamus (Oesterling, 1991, Reissmann, et al., 2000,

**3.5 Late - onset hypogonadism, chronic prostatitis, BPH and prostate cancer** 

usually seen in urine samples under a microscope.

Debruyne, et al., 2008).

hormonal changes in men, and practically to testosterone deficiency.

#### Fig. 11.


#### Fig. 12.

Fig. 13.

Fig. 11.

Fig. 12.

Fig. 13.

#### **3.5 Late - onset hypogonadism, chronic prostatitis, BPH and prostate cancer**

It should come as no surprise to anyone that chronic prostatitis, benign prostatic hyperplasia (BPH), and prostate cancer (PC) are consequences of hormonal deficits. Prostate-specific antigen (PSA), a marker that has been used for many years in early prostate cancer detection, turned out to be less than 100% accurate to diagnose prostate malignancy. PSA was originally supposed to be a unique marker for prostate cancer. It was supposed to be just what its name implies – a specific marker. Over the past few decades an enormous amount of material has been written about the role of PSA in diagnosis of prostate cancer. Doctors started to use other PSA values - such as PSA velocity, PSA density, and percentfree PSA - to get a more accurate idea of what was happening within the prostate. When the PSA reaches or exceeds the level of 0.5 ng/mL, it is strongly recommended to take all-out diagnostic measures, in order to ascertain the absence of PC (Gould, et al., 2006). Practically in such cases, the biopsy of the prostate should be done. After a prostate biopsy, the PSA continues to rise, so the biopsy is repeated. This method often cannot give definite confirmation of prostate cancer, which has been the first dilemma. The other dilemma: what if the blood tests detect much lower levels of PSA? What if the reduction amounts to 2-3 fold? Long-term clinical experience shows that a sudden significant increase in PSA level is possible with any surgery, after virus infection regardless of its location, or with regard to a severe exacerbation of chronic inflammatory diseases. A sudden increase in one's PSA level should not escape the attention, but it is not equivalent to the occurrence or the development of prostate cancer. Changes in the PSA levels may be the consequence of the high levels of prostaglandins, which induce non-specific inflammations of the prostate usually seen in urine samples under a microscope.

Harbitz stated over 30 years ago that male gonadal dysfunction (endocrine testicular failure) leads to adenomas and to the prostate cancer (Harbitz & Haugen, 1974). It is an undeniable fact that testosterone levels peak in a man at approximately age 30, but this does not yet cause BPH or prostate cancer. And the older the man, and the greater the chances of testosterone deficiency, the higher the prevalence of benign prostatic hyperplasia and of PC. It can no longer be doubted, questioned, whether BPH and PC are closely related to the hormonal changes in men, and practically to testosterone deficiency.

There are publications, which say that there was never any association between testosterone treatment, when subjects were all healthy men, and the increase of prostate volume and serum PSA level. They also say that in the treatment of men with hypogonadism, the therapy led to moderate prostate enlargement and to a 15% increase in PSA (Algarte-Genin, et al., 2004, Behre, et al., 1994, Gould & Kirby, 2006). Among my own 1200 patients under treatment for hypogonadism, more than two-fold increase in endogenous testosterone concentrations after 37 months of hCG therapy resulted in a 40% decrease in their PSA levels. The aforementioned decrease is closely related not only to concentration of testosterone, but to the level of estradiol, which stimulates LHRH mRNA synthesis and increases pituitary LH synthesis. Testosterone deficiency does not cause prostate pathology. However, the losses of testosterone and long-term E2 deficiency result in the situation in which the hypothalamus and pituitary gland can become prostate's greatest enemies. Therefore, modern hormonal medications used to treat BPH have selfstimulation points in the lateral hypothalamus (Oesterling, 1991, Reissmann, et al., 2000, Debruyne, et al., 2008).

Late - Onset Hypogonadism - New Point of View 135

blockers do not solve the problem of treatment for BPH.

cause hot flashes, decreased libido and impotence (Oesterling, 1991).

testosterone undoubtedly can positively affect the biology of man?

using 5 alpha reductase inhibitors in the treatment of BPH.

which inhibit LH synthesis or LHRH synthesis.

**3.5.2 LOH and prostate cancer** 

markedly reduced.

Fig. 18. Fig. 19. Fig. 20.

19), and after 6 months of hCG therapy (Fig. 20). Arrows indicate regions of prostate adenoma − hyperplasia of the transition zone − which as a result of hCG treatment was

Fig. 18. − 20. Prostate MRI before hCG therapy (Fig. 18), after 3 months of hCG therapy (Fig.

Some experts now recommend alpha-blockers and hormones as first-line treatment for patients with moderate to severe BPH symptoms. It must be emphatically stated that alphablockers therapy appears to have no effect on the disease; it does not hinder its development; it only mitigates symptoms of BPH and facilitates urination. But after using alpha-blockers for a number of years adenoma can enlarge to the extent that surgical treatment becomes necessary. At the same time, there will be irreversible pathological changes of the bladder wall, which disrupt the normal function thereof. Thus, alpha-

In hormonal therapy of BPH the agents used are: 1.- 5α-reductase inhibitors, and 2. - drugs,

Ad. 1. 5-alpha reductase inhibitors block the conversion of testosterone to dihydroxytestosterone. Their effectiveness in the management of BPH is debatable. Laurie Barclay, MD (Pfizer), issued a statement used in Pfizer commercial that Finasteride was no better than placebo in treating BPH symptoms. In fact, 5 alpha reductase inhibitors may

In my opinion, this drug works, but its metabolic and functional consequences mean that the balance of potential profit versus loss can be harmful for men. Therefore I have not been

Ad. 2. In the treatment of BPH, other drugs have been used as well to decrease LH synthesis for more than 20 years. This effect has been achieved by LHRH analogues or LHRH agonists (Oesterling, 1991, Reissmann, et al., 2000, Debruyne, et al., 2008). This therapy, however, has significant disadvantages. It blocks testosterone endosynthesis, which leads to metabolic and functional complications. A fundamental question arises: What kind of man will deprive himself of potency and disrupt his normal body chemistry and metabolism in order to improve the health of his prostate? And so, shall we provide such a treatment, if it can block LH synthesis, and, while at the same time, more than a twofold increase in

Half a century ago arose the bizarre notion that testosterone was really dangerous and that it could cause prostate cancer. Years later, it became clear that there was no credible

#### **3.5.1 Late - onset hypogonadism, BPH and chronic prostatitis**

The symptoms of BPH are practically only recognized when there are irreversible anatomical changes in the prostate, causing LUTS. Patients with BPH often see the doctor when they already have symptoms of LUTS. Meanwhile, the symptoms of chronic prostatitis will be in place long before that. Antibiotic or other medications are rather ineffective in those patients and the hormone therapy can help to relieve clinical symptoms. The effects of hormonal disbalance, including testosterone deficiency, can be seen on transabdominal and transrectal ultrasound scans.

If hypoechoic areas in the transition zone of the prostate (marked with dots in Figures 14÷17) are noted in ultrasound, it is a common sign of deficient testosterone levels. Such changes are occasionally described as inflammation and are not paid due attention to.

Figure 14.÷17. Ultrasound scan images of the prostate of testosterone deficiency patients. Figure 14 & 15. Transrectal ultrasonography. Figure 16 & 17. Abdominal examination. Hypoechoic area marked by dots.

In my 30 years of being an urologist, I have treated hundreds of men diagnosed with prostatitis by other physicians. Antibiotics treatment could last several weeks or even months. Worst of all, it was not effective. Treatment with hCG led to the freedom from symptoms within 1-3 weeks. These symptoms were associated with the spread of prostate adenoma which, as it can lift the bladder, causes prostate inflammation symptoms. During hCG therapy, there is a reduction in adenoma, as demonstrated by MRI (Figures 18÷20).

Fig. 18. Fig. 19. Fig. 20.

The symptoms of BPH are practically only recognized when there are irreversible anatomical changes in the prostate, causing LUTS. Patients with BPH often see the doctor when they already have symptoms of LUTS. Meanwhile, the symptoms of chronic prostatitis will be in place long before that. Antibiotic or other medications are rather ineffective in those patients and the hormone therapy can help to relieve clinical symptoms. The effects of hormonal disbalance, including testosterone deficiency, can be seen on

If hypoechoic areas in the transition zone of the prostate (marked with dots in Figures 14÷17) are noted in ultrasound, it is a common sign of deficient testosterone levels. Such changes are occasionally described as inflammation and are not paid due attention to.

Figure 14.÷17. Ultrasound scan images of the prostate of testosterone deficiency patients. Figure 14 & 15. Transrectal ultrasonography. Figure 16 & 17. Abdominal examination.

In my 30 years of being an urologist, I have treated hundreds of men diagnosed with prostatitis by other physicians. Antibiotics treatment could last several weeks or even months. Worst of all, it was not effective. Treatment with hCG led to the freedom from symptoms within 1-3 weeks. These symptoms were associated with the spread of prostate adenoma which, as it can lift the bladder, causes prostate inflammation symptoms. During hCG therapy, there is a reduction in adenoma, as demonstrated by MRI (Figures 18÷20).

Fig.14. Fig. 15.

Fig. 16. Fig. 17.

Hypoechoic area marked by dots.

**3.5.1 Late - onset hypogonadism, BPH and chronic prostatitis** 

transabdominal and transrectal ultrasound scans.

Fig. 18. − 20. Prostate MRI before hCG therapy (Fig. 18), after 3 months of hCG therapy (Fig. 19), and after 6 months of hCG therapy (Fig. 20). Arrows indicate regions of prostate adenoma − hyperplasia of the transition zone − which as a result of hCG treatment was markedly reduced.

Some experts now recommend alpha-blockers and hormones as first-line treatment for patients with moderate to severe BPH symptoms. It must be emphatically stated that alphablockers therapy appears to have no effect on the disease; it does not hinder its development; it only mitigates symptoms of BPH and facilitates urination. But after using alpha-blockers for a number of years adenoma can enlarge to the extent that surgical treatment becomes necessary. At the same time, there will be irreversible pathological changes of the bladder wall, which disrupt the normal function thereof. Thus, alphablockers do not solve the problem of treatment for BPH.

In hormonal therapy of BPH the agents used are: 1.- 5α-reductase inhibitors, and 2. - drugs, which inhibit LH synthesis or LHRH synthesis.

Ad. 1. 5-alpha reductase inhibitors block the conversion of testosterone to dihydroxytestosterone. Their effectiveness in the management of BPH is debatable. Laurie Barclay, MD (Pfizer), issued a statement used in Pfizer commercial that Finasteride was no better than placebo in treating BPH symptoms. In fact, 5 alpha reductase inhibitors may cause hot flashes, decreased libido and impotence (Oesterling, 1991).

In my opinion, this drug works, but its metabolic and functional consequences mean that the balance of potential profit versus loss can be harmful for men. Therefore I have not been using 5 alpha reductase inhibitors in the treatment of BPH.

Ad. 2. In the treatment of BPH, other drugs have been used as well to decrease LH synthesis for more than 20 years. This effect has been achieved by LHRH analogues or LHRH agonists (Oesterling, 1991, Reissmann, et al., 2000, Debruyne, et al., 2008). This therapy, however, has significant disadvantages. It blocks testosterone endosynthesis, which leads to metabolic and functional complications. A fundamental question arises: What kind of man will deprive himself of potency and disrupt his normal body chemistry and metabolism in order to improve the health of his prostate? And so, shall we provide such a treatment, if it can block LH synthesis, and, while at the same time, more than a twofold increase in testosterone undoubtedly can positively affect the biology of man?

#### **3.5.2 LOH and prostate cancer**

Half a century ago arose the bizarre notion that testosterone was really dangerous and that it could cause prostate cancer. Years later, it became clear that there was no credible

Late - Onset Hypogonadism - New Point of View 137

them have had some form of prostate cancer within 3 to 36 months. The results clearly show that there is a greater decrease in endogenous testosterone synthesis, but at the same time there is an increased risk of prostate cancer. The patients in question had very high LH/PSA ratio. Therefore prostate cancer is not caused by testosterone, but, to the contrary, it is a consequence of the development of testosterone deficiency. Moreover, there is a correlation between testosterone levels and the degree of PC malignancy (Hoffman, et al., 2000). It has been known for years that patients diagnosed with prostate cancer and with high Gleason scores, have lower than normal levels of testosterone. Lower testosterone levels result in greater PC malignancy (Schatzl, et al., 2001). Patients with high Gleason score prostate

There is also no evidence that men with prostate cancer have a much higher testosterone concentration than the normal population. On the other hand, low testosterone values found in men with prostate cancer are associated with poorer prognosis, with a lower degree of differentiation of PC, and with a higher degree of PC severity (Hoffman, et al., 2000, Morgentaler, 2006, Yano, et al., 2007). In men treated with testosterone, prostate cancer was detected in biopsies of 1% of patients, while in men with the prevalence of hypogonadism − in 14.3%. There are some reports that testosterone therapy may reduce the risk of prostate cancer (Fowler & Whitmore, 1981, Prout & Brewer, 1967). Only some reports, because who

For many years, we have been reassured that only androgens have a key role in both stimulating and maintaining sexual function in men. It was believed that testosterone and the existence of a normal level of libido were inseparably connected (Shabsigh, 2003, Morales, et al., 2004). Nevertheless, sexual dysfunction in men is directly associated not only with testosterone but also with estradiol (E-2) and with other neurohormonal factors. Erectile dysfunction was linked to the development of benign prostatic hyperplasia (BPH), without even taking into account the fact that both benign prostatic hyperplasia (BPH) and erectile dysfunction (ED) were often caused by the hormone deficit. The effects of women's hormone replacement therapy on their psychological and sexual functioning are still the subject of the research in the borderline field between medicine and psychology. Female hormone therapy in treating menopause is common around the world. Meanwhile, several years ago it was stated that the effects of this therapy on men were not known. Hormone replacement therapy for men has been lagging for at least 20 years compared with hormone replacement therapy in women (Tenover, 1999). There is a decline in testosterone production in elderly men that can lead to a decrease in sexual desire (Kaufman & T'Sjoen, 2002). Androgen therapy can stop and even reverse this degenerating process (Hajjar, et al., 1997, Morales, et al., 2004). Information on the importance of testosterone in male sexuality is often divergent. It is known that surgical and pharmacological castration leads to impotence. On the other hand, it is recognized that testosterone deficiency is considered to be of little importance in the development of erectile dysfunction or in life force. (Anderson,

There was no sexual orientation change in men who underwent surgical castration. Sexual desire was preserved by the majority of the patients, but interest in sex decreased, which was associated with decreased frequency/intensity of orgasms (Zverina, et al., 1990). Testosterone replacement therapy suitable for men with primary testicular failure as a result

would dare to publish anything incompatible with the study by Nobel laureates?

**3.6 Late - onset hypogonadism, sexual drive, potency and libido** 

2003, Montorsi et al., 2003).

cancer have lower testosterone and estradiol serum levels.

scientific basis for the claim that high levels of testosterone accelerated development of prostate cancer. In fact, the opposite is true (Bonczyk, et al., 2008). The theory that testosterone may stimulate the growth of prostate cancer originates from 1941 when Huggins and Hodges reported that carcinoma was androgen-dependent. They demonstrated the importance of lowering testosterone levels in prostate cancer progression. Huggins and Hodges won the 1966 Nobel Peace Prize for their groundbreaking work revealing that castration causes regression of prostate cancer; while testosterone may cause its progression (Huggins & Hodges, 1941). It is now known that a multiplicity of data contradict Huggins and Hodges's contention that testosterone accelerates prostate cancer growth. Apparently it was negligent misinterpretation of one patient observation (Morgentaler, 2006). And they won the Nobel Prize. This caused a distorted look at the testosterone-prostate cancer link over the last half century. Even 15 years ago in Poland, testosterone was considered a carcinogen by Polish Ministry of Health and Social Welfare (Official Law Daily, 1996). There is no evidence that among men with prostate cancer, serum testosterone concentration is higher than in the rest of the population. On the other hand, low testosterone values coexisting with prostate cancer are associated with worse prognosis, a lower degree of differentiation, and a higher degree of severity of cancer (Morgentaler, 2006, Yano, et al., 2007). Moreover, there is no convincing evidence showing the effect of testosterone on the development of pre-cancerous prostate condition. Rhoden et al. determined that the risk of the development of prostate cancer in hypogonadal patients receiving testosterone, with and without accompanying HG-PIN, does not increase with hormone therapy. After twelve months, the risk was 1,2% in men without HG-PIN, and 5% in men with HGPIN detected. The aforementioned values do not exceed those in the general population (Rhoden & Morgentaler, 2003). At the same time, one can find statements that there are two classic contraindications for the administration of testosterone, namely suspected or histologically proven prostate cancer and symptomatic benign prostatic hyperplasia (Montorsi, 2007). While I understand precautions that most men with prostate cancer should avoid testosterone therapy, even though I use it for my patients, a total ban on the use of testosterone to treat BPH appears to be an anachronism.

25 years ago, Fowler and Whitmore proposed the concept of "saturation" in order to explain the relationship between prostate cancer and serum testosterone levels. It explains why the increase in serum testosterone levels in patients treated with that hormone does not cause the disease (Fowler & Whitmore, 1981). The correctness of this concept is confirmed by observations of patients, despite treatment with T, there was no increase in PSA or prostate volume. In men suffering from hypogonadism the treatment did not lead to excessive growth in prostate size, and it led to an only 15% increase in PSA (Algarte-Genin, et al., 2004, Gould & Kirby, 2006). Currently, it is believed that exogenous testosterone treatment in patients with PC, which does not follow androgen ablation, should not worsen prognosis of cancer (Bonczyk, et al., 2008). It has long been feared that the Late Onset Hypogonadism (LOH) treatment by administering testosterone may increase the risk of high-grade prostate cancer. Numerous studies show that there is no PSA increase during testosterone therapy. Among my own 1200 patients under treatment for hypogonadism, more than two-fold increase in endogenous testosterone concentrations after 37 months of hCG therapy resulted in a 40% decrease in their PSA levels. In each age group − and the older the man, the more severe it is − there are patients who present insufficient endosynthesis, but they constitute only a few percent of total number of patients. During the extended follow-up of these patients, it has been found that 14% of

scientific basis for the claim that high levels of testosterone accelerated development of prostate cancer. In fact, the opposite is true (Bonczyk, et al., 2008). The theory that testosterone may stimulate the growth of prostate cancer originates from 1941 when Huggins and Hodges reported that carcinoma was androgen-dependent. They demonstrated the importance of lowering testosterone levels in prostate cancer progression. Huggins and Hodges won the 1966 Nobel Peace Prize for their groundbreaking work revealing that castration causes regression of prostate cancer; while testosterone may cause its progression (Huggins & Hodges, 1941). It is now known that a multiplicity of data contradict Huggins and Hodges's contention that testosterone accelerates prostate cancer growth. Apparently it was negligent misinterpretation of one patient observation (Morgentaler, 2006). And they won the Nobel Prize. This caused a distorted look at the testosterone-prostate cancer link over the last half century. Even 15 years ago in Poland, testosterone was considered a carcinogen by Polish Ministry of Health and Social Welfare (Official Law Daily, 1996). There is no evidence that among men with prostate cancer, serum testosterone concentration is higher than in the rest of the population. On the other hand, low testosterone values coexisting with prostate cancer are associated with worse prognosis, a lower degree of differentiation, and a higher degree of severity of cancer (Morgentaler, 2006, Yano, et al., 2007). Moreover, there is no convincing evidence showing the effect of testosterone on the development of pre-cancerous prostate condition. Rhoden et al. determined that the risk of the development of prostate cancer in hypogonadal patients receiving testosterone, with and without accompanying HG-PIN, does not increase with hormone therapy. After twelve months, the risk was 1,2% in men without HG-PIN, and 5% in men with HGPIN detected. The aforementioned values do not exceed those in the general population (Rhoden & Morgentaler, 2003). At the same time, one can find statements that there are two classic contraindications for the administration of testosterone, namely suspected or histologically proven prostate cancer and symptomatic benign prostatic hyperplasia (Montorsi, 2007). While I understand precautions that most men with prostate cancer should avoid testosterone therapy, even though I use it for my patients, a total ban on

the use of testosterone to treat BPH appears to be an anachronism.

25 years ago, Fowler and Whitmore proposed the concept of "saturation" in order to explain the relationship between prostate cancer and serum testosterone levels. It explains why the increase in serum testosterone levels in patients treated with that hormone does not cause the disease (Fowler & Whitmore, 1981). The correctness of this concept is confirmed by observations of patients, despite treatment with T, there was no increase in PSA or prostate volume. In men suffering from hypogonadism the treatment did not lead to excessive growth in prostate size, and it led to an only 15% increase in PSA (Algarte-Genin, et al., 2004, Gould & Kirby, 2006). Currently, it is believed that exogenous testosterone treatment in patients with PC, which does not follow androgen ablation, should not worsen prognosis of cancer (Bonczyk, et al., 2008). It has long been feared that the Late Onset Hypogonadism (LOH) treatment by administering testosterone may increase the risk of high-grade prostate cancer. Numerous studies show that there is no PSA increase during testosterone therapy. Among my own 1200 patients under treatment for hypogonadism, more than two-fold increase in endogenous testosterone concentrations after 37 months of hCG therapy resulted in a 40% decrease in their PSA levels. In each age group − and the older the man, the more severe it is − there are patients who present insufficient endosynthesis, but they constitute only a few percent of total number of patients. During the extended follow-up of these patients, it has been found that 14% of them have had some form of prostate cancer within 3 to 36 months. The results clearly show that there is a greater decrease in endogenous testosterone synthesis, but at the same time there is an increased risk of prostate cancer. The patients in question had very high LH/PSA ratio. Therefore prostate cancer is not caused by testosterone, but, to the contrary, it is a consequence of the development of testosterone deficiency. Moreover, there is a correlation between testosterone levels and the degree of PC malignancy (Hoffman, et al., 2000). It has been known for years that patients diagnosed with prostate cancer and with high Gleason scores, have lower than normal levels of testosterone. Lower testosterone levels result in greater PC malignancy (Schatzl, et al., 2001). Patients with high Gleason score prostate cancer have lower testosterone and estradiol serum levels.

There is also no evidence that men with prostate cancer have a much higher testosterone concentration than the normal population. On the other hand, low testosterone values found in men with prostate cancer are associated with poorer prognosis, with a lower degree of differentiation of PC, and with a higher degree of PC severity (Hoffman, et al., 2000, Morgentaler, 2006, Yano, et al., 2007). In men treated with testosterone, prostate cancer was detected in biopsies of 1% of patients, while in men with the prevalence of hypogonadism − in 14.3%. There are some reports that testosterone therapy may reduce the risk of prostate cancer (Fowler & Whitmore, 1981, Prout & Brewer, 1967). Only some reports, because who would dare to publish anything incompatible with the study by Nobel laureates?

#### **3.6 Late - onset hypogonadism, sexual drive, potency and libido**

For many years, we have been reassured that only androgens have a key role in both stimulating and maintaining sexual function in men. It was believed that testosterone and the existence of a normal level of libido were inseparably connected (Shabsigh, 2003, Morales, et al., 2004). Nevertheless, sexual dysfunction in men is directly associated not only with testosterone but also with estradiol (E-2) and with other neurohormonal factors. Erectile dysfunction was linked to the development of benign prostatic hyperplasia (BPH), without even taking into account the fact that both benign prostatic hyperplasia (BPH) and erectile dysfunction (ED) were often caused by the hormone deficit. The effects of women's hormone replacement therapy on their psychological and sexual functioning are still the subject of the research in the borderline field between medicine and psychology. Female hormone therapy in treating menopause is common around the world. Meanwhile, several years ago it was stated that the effects of this therapy on men were not known. Hormone replacement therapy for men has been lagging for at least 20 years compared with hormone replacement therapy in women (Tenover, 1999). There is a decline in testosterone production in elderly men that can lead to a decrease in sexual desire (Kaufman & T'Sjoen, 2002). Androgen therapy can stop and even reverse this degenerating process (Hajjar, et al., 1997, Morales, et al., 2004). Information on the importance of testosterone in male sexuality is often divergent. It is known that surgical and pharmacological castration leads to impotence. On the other hand, it is recognized that testosterone deficiency is considered to be of little importance in the development of erectile dysfunction or in life force. (Anderson, 2003, Montorsi et al., 2003).

There was no sexual orientation change in men who underwent surgical castration. Sexual desire was preserved by the majority of the patients, but interest in sex decreased, which was associated with decreased frequency/intensity of orgasms (Zverina, et al., 1990). Testosterone replacement therapy suitable for men with primary testicular failure as a result

Late - Onset Hypogonadism - New Point of View 139

Fig. 21. Average IIEF-5 score before treatment

Fig. 22. Average IIEF-5 score during treatment

According to the Arizona Sexual Experiences Scale (ASEX), there was also a significant improvement in sexual health, from an average of 17.39 points (SD=4.24) before treatment to 12.45 points (SD=3.57). The average ASEX values distribution has been shown in Table 8.

of surgery conduces to a return to good sexual health and to good psycho-social outcomes (Fossa, et al., 1999). A drop in men's testosterone levels results in reduced libido and sexual potency. The implementation of hormonal therapy in hipogonadal men at the time when testosterone levels increase significantly, approaching the upper limit for normal, causes regular nocturnal erections. It also increases the number of spontaneous erections and it increases sexual activity (Burries, et al., 1992, Tariq, 2002). Other authors also propose that libido is closely tied to testosterone levels and that hormone replacement therapy increases the frequency of sexual thoughts and significantly improves one's libido (Davidson, et al., 1982, Kwan, et al., 1983). Testosterone induces nitric oxide synthesis in vascular endothelium through its influence on arginase activity. This leads to the opening of vascular pathways; facilitates blood flow into the corpora cavernosa; and enhances penile erection. The same mechanism is used in treatment of erectile dysfunction (ED) with PDE-5 inhibitors. It is currently the primary means of treating ED.

However, such treatment might not be effective when there is a decrease in male libido (the decrease being one of the symptoms of andropause) as a result of a decrease in the level of testosterone which is a hormone produced in a man's testicles. The occurrence of erectile dysfunction causes an increase in depression. Meanwhile, the treatment of depression through the introduction of SSRIs (selective serotonin reuptake inhibitors) affects the deterioration of erectile function in a secondary way (Hsu & Shen, 1995, Keller, et al., 1997). Thus, Andropause Erectile Dysfunction Treatment and male depression treatment are often ineffective. It is worth remembering, however, that andropause can start from any age but generally around age 30, a relatively young age, when a man still has some 40 years of life. Erectile dysfunction, especially in young men, has not yet been linked with their hormonal status.

I have found that during hormone therapy my patients are affected by changes in their sexual health. A thorough analysis of the problem became the subject of the doctoral dissertation of one of my assistants (Czyżowska, 2009).

My research on 88 men aged 20-68 years, (mean age 45), clearly shows an increase in testosterone endosynthesis at the time of hCG Hormone Therapy, on average from 17.93 nmil/L to 40.86 nmol/L. At the same time, a significant increase in E-2 was found in those examined, on average from 168.72 pmol/L to the value of 332.44 pmol/L. The change in hormone levels of those examined resulted in a significant improvement in their sexual performance, in libido and in erectile potency, evaluated using the International Index of Erectile Function (IIEF-5) questionnaire. The average value of IIEF-5 before therapy was 13.4 points (SD = 5.0), and during therapy − 19.88 points (SD = 4.3). The data are shown in numbers in Table 7, and graphically in Figures 21÷22.


Table 7. Average hormone concentrations and the IIEF-5 score before and during treatment

of surgery conduces to a return to good sexual health and to good psycho-social outcomes (Fossa, et al., 1999). A drop in men's testosterone levels results in reduced libido and sexual potency. The implementation of hormonal therapy in hipogonadal men at the time when testosterone levels increase significantly, approaching the upper limit for normal, causes regular nocturnal erections. It also increases the number of spontaneous erections and it increases sexual activity (Burries, et al., 1992, Tariq, 2002). Other authors also propose that libido is closely tied to testosterone levels and that hormone replacement therapy increases the frequency of sexual thoughts and significantly improves one's libido (Davidson, et al., 1982, Kwan, et al., 1983). Testosterone induces nitric oxide synthesis in vascular endothelium through its influence on arginase activity. This leads to the opening of vascular pathways; facilitates blood flow into the corpora cavernosa; and enhances penile erection. The same mechanism is used in treatment of erectile dysfunction (ED) with PDE-5

However, such treatment might not be effective when there is a decrease in male libido (the decrease being one of the symptoms of andropause) as a result of a decrease in the level of testosterone which is a hormone produced in a man's testicles. The occurrence of erectile dysfunction causes an increase in depression. Meanwhile, the treatment of depression through the introduction of SSRIs (selective serotonin reuptake inhibitors) affects the deterioration of erectile function in a secondary way (Hsu & Shen, 1995, Keller, et al., 1997). Thus, Andropause Erectile Dysfunction Treatment and male depression treatment are often ineffective. It is worth remembering, however, that andropause can start from any age but generally around age 30, a relatively young age, when a man still has some 40 years of life. Erectile dysfunction, especially in young men, has not yet been linked with their hormonal

I have found that during hormone therapy my patients are affected by changes in their sexual health. A thorough analysis of the problem became the subject of the doctoral

My research on 88 men aged 20-68 years, (mean age 45), clearly shows an increase in testosterone endosynthesis at the time of hCG Hormone Therapy, on average from 17.93 nmil/L to 40.86 nmol/L. At the same time, a significant increase in E-2 was found in those examined, on average from 168.72 pmol/L to the value of 332.44 pmol/L. The change in hormone levels of those examined resulted in a significant improvement in their sexual performance, in libido and in erectile potency, evaluated using the International Index of Erectile Function (IIEF-5) questionnaire. The average value of IIEF-5 before therapy was 13.4 points (SD = 5.0), and during therapy − 19.88 points (SD = 4.3). The data are shown in

Testosterone 17.937 nmol/L, 7.11 40.89 nmol/L, 12.55 p<0.0001 Estradiol 168.72 pmol/L, 77.9 332.44 pmol/L, 141.72 p<0.0001 IIEF-5 13.4, 5.0 19.876, 4.3 p<0.001

Table 7. Average hormone concentrations and the IIEF-5 score before and during treatment

During therapy

(average, SD) Significance

inhibitors. It is currently the primary means of treating ED.

dissertation of one of my assistants (Czyżowska, 2009).

numbers in Table 7, and graphically in Figures 21÷22.

Before therapy (average, SD)

status.

Fig. 21. Average IIEF-5 score before treatment

Fig. 22. Average IIEF-5 score during treatment

According to the Arizona Sexual Experiences Scale (ASEX), there was also a significant improvement in sexual health, from an average of 17.39 points (SD=4.24) before treatment to 12.45 points (SD=3.57). The average ASEX values distribution has been shown in Table 8.

Late - Onset Hypogonadism - New Point of View 141

damage to mechanisms blocking the blood flow from the corpora cavernosa, how do PDE-5 inhibitors pour the proverbial oil on the troubled waters? And at the same time testosterone

Fig. 23. Aging changes in the cavernosal tissue (Fig. 23-V) can also be induced by androgen deprivation (Fig. 23-E). The ratio of smooth muscle compartment to connective tissue which is normally 1:1 (Fig. 23 C & T) would suffer a shifting till 1:5 (Fig. 23 V & E) with a higher mRNA concentration as an expression of increasing number of alpha adrenoceptors

Testosterone deficiency is closely linked to the reduction in libido. And when one's libido is lowered, the effectiveness of PDE-5 inhibitors is also limited. In such situations, the preventive and curative procedure is to maintain adequate testosterone level in the body. What is more, in patients receiving PDE-5 inhibitors, the increase in the concentrations of testosterone can improve the penile vascular blood flow, which is yet another argument for

It is also known that diabetes can increase the problem of erectile dysfunction. It was previously believed that diabetes lead to lower testosterone levels. In contrast, quite the opposite is happening. A drop in testosterone levels due to a decrease in glucose uptake facilitated by anabolism reduction causes diabetes. Increased levels of testosterone significantly improve glucose tolerance and reduce one's insulin levels and the HbA1c level. Testosterone deficit can thus affect sexuality, both at a particular moment and through changes in metabolic processes leading to vascular lesions. There may also be a significant delay in the increase/drop in testosterone, even for many years, which may lead to erectile dysfunction (Gomuła, 2006). Some argue that the very fact of the occurrence of erectile dysfunction, rather than the testosterone concentration level, is the main indication for hormone replacement (Shabsigh, 2003). But the mechanism of erection is based not only on testosterone levels. It is not based merely on the E-2 either. Because neither testosterone nor E-2 alone determine the adequacy of an erection. If the mechanisms of erection depended on changes in serum testosterone levels, one would

restores penile smooth muscle.

according to Yassin (Yassin & Treish, 2004).

using the aforementioned procedure.


Table 8. Average hormone concentrations and the Arizona Sexual Experiences Scale measurement before and during therapy.

As noted above, serum concentration of E-2 plays a dominant role in male sexual health. Low levels of aromatase, which result in extremely low E-2 concentrations, may, nevertheless, be important in male sexual performance without affecting sexual orientation and gender identity (Gomuła, 2006, Gomuła, 2007). In patients with congenital absence of aromatase only the low-dose E-2 substitution results in significant changes in sexual behavior. Estradiol administration leads to increased erotic fantasies, masturbation or sexual activity Carani, et al., 1999).

My own research clearly shows that E-2 is required to maintain sexual functions in adult men (Gomuła, 2007). The manifestations thereof have been observed after prolonged hormonal therapy. Androgen deficiency patients had their testosterone levels increased for therapeutic purposes. During the therapy, a parallel increase occurred in serum E-2 concentrations, as the effect of the all natural aromatase. Some patients had such high E-2 levels that they exceeded the normal physiological range. In order to reduce E-2 concentration, my patients received preparations blocking aromatase activity. As a result of this therapy, men characterized by high concentrations of testosterone (falling in the upper limits of normal), whose E-2 was detected at very low levels, had a total loss of libido. At the same time those men suffered from erectile dysfunction, which could even lead to inability to initiate or maintain an erection. Stopping the drug which blocked aromatase resolved the symptoms and resulted in a rapid return of high concentrations of E-2. Some authors report that in the activation of male sexual behavior the brain level conversion of testosterone to estradiol is of major importance and that testosterone's effects are not in themselves so important (Balthazar & Ball, 1998). Testosterone has a significant effect on the smooth muscle in the corpora cavernosa. Androgens may significantly affect the ultrastructure of the corpora cavernosa and these changes are responsible for erectile dysfunction (Traish & Kim, 2005).

In young men, the ratio between smooth muscle and stroma in the corpus cavernosum is 1:1. Long-term hypogonadism causes the ratio of contents in muscle tissue to be 1:5, which results in smooth muscle atrophy and in fibrotic changes of the corpora cavernosa (Yassin & Treish, 2004).

Fig. 23 represents normal cavernosal histological appearance and its loss as a result of testosterone deficiency, according to Yassin (Yassin & Treish, 2004).

Androgen replacement can lead especially in hypogonadism patients to recovery process within the trabecular tissue. This result means that testosterone therapy supports the "recovery process" not only in striated muscles in human body /reversible process/ (Yassin & Treish, 2004).

The basic question arises as to what should the first step be in a successful erectile dysfunction prevention and in a successful erectile dysfunction treatment? If the decrease in testosterone leads to penile tissue fibrosis, smooth muscle atrophy in corpora cavernosa, and

Testosterone 17.937 nmol/L, 7.11 40.89 nmol/L, 12.55 Estradiol 168.72 pmol/L, 77.9 332.44 pmol/L, 141.72 Scale – ASEX 3.9 3.4 3.6 3.1 3.3 2.6 2.3 2.6 2.5 2.5

Table 8. Average hormone concentrations and the Arizona Sexual Experiences Scale

As noted above, serum concentration of E-2 plays a dominant role in male sexual health. Low levels of aromatase, which result in extremely low E-2 concentrations, may, nevertheless, be important in male sexual performance without affecting sexual orientation and gender identity (Gomuła, 2006, Gomuła, 2007). In patients with congenital absence of aromatase only the low-dose E-2 substitution results in significant changes in sexual behavior. Estradiol administration leads to increased erotic fantasies, masturbation or sexual

My own research clearly shows that E-2 is required to maintain sexual functions in adult men (Gomuła, 2007). The manifestations thereof have been observed after prolonged hormonal therapy. Androgen deficiency patients had their testosterone levels increased for therapeutic purposes. During the therapy, a parallel increase occurred in serum E-2 concentrations, as the effect of the all natural aromatase. Some patients had such high E-2 levels that they exceeded the normal physiological range. In order to reduce E-2 concentration, my patients received preparations blocking aromatase activity. As a result of this therapy, men characterized by high concentrations of testosterone (falling in the upper limits of normal), whose E-2 was detected at very low levels, had a total loss of libido. At the same time those men suffered from erectile dysfunction, which could even lead to inability to initiate or maintain an erection. Stopping the drug which blocked aromatase resolved the symptoms and resulted in a rapid return of high concentrations of E-2. Some authors report that in the activation of male sexual behavior the brain level conversion of testosterone to estradiol is of major importance and that testosterone's effects are not in themselves so important (Balthazar & Ball, 1998). Testosterone has a significant effect on the smooth muscle in the corpora cavernosa. Androgens may significantly affect the ultrastructure of the corpora cavernosa and these changes are

In young men, the ratio between smooth muscle and stroma in the corpus cavernosum is 1:1. Long-term hypogonadism causes the ratio of contents in muscle tissue to be 1:5, which results in smooth muscle atrophy and in fibrotic changes of the corpora cavernosa (Yassin &

Fig. 23 represents normal cavernosal histological appearance and its loss as a result of

Androgen replacement can lead especially in hypogonadism patients to recovery process within the trabecular tissue. This result means that testosterone therapy supports the "recovery process" not only in striated muscles in human body /reversible process/ (Yassin

The basic question arises as to what should the first step be in a successful erectile dysfunction prevention and in a successful erectile dysfunction treatment? If the decrease in testosterone leads to penile tissue fibrosis, smooth muscle atrophy in corpora cavernosa, and

measurement before and during therapy.

responsible for erectile dysfunction (Traish & Kim, 2005).

testosterone deficiency, according to Yassin (Yassin & Treish, 2004).

activity Carani, et al., 1999).

Treish, 2004).

& Treish, 2004).

During therapy (average, SD)

Before therapy (average, SD)

damage to mechanisms blocking the blood flow from the corpora cavernosa, how do PDE-5 inhibitors pour the proverbial oil on the troubled waters? And at the same time testosterone restores penile smooth muscle.

Fig. 23. Aging changes in the cavernosal tissue (Fig. 23-V) can also be induced by androgen deprivation (Fig. 23-E). The ratio of smooth muscle compartment to connective tissue which is normally 1:1 (Fig. 23 C & T) would suffer a shifting till 1:5 (Fig. 23 V & E) with a higher mRNA concentration as an expression of increasing number of alpha adrenoceptors according to Yassin (Yassin & Treish, 2004).

Testosterone deficiency is closely linked to the reduction in libido. And when one's libido is lowered, the effectiveness of PDE-5 inhibitors is also limited. In such situations, the preventive and curative procedure is to maintain adequate testosterone level in the body. What is more, in patients receiving PDE-5 inhibitors, the increase in the concentrations of testosterone can improve the penile vascular blood flow, which is yet another argument for using the aforementioned procedure.

It is also known that diabetes can increase the problem of erectile dysfunction. It was previously believed that diabetes lead to lower testosterone levels. In contrast, quite the opposite is happening. A drop in testosterone levels due to a decrease in glucose uptake facilitated by anabolism reduction causes diabetes. Increased levels of testosterone significantly improve glucose tolerance and reduce one's insulin levels and the HbA1c level. Testosterone deficit can thus affect sexuality, both at a particular moment and through changes in metabolic processes leading to vascular lesions. There may also be a significant delay in the increase/drop in testosterone, even for many years, which may lead to erectile dysfunction (Gomuła, 2006). Some argue that the very fact of the occurrence of erectile dysfunction, rather than the testosterone concentration level, is the main indication for hormone replacement (Shabsigh, 2003). But the mechanism of erection is based not only on testosterone levels. It is not based merely on the E-2 either. Because neither testosterone nor E-2 alone determine the adequacy of an erection. If the mechanisms of erection depended on changes in serum testosterone levels, one would

Late - Onset Hypogonadism - New Point of View 143

enough reason to conduct a thorough analysis of the problem, which turned out to be the

Depression is a serious medical condition where a person may feel "down" or "hopeless" for weeks or more. According to the National Institute of Mental Health, the signs and symptoms of depression include: persistent sad, anxious, or "empty" mood; feelings of hopelessness, pessimism; feelings of guilt, worthlessness, helplessness; loss of interest or pleasure in hobbies and activities that were once enjoyed, including sex; decreased energy, fatigue, being "slowed down"; difficulty concentrating, remembering, making decisions; insomnia, early-morning awakening, or oversleeping; appetite and/or weight loss, or overeating and weight gain; thoughts of death or suicide, suicide attempts; restlessness, irritability; persistent physical symptoms that do not respond to treatment, such as headaches, digestive disorders, and chronic pain. Treatment of sexual disorders caused by depression with antidepressants based on Selective Serotonin Reuptake Inhibitor (SSRI) has a secondary detrimental effect on erection (Hsu & Shen, 1995, Keller, et al., 1997). It results in a vicious circle – antidepressants increase erection disorders, while sexual life disorders

Evidence proving therapeutical effect of estradiol in depression disorders has been found amongst women, however the research has not proven explicit relation between the estradiol level and depression among men (Studd & Panay, 2004). Neurosteroids produced in the central nervous system from cholesterol or other steroidal precursors are responsible for direct functioning within the brain, while alterations in their production typical for menopause in women and men cause certain aging symptoms. They directly influence, for example, body temperature regulation (hence the heat waves and cold sweats symptoms), memory function and emotions. Situational factors experienced by the given person, such as

Research results demonstrated that hormone therapy for men, with hCG inducing testosterone endosynthesis causing over a double increase of testosterone and E-2 concentrations, result in substantial improvement of psychological condition and not only relieve depressive states, but

Based on the author's own research on 88 men aged between 20 and 68 years, (mean age 45 years), during the hormone therapy with hCG testosterone endosynthesis increase was achieved from E-2 average value 168.72 pmol/L up to 332.44 pmol/L. The change of hormonal state of the subjects resulted in substantial improvement of their psychological state assessed on the basis of extended Beck Depression Inventory /BDI/ (Beck, 1967). The classic BDI evaluates exclusively depressive state. Condition above zero is not assessed, only depression is included. Accepting BDI as a good test however not designed to evaluate joy of life state, I have developed the questionnaire adding reflection towards opposite direction, i.e. towards joy of life. Implementing such a tool for analysis of patients' psychological state enabled the change of hormonal condition to be demonstrated as not only what causes relief from depression, but also progress to the

Restoring man's normal concentration of testosterone and E-2 results in significant improvement of his mental health; depressive states subside. Furthermore, patients who prior to treatments were below state "0", in depression, during the therapy note states

significantly above state "0"; they move to the joy of life state, as presented in Fig. 25.

topic of the PhD dissertation of one of my assistants (Czyżowska, 2009).

intensify depression. The circle is closed. How can it be broken?

stress, secondarily disturb their secretions of hormones (Carruthers, 2004).

also causes a progression from depression to the state of joy of life.

state of joy of life.

have to wait many hours for an erection. Testosterone concentrations and E2 concentration increase as a result of natural endosyntesis. This lasts about 6 hours because that much time elapses from the original signals for spectral contrast in visual cortex during the first stage of sleep, rapid eye movement (REM). And the highest concentration of testosterone for a man is at approx. 4-5 am, after about six hours of sleep. The mechanisms of erection depend on such factors as the concentration of hormones, but they do not depend on them in a direct way. To obtain or maintain an adequate erection, one needs adequate levels of testosterone and of E-2. Therefore, aromatase, which converts testosterone to E-2, is also essential. Arginase, which induces the synthesis of nitric oxide in vascular endothelial cells, also plays an important role. 4-hydroxyestradiol derivative of estradiol is a substance that at the level of brain activity plays perhaps the most important role therein. 4-hydroxyestradiol has this property that it may saturate dopamine receptors in the brain. At high saturation level, dopamine stimulates these receptors, causing penile erection. Release of dopamine, which is one of many neurotransmitters, occurs rapidly. These two mechanisms are shown in Figure 24.

Fig. 24. Erection occurs as a result of the involvement of neurotransmiters, secreted at the time counted in seconds, and does not depend on hormonal changes, which can last for hours.

It clearly shows that we need to revise our views on the impact of hormones on our sexuality, potency, and libido, because not just hormones but also brain neurotransmitters have an impact on male sexuality.

#### **3.7 Late - onset hypogonadism and depression**

I have observed in my patients a change in their mental condition while they were under hormone therapy. A state of depression transformed itself into a state of joy. This gave

have to wait many hours for an erection. Testosterone concentrations and E2 concentration increase as a result of natural endosyntesis. This lasts about 6 hours because that much time elapses from the original signals for spectral contrast in visual cortex during the first stage of sleep, rapid eye movement (REM). And the highest concentration of testosterone for a man is at approx. 4-5 am, after about six hours of sleep. The mechanisms of erection depend on such factors as the concentration of hormones, but they do not depend on them in a direct way. To obtain or maintain an adequate erection, one needs adequate levels of testosterone and of E-2. Therefore, aromatase, which converts testosterone to E-2, is also essential. Arginase, which induces the synthesis of nitric oxide in vascular endothelial cells, also plays an important role. 4-hydroxyestradiol derivative of estradiol is a substance that at the level of brain activity plays perhaps the most important role therein. 4-hydroxyestradiol has this property that it may saturate dopamine receptors in the brain. At high saturation level, dopamine stimulates these receptors, causing penile erection. Release of dopamine, which is one of many

neurotransmitters, occurs rapidly. These two mechanisms are shown in Figure 24.

Fig. 24. Erection occurs as a result of the involvement of neurotransmiters, secreted at the time counted in seconds, and does not depend on hormonal changes, which can last for hours.

It clearly shows that we need to revise our views on the impact of hormones on our sexuality, potency, and libido, because not just hormones but also brain neurotransmitters

I have observed in my patients a change in their mental condition while they were under hormone therapy. A state of depression transformed itself into a state of joy. This gave

have an impact on male sexuality.

**3.7 Late - onset hypogonadism and depression** 

enough reason to conduct a thorough analysis of the problem, which turned out to be the topic of the PhD dissertation of one of my assistants (Czyżowska, 2009).

Depression is a serious medical condition where a person may feel "down" or "hopeless" for weeks or more. According to the National Institute of Mental Health, the signs and symptoms of depression include: persistent sad, anxious, or "empty" mood; feelings of hopelessness, pessimism; feelings of guilt, worthlessness, helplessness; loss of interest or pleasure in hobbies and activities that were once enjoyed, including sex; decreased energy, fatigue, being "slowed down"; difficulty concentrating, remembering, making decisions; insomnia, early-morning awakening, or oversleeping; appetite and/or weight loss, or overeating and weight gain; thoughts of death or suicide, suicide attempts; restlessness, irritability; persistent physical symptoms that do not respond to treatment, such as headaches, digestive disorders, and chronic pain. Treatment of sexual disorders caused by depression with antidepressants based on Selective Serotonin Reuptake Inhibitor (SSRI) has a secondary detrimental effect on erection (Hsu & Shen, 1995, Keller, et al., 1997). It results in a vicious circle – antidepressants increase erection disorders, while sexual life disorders intensify depression. The circle is closed. How can it be broken?

Evidence proving therapeutical effect of estradiol in depression disorders has been found amongst women, however the research has not proven explicit relation between the estradiol level and depression among men (Studd & Panay, 2004). Neurosteroids produced in the central nervous system from cholesterol or other steroidal precursors are responsible for direct functioning within the brain, while alterations in their production typical for menopause in women and men cause certain aging symptoms. They directly influence, for example, body temperature regulation (hence the heat waves and cold sweats symptoms), memory function and emotions. Situational factors experienced by the given person, such as stress, secondarily disturb their secretions of hormones (Carruthers, 2004).

Research results demonstrated that hormone therapy for men, with hCG inducing testosterone endosynthesis causing over a double increase of testosterone and E-2 concentrations, result in substantial improvement of psychological condition and not only relieve depressive states, but also causes a progression from depression to the state of joy of life.

Based on the author's own research on 88 men aged between 20 and 68 years, (mean age 45 years), during the hormone therapy with hCG testosterone endosynthesis increase was achieved from E-2 average value 168.72 pmol/L up to 332.44 pmol/L. The change of hormonal state of the subjects resulted in substantial improvement of their psychological state assessed on the basis of extended Beck Depression Inventory /BDI/ (Beck, 1967).

The classic BDI evaluates exclusively depressive state. Condition above zero is not assessed, only depression is included. Accepting BDI as a good test however not designed to evaluate joy of life state, I have developed the questionnaire adding reflection towards opposite direction, i.e. towards joy of life. Implementing such a tool for analysis of patients' psychological state enabled the change of hormonal condition to be demonstrated as not only what causes relief from depression, but also progress to the state of joy of life.

Restoring man's normal concentration of testosterone and E-2 results in significant improvement of his mental health; depressive states subside. Furthermore, patients who prior to treatments were below state "0", in depression, during the therapy note states significantly above state "0"; they move to the joy of life state, as presented in Fig. 25.

Late - Onset Hypogonadism - New Point of View 145

disorders (Sawada, et al., 1998). HRT in men has been used only recently and never so

Regardless of the fact that the relation between dopamine and androgens at the level of the central nervous system is still not absolutely clear, on the basis of my own clinical observations it can be stated categorically that in men there exists a strong correlation between the hormonal system and PD symptoms. This statement is supported by the initial

The preliminary study comprised of 16 men suffering from PD and LOH, aged 64 –78 years. The hCG therapy used was to induce testosterone endosynthesis. The follow-up lasted from

Following the therapy, the serum testosterone concentration increased from the mean baseline level of 12.3 nmol/l to 34.8 nmol/L. So far, the main parameter that has been considered for HRT in men has been the testosterone concentration. When the hCG was administered, though, the testosterone concentration increase was accompanied by simultaneous increase of estradiol level. In the analysed group of patients, the increase of

The addition of the hormonal therapy was supplemental to the standard long-term pharmacological anti-PD treatment. As a result of this addition, a dramatic improvement of

Before the hormonal therapy began, the patients complained of motor latency and insufficiency, hand tremors, muscular tremor and rigidity in legs. During the treatment

Just stating the fact, though, without being able to document it, may justly be questionable. Numerical assessment and analysis of parameters from various questionnaires are ambiguous. That is why every possible effort has been made to present undeniable proof and evidence of beneficial changes in the clinical condition of PD patients. The first evidence for the relationship between LOH and clinical symptoms of PD is the film which was shown during ISSAM Seminar 'Androgen Deficiency in the Adult Male' at the 4th World

Dr Malcolm Carruthers, a Congress participant, wrote in his book, published a few months after the Congress:"The results showed that after treatment, Parkinsonian symptoms greatly improved. Physical symptoms of slow, limited movement (bradykinesia), hand tremor, muscular tremor and rigidity of the legs clearly improved or disappeared, as did standing and walking alone, and ease of movement. Daily activities of living such as eating, dressing, personal hygiene and handwriting were all made easier. Clear improvements in many symptom areas were also documented by the film evidence" (Carruthers, 2004). An excerpt

The mental condition of the patients also improved substantially. The patients who had been slow, even dull, depressive, unwilling to contact with others, once the therapy

After the hCG therapy was introduced, PD and other accompanying symptoms improved noticeably. Dyskinesis and involuntary body functions, being the adverse effects of

If any patients had not been able to stand up, that problem disappeared. Now the patients could walk by themselves. They also reported that the way they moved had improved. Without any help of others, they could walk on an uneven ground or even stroll in woods.

widely as in women, thus we have less experience for HRT in men.

1 to 4 years.

PD symptoms was observed.

Congress of The Aging Male in Prague.

of the film can be seen on www.medan.pl.

commenced, changed dramatically.

examinations and observation of PD patients who consider to undergo HRT.

the mean estradiol level was from the baseline of 46 pmol/L to 189 pmol/L.

these symptoms clearly improved or completely disappeared.

treatment with L-dopa, discontinued or occurred sporadically.

Fig. 25. Psychological state according to BDI: before the therapy – red, after the therapy – green. Patients who suffered from depression are in the joy of life area during the treatment.

The facts above presented explicitly demonstrate a close correlation between hormonal and psychological states of man.

#### **3.8 Late - onset hypogonadism and Parkinson's disease**

Although Parkinson's disease (PD) is diagnosed in only 0.1% of the total population, in the population older than 70 years of age its prevalence reaches 1%. The mean age of developing the disease is 58 years, and PD affects men more frequently than women /1.6:1/ (Edwards, et al., 2002, Nelson, 2002). The main cause of PD is the degeneration of dopaminergic neurons of substantia nigra in the nigro-striatal system of the brain. The development of the disease is believed to be triggered by both genetic and environmental factors. So far there have been very few reports in the literature, linking PD with the hormonal condition of a patient. Yet numerous symptoms resulting from the deficiency of such sex hormones as testosterone in men and progesterone-estrogen group in women, may co-exist with motor and other PD symptoms. These include impotence, lowered joy of life, lack of energy and mental depression (Edwards, et al., 2002, Nelson, 2002). Experimental studies have shown that estrogens can protect hippocampus neurons against beta-amyloid protein, which is implicated in the development of PD. The estrogens influence a creation of new synapses and improve the survival of neurons in hippocampus region (Serwin, 1994, Okun, et al., 2001).

Recent studies suggest the existence of morphogenic influence of estrogens on neurons and on plasticity of synapses. Estrogens appear to provoke synapsogenesis in hypothalamic cells. Apart from that, nonspecific systems activating brain neuroplasticity processes seem to be modulated as well (Skibińska & Kossuth, 2003).

The neuroprotective action of estradiol is connected with oxidation processes. Oxidative stress is known to be important in the process of dopaminergic neuronal degeneration in Parkinson's disease, whereas estrogens have neuroprotective effects in neurodegenerative

Fig. 25. Psychological state according to BDI: before the therapy – red, after the therapy – green. Patients who suffered from depression are in the joy of life area during the treatment. The facts above presented explicitly demonstrate a close correlation between hormonal and

Although Parkinson's disease (PD) is diagnosed in only 0.1% of the total population, in the population older than 70 years of age its prevalence reaches 1%. The mean age of developing the disease is 58 years, and PD affects men more frequently than women /1.6:1/ (Edwards, et al., 2002, Nelson, 2002). The main cause of PD is the degeneration of dopaminergic neurons of substantia nigra in the nigro-striatal system of the brain. The development of the disease is believed to be triggered by both genetic and environmental factors. So far there have been very few reports in the literature, linking PD with the hormonal condition of a patient. Yet numerous symptoms resulting from the deficiency of such sex hormones as testosterone in men and progesterone-estrogen group in women, may co-exist with motor and other PD symptoms. These include impotence, lowered joy of life, lack of energy and mental depression (Edwards, et al., 2002, Nelson, 2002). Experimental studies have shown that estrogens can protect hippocampus neurons against beta-amyloid protein, which is implicated in the development of PD. The estrogens influence a creation of new synapses and improve the survival of neurons in hippocampus region (Serwin, 1994,

Recent studies suggest the existence of morphogenic influence of estrogens on neurons and on plasticity of synapses. Estrogens appear to provoke synapsogenesis in hypothalamic cells. Apart from that, nonspecific systems activating brain neuroplasticity processes seem to

The neuroprotective action of estradiol is connected with oxidation processes. Oxidative stress is known to be important in the process of dopaminergic neuronal degeneration in Parkinson's disease, whereas estrogens have neuroprotective effects in neurodegenerative

psychological states of man.

Okun, et al., 2001).

**3.8 Late - onset hypogonadism and Parkinson's disease** 

be modulated as well (Skibińska & Kossuth, 2003).

disorders (Sawada, et al., 1998). HRT in men has been used only recently and never so widely as in women, thus we have less experience for HRT in men.

Regardless of the fact that the relation between dopamine and androgens at the level of the central nervous system is still not absolutely clear, on the basis of my own clinical observations it can be stated categorically that in men there exists a strong correlation between the hormonal system and PD symptoms. This statement is supported by the initial examinations and observation of PD patients who consider to undergo HRT.

The preliminary study comprised of 16 men suffering from PD and LOH, aged 64 –78 years. The hCG therapy used was to induce testosterone endosynthesis. The follow-up lasted from 1 to 4 years.

Following the therapy, the serum testosterone concentration increased from the mean baseline level of 12.3 nmol/l to 34.8 nmol/L. So far, the main parameter that has been considered for HRT in men has been the testosterone concentration. When the hCG was administered, though, the testosterone concentration increase was accompanied by simultaneous increase of estradiol level. In the analysed group of patients, the increase of the mean estradiol level was from the baseline of 46 pmol/L to 189 pmol/L.

The addition of the hormonal therapy was supplemental to the standard long-term pharmacological anti-PD treatment. As a result of this addition, a dramatic improvement of PD symptoms was observed.

Before the hormonal therapy began, the patients complained of motor latency and insufficiency, hand tremors, muscular tremor and rigidity in legs. During the treatment these symptoms clearly improved or completely disappeared.

Just stating the fact, though, without being able to document it, may justly be questionable. Numerical assessment and analysis of parameters from various questionnaires are ambiguous. That is why every possible effort has been made to present undeniable proof and evidence of beneficial changes in the clinical condition of PD patients. The first evidence for the relationship between LOH and clinical symptoms of PD is the film which was shown during ISSAM Seminar 'Androgen Deficiency in the Adult Male' at the 4th World Congress of The Aging Male in Prague.

Dr Malcolm Carruthers, a Congress participant, wrote in his book, published a few months after the Congress:"The results showed that after treatment, Parkinsonian symptoms greatly improved. Physical symptoms of slow, limited movement (bradykinesia), hand tremor, muscular tremor and rigidity of the legs clearly improved or disappeared, as did standing and walking alone, and ease of movement. Daily activities of living such as eating, dressing, personal hygiene and handwriting were all made easier. Clear improvements in many symptom areas were also documented by the film evidence" (Carruthers, 2004). An excerpt of the film can be seen on www.medan.pl.

The mental condition of the patients also improved substantially. The patients who had been slow, even dull, depressive, unwilling to contact with others, once the therapy commenced, changed dramatically.

After the hCG therapy was introduced, PD and other accompanying symptoms improved noticeably. Dyskinesis and involuntary body functions, being the adverse effects of treatment with L-dopa, discontinued or occurred sporadically.

If any patients had not been able to stand up, that problem disappeared. Now the patients could walk by themselves. They also reported that the way they moved had improved. Without any help of others, they could walk on an uneven ground or even stroll in woods.

Late - Onset Hypogonadism - New Point of View 147

These facts led to the notion that diminshing levels of testosterone in aging men, which lead to late onset hypogonadism (LOH), are responsible for such differences and higher mortality of men. Indeed, the nested case-control study on 11'600 men aged 40 to 79 years (EPIC-Norfolk UK) showed inverse relationship of endogenous testosterone concentration and mortality due to cardiovascular disease and all causes including CAD and cancer. This study clearly suggested that high endogenous testosterone concentrations appear to lower mortality rates of

In the "Hypogonadism in Males (HIM)" study (Mulligan, et al. 2006) on 2162 male patients over 45 years of age and visiting their primary care physicians for general reasons, it was found that 38.7% of them were hypogonadal and 3.7% of them were receiving testosterone. Among men not receiving testosterone, odds ratios for having hypogonadism were significantly higher in men with arterial hypertension (1.84), hyperlipidemia (1.47), diabetes (2.09), obesity (2.38). Conversely, late onset hypogonadism (LOH) and the Testosterone Deficiency (TD) connection with cardiovascular disease is evident from TD associations with diabetes (Dhindsa, et al. 2010) and metabolic syndrome (Kalyani & Dobs, 2000) and such findings as an inverse relationship between testosterone level and aortic atherosclerosis, which was seen among middle-aged and older men in the Rotterdam Study

Low free testosterone has also been associated with abdominal aortic aneurysm in community-dwelling men aged 70-88 years (Yeap et al., 2010). Furthermore, for men aged 50 to 91 years of age who were followed for 20 years in the Rancho Bernardo Study (Barrett-Connor et al., 1999), serum testosterone levels were inversely related to weight, body mass index, arterial blood pressure, serum insulin and plasma glucose. Nevertheless, as these factors may be relevant to mortality there was clear evidence that men with serum testosterone concentration below 25th percentile had a 40% higher risk of death, independent of obesity, lifestyle choices (eg. exercise, smoking) and age (Laughlin, et al., 2008, Tivestena et al., 2009). Another 5-year retrospect study on male veterans (N=858), compared mortality of men with normal (± 450 ng/dL) T levels to those with low (<250 ng/dL) total testosterone and low (< 0.75ng/dL) free testosterone levels (Shores, et al., 2006). The results showed that mortality for subjects with low T was almost twice as high (34.9%) as for those with normal T levels was 20.1%. There is an obvious link between mortality and morbidity due to coronary CAD and other cardiovascular diseases and LOH due to low free testosterone levels in serum of aging men (Wu & von Eckardstein, 2003, Wu, et al., 2010). This is due to existing strong correlation of testosterone defficiency with glucose tolerance, visceral obesity, serum lipid disorders and elevated arterial pressure (Kalyani & Dobs, 2007). These factors usually lead to vascular disorders. The clinical studies (English, et al., (1997) showed that low natural androgen concentration can cause deletorious changes in atherogenic lipid profile, high fibrinogen and a hypercoagulable state, an increase in insulin resistance and hyperinsulinemia, higher systolic and diastolic blood pressures. The biologically plausible mechanism of testosterone protective action against cardiovascular disorers can be explained by the fact that testosterone has direct vasoactive properties (English, et al., (1997). In animal models of isolated coronary, femoral and pulmonary arteries testosterone showed a dose-dependent vasodilatory effect (Channer & Jones, 2003). It is caused by a direct effect on the vascular smooth muscle, by either an effect on potassium or calcium channels (Deenadayalu, V.P., et al. 2001, English, K.M., 2002). In man, testosterone has been shown to cause dose-dependent vasodilation both in vitro and in vivo. During cardiac surgery on man, the coronary artery diameter and subsequent

men due to all causes, cardiovascular disease and cancer (Khaw, K.T., et al., 2007).

(Hak, et al., 2002).

The patients were able to eat their meals without any help, they could normally use a spoon, a knife and a fork because the hand tremor stopped. They could do their shirt and coat buttons up, and needed no help from others to get dressed or to wash themselves.

Another important objective element of improvement was the disappearance of sialosis. Their handwriting also improved, became clearer, with no traces of tremor or micrographism.

In 5 out of 16 patients described here, the improvement of their quality of life (QOL) also included the sexual function. Both their libido and potency improved. They resumed sexual activity, in which they had been inactive for a long time.

The hormonal therapy enabled them to live as before they fell ill, without having to be helped by others. Their quality of life benefited from that situation too. Their caregiving families felt extremely relieved. PD patients' families voluntarily admitted that the hormonal therapy was effective not only for PD sufferers but it improved quality of life of their families as well.

Initially, the PD-treatment regime had been kept. Neither the drugs nor their doses had been changed. After a few months of hCG administration, though, it was possible to lower the dosage or even withdraw certain anti-parkinsonism drugs, without aggravation of PD symptoms.

In the study presented, there was no control group. Yet, when the hormone-stimulation therapy was stopped, the patients' condition and symptoms clearly deteriorated, returning to what they had been like before the hormonal treatment was administered. Once the hormonal therapy was restarted, parkinsonian symptoms disappeared again. That fact was observed by patients and by their families as well.

Parkinson's disease and hormonal deficiency are inextricably linked. Both men and women suffer from Parkinson's disease because of deficit of estradiol, and more specifically because of the deficit of 4-hydroxyestradiol (dihydroxyestradiol), which is a derivative of estradiol. The molecular structure drawings, which illustrate how dopamine and 4-hydroxyestradiol are attached to the same key site to dopamine receptor in the brain, as given above, are in the subsection *2.1. Late Onset Hypogonadism is not just testosterone deficiency*. And it is only 4 hydroxyestradiol - a chemical compound, which due to its chemical structure, may saturate dopamine E-2 receptors in the brain. Therefore, in the treatment of Parkinson's disease special attention should be paid not only to testosterone levels, but particularly to the concentration of E-2. The desirable concentration of E-2 is approximately 250-300 pmol/l. This implies, however, maintaining also excessively high levels of testosterone, which cause hyperactivity in patients. In such cases, apart from hCG injections, I resort to microdoses of transdermal E-2 treatment in order to obtain the desired physiological serum levels of testosterone.

#### **3.9 LOH and mortality & morbidity from coronary artery disease**

Cardiovascular diseases, which include CAD, are now the biggest killer in the developed countries. The mortality rate from CAD varies by a factor of five in different populations in the world. Yet, in spite of such a high range in mortality rate, the ratio of male-to female deaths is relatively constant at more than three-to-one (Tunstall-Pedoe, 1999). This cannot be explained solely by differences in the risk factors for cardiovascular disease, between the sexes (Rayner, et al., 1998). The remarkably lower prevalence of CAD is particularily prominent in pre-menopausal women, but increases slightly after the menopause (Kalin, & Zurnoff 1990).

The patients were able to eat their meals without any help, they could normally use a spoon, a knife and a fork because the hand tremor stopped. They could do their shirt and coat

Another important objective element of improvement was the disappearance of sialosis. Their handwriting also improved, became clearer, with no traces of tremor or

In 5 out of 16 patients described here, the improvement of their quality of life (QOL) also included the sexual function. Both their libido and potency improved. They resumed sexual

The hormonal therapy enabled them to live as before they fell ill, without having to be helped by others. Their quality of life benefited from that situation too. Their caregiving families felt extremely relieved. PD patients' families voluntarily admitted that the hormonal therapy was effective not only for PD sufferers but it improved quality of life of

Initially, the PD-treatment regime had been kept. Neither the drugs nor their doses had been changed. After a few months of hCG administration, though, it was possible to lower the dosage or even withdraw certain anti-parkinsonism drugs, without aggravation of PD

In the study presented, there was no control group. Yet, when the hormone-stimulation therapy was stopped, the patients' condition and symptoms clearly deteriorated, returning to what they had been like before the hormonal treatment was administered. Once the hormonal therapy was restarted, parkinsonian symptoms disappeared again. That fact was

Parkinson's disease and hormonal deficiency are inextricably linked. Both men and women suffer from Parkinson's disease because of deficit of estradiol, and more specifically because of the deficit of 4-hydroxyestradiol (dihydroxyestradiol), which is a derivative of estradiol. The molecular structure drawings, which illustrate how dopamine and 4-hydroxyestradiol are attached to the same key site to dopamine receptor in the brain, as given above, are in the subsection *2.1. Late Onset Hypogonadism is not just testosterone deficiency*. And it is only 4 hydroxyestradiol - a chemical compound, which due to its chemical structure, may saturate dopamine E-2 receptors in the brain. Therefore, in the treatment of Parkinson's disease special attention should be paid not only to testosterone levels, but particularly to the concentration of E-2. The desirable concentration of E-2 is approximately 250-300 pmol/l. This implies, however, maintaining also excessively high levels of testosterone, which cause hyperactivity in patients. In such cases, apart from hCG injections, I resort to microdoses of transdermal E-2

treatment in order to obtain the desired physiological serum levels of testosterone.

Cardiovascular diseases, which include CAD, are now the biggest killer in the developed countries. The mortality rate from CAD varies by a factor of five in different populations in the world. Yet, in spite of such a high range in mortality rate, the ratio of male-to female deaths is relatively constant at more than three-to-one (Tunstall-Pedoe, 1999). This cannot be explained solely by differences in the risk factors for cardiovascular disease, between the sexes (Rayner, et al., 1998). The remarkably lower prevalence of CAD is particularily prominent in pre-menopausal women, but increases slightly after the menopause (Kalin, &

**3.9 LOH and mortality & morbidity from coronary artery disease** 

buttons up, and needed no help from others to get dressed or to wash themselves.

activity, in which they had been inactive for a long time.

observed by patients and by their families as well.

micrographism.

their families as well.

symptoms.

Zurnoff 1990).

These facts led to the notion that diminshing levels of testosterone in aging men, which lead to late onset hypogonadism (LOH), are responsible for such differences and higher mortality of men. Indeed, the nested case-control study on 11'600 men aged 40 to 79 years (EPIC-Norfolk UK) showed inverse relationship of endogenous testosterone concentration and mortality due to cardiovascular disease and all causes including CAD and cancer. This study clearly suggested that high endogenous testosterone concentrations appear to lower mortality rates of men due to all causes, cardiovascular disease and cancer (Khaw, K.T., et al., 2007).

In the "Hypogonadism in Males (HIM)" study (Mulligan, et al. 2006) on 2162 male patients over 45 years of age and visiting their primary care physicians for general reasons, it was found that 38.7% of them were hypogonadal and 3.7% of them were receiving testosterone. Among men not receiving testosterone, odds ratios for having hypogonadism were significantly higher in men with arterial hypertension (1.84), hyperlipidemia (1.47), diabetes (2.09), obesity (2.38). Conversely, late onset hypogonadism (LOH) and the Testosterone Deficiency (TD) connection with cardiovascular disease is evident from TD associations with diabetes (Dhindsa, et al. 2010) and metabolic syndrome (Kalyani & Dobs, 2000) and such findings as an inverse relationship between testosterone level and aortic atherosclerosis, which was seen among middle-aged and older men in the Rotterdam Study (Hak, et al., 2002).

Low free testosterone has also been associated with abdominal aortic aneurysm in community-dwelling men aged 70-88 years (Yeap et al., 2010). Furthermore, for men aged 50 to 91 years of age who were followed for 20 years in the Rancho Bernardo Study (Barrett-Connor et al., 1999), serum testosterone levels were inversely related to weight, body mass index, arterial blood pressure, serum insulin and plasma glucose. Nevertheless, as these factors may be relevant to mortality there was clear evidence that men with serum testosterone concentration below 25th percentile had a 40% higher risk of death, independent of obesity, lifestyle choices (eg. exercise, smoking) and age (Laughlin, et al., 2008, Tivestena et al., 2009). Another 5-year retrospect study on male veterans (N=858), compared mortality of men with normal (± 450 ng/dL) T levels to those with low (<250 ng/dL) total testosterone and low (< 0.75ng/dL) free testosterone levels (Shores, et al., 2006). The results showed that mortality for subjects with low T was almost twice as high (34.9%) as for those with normal T levels was 20.1%. There is an obvious link between mortality and morbidity due to coronary CAD and other cardiovascular diseases and LOH due to low free testosterone levels in serum of aging men (Wu & von Eckardstein, 2003, Wu, et al., 2010). This is due to existing strong correlation of testosterone defficiency with glucose tolerance, visceral obesity, serum lipid disorders and elevated arterial pressure (Kalyani & Dobs, 2007). These factors usually lead to vascular disorders. The clinical studies (English, et al., (1997) showed that low natural androgen concentration can cause deletorious changes in atherogenic lipid profile, high fibrinogen and a hypercoagulable state, an increase in insulin resistance and hyperinsulinemia, higher systolic and diastolic blood pressures. The biologically plausible mechanism of testosterone protective action against cardiovascular disorers can be explained by the fact that testosterone has direct vasoactive properties (English, et al., (1997). In animal models of isolated coronary, femoral and pulmonary arteries testosterone showed a dose-dependent vasodilatory effect (Channer & Jones, 2003). It is caused by a direct effect on the vascular smooth muscle, by either an effect on potassium or calcium channels (Deenadayalu, V.P., et al. 2001, English, K.M., 2002). In man, testosterone has been shown to cause dose-dependent vasodilation both in vitro and in vivo. During cardiac surgery on man, the coronary artery diameter and subsequent

Late - Onset Hypogonadism - New Point of View 149

totally infertile (WHO, 1990, Wallace, et al., 1993, Anderson & Wu, 1996, Zhang et al., 1999, Baird, 2002, Anderson & Waites, 2003, Si-Tian, et al., 2004). Furthermore, long-time testosterone therapy may result in irreversible testicular atrophy. From this moment on, a man becomes dependent on taking testosterone. The discontinuation of testosterone intake

The effect of the treatment of 908 patients taking hCG was described in 2010 (Gomula & Rabijewski, 2010). Below, I present a further study of 1200 men (age range 20-89 years;

During the therapy with hCG (2 x 5000 i.u. per week) there was an average increase in serum concentrations of total testosterone from 18.4 nmol/L to 38.59 nmol/L. It was noted that during the hCG therapy, there was no increase of SHBG. To the contrary, the SHBG concentrations even showed a slight decrease. This meant that as the result of hCG treatment, free and bioavailable testosterone concentrations increased. The rise was

There was an average increase in free testosterone concentrations from 0.0829 ng/mL (1.98%) to 0.201 ng/mL (2.29%) The bio-available testosterone concentration also increased: − on average from 1.94 ng/mL (46.4%) to 4.71 ng /mL (53.6%). At the same time, there was a steady increase in the average concentration of estradiol, from 138.6 pmol/L to 280.9 pmol/L. In paralel, the average PSA level decreased by 40% (from 3.09 ng/mL to 1.83

 T T-f T-ba E-2 SHBG PSA-t Before 18.4 0.0829 (1.98%) 1.94 (46.4 %) 138.6 35.1 3.09 During 38.59 0.201 (2.29%) 4.71 (53.6 %) 280.9 34.86 1.83 T= total testosterone (in nmol/L), T-f = free testosterone (in ng/mL), T-ba = bioavailable testosterone (in ng/mL), E-2=estradiol (in pmol/L), SHBG=sex hormone binding globulin (in nmol/L), PSA-t = total

During LOH treatment with testosterone there was also no significant PSA increase, which was regarded as evidence further supporting the safety of this therapy (Wang, et al., 2009). But achieving a 40% reduction in PSA during the hCG therapy, with a simultaneous increase of more than 2-fold in serum testosterone levels as reported above show for the first time that the hCG therapy is not only highly effective for all age groups, but quite safe

There is clear evidence now that hCG therapy is safe and effective as a treatment of choice for men with late onset hypogonadism. During many years of continuous use of preparations, where hCG was the active ingredient, for LOH therapy, none of untoward side-effects were observed nor reported. It has also been confirmed by the files at the Division for Monitoring of Adverse Actions of the High Authority for Registration of Drugs and other Medical Products in Poland. The reports in these files have shown that in the last 14 years there has not been any case reporting a man showing the potential side effects of preparations used in the treatment, in which the hCG is the active ingredient (Reports from

leads to metabolic disorders, which may cause many other diseases to develop.

proportional to that in total testosterone, as was reported (Fiers & Kaufman, 1999).

ng/mL) after 37 months of therapy. These results are shown in Table 9, below.

Table 9. Testosterone (total, free, bioavailable), estradiol, SHBG & PSA before and

mean, 54). Mean follow-up period of the patients was over 37 months.

**4.1 Late - onset hypogonadism and hCG therapy** 

Prostate-Specific Antigen (in ng/mL).

as well.

during/after 3 years of hCG therapy

coronary flow immediately increased when testosterone was injected via intracoronary indwelling catheter (Webb, C.M. et al., 1999).

During the non-invasive therapy of LOH when the increased T concentration levels can be sustained for longer periods. T can not only protect man against atherogenic factors but also may cause long-term coronary dilatation. This is due to testosterone interaction with arginase activity, which results in increased synthesis of nitric oxide in endothelial cells in the entire vascular system. Nitric oxide is a well known vasodilator, which acts systemically. The synthesis of NO is also used in the treatment of erectile dysfunction (ED) by applying fosfodiesterase 5 inhibitors (PDE-5-I). Interestingly, nowadays these PDE-5 inhibitors become popular as a treatment of choice for some cardiovascular diseases.

Large scale clinical studies on long term effects of treatment of LOH with hCG on CAD were not yet reported, but case reports from my colleagues in cardiology who clinically follow some of my patients with exercise tests, are positive, including diminshing or disappearance of so called angina pectoris pain.

#### **3.10 Late - onset hypogonadism and immune resistance**

I have no scientific evidence to support any representation of a change in the immunity in my patients undergoing hormone therapy. Conducting such research is practically very difficult, perhaps impossible. Antibiotics, aspirin, and anti-inflammatory drugs should not be taken for at least a year by patients who have their immune status tested. This is impossible!!! But I can say with conviction that men whose testosterone levels increased 2 fold during hCG therapy as well as those already experiencing androversion (for androversion see 3.3 LOH and androversion) claimed that they did not get colds and that they had no infections, which they had had quite often before.

#### **4. The treatment of late - onset hypogonadism**

Now, the most widely practiced therapy for men with LOH are injections of testosterone esters or transcutaneous applications of 1% and 2% testosterone gel preparations. This is because many years ago it was erroneously believed that, with age, all men lose their ability for endo-synthesis of testosterone.

Another method known over the past 50 years, presently gaining "renaissance" of recognition, has been the stimulation of testosterone endo-synthesis by administering hCG (Gould, 1951, Gomula, 2001, Gomuła, 2002-a, Gomuła, 2002-b, Gomuła & Twarkowski, 2002, Gomuła, 2006, Gomuła, 2007).

The serum testosterone concentrations observed in patients treated with hCG increased by at least 150% and up to 200% (Gould, 1951, Janczewski, et al., 1966, Gomuła, 2001, Gomuła, 2002-a, Liu et al., 2002, Gomuła, 2007, Gomuła & Rabijewski, 2010).

Yet, if applying hCG makes it possible to almost double testosterone concentration, why to inject testosterone esters to men when they still have the ability for endo-synthesis of their own, natural testosterone?

The hCG therapy improves testicular function, while treatment with synthetic testosterone inhibits it. Years ago, there was a trend to produce oral contraceptives for men but this idea was soon abandoned. That oral contraceptive for men was supposed to be testosterone ester pill. The information that taking synthetic testosterone not only inhibits spermatogenesis, but also inhibits male's own testosterone endo-synthesis, is now almost labeled as "classified". In fact, after three months of taking synthetic testosterone, a man becomes

coronary flow immediately increased when testosterone was injected via intracoronary

During the non-invasive therapy of LOH when the increased T concentration levels can be sustained for longer periods. T can not only protect man against atherogenic factors but also may cause long-term coronary dilatation. This is due to testosterone interaction with arginase activity, which results in increased synthesis of nitric oxide in endothelial cells in the entire vascular system. Nitric oxide is a well known vasodilator, which acts systemically. The synthesis of NO is also used in the treatment of erectile dysfunction (ED) by applying fosfodiesterase 5 inhibitors (PDE-5-I). Interestingly, nowadays these PDE-5 inhibitors

Large scale clinical studies on long term effects of treatment of LOH with hCG on CAD were not yet reported, but case reports from my colleagues in cardiology who clinically follow some of my patients with exercise tests, are positive, including diminshing or disappearance

I have no scientific evidence to support any representation of a change in the immunity in my patients undergoing hormone therapy. Conducting such research is practically very difficult, perhaps impossible. Antibiotics, aspirin, and anti-inflammatory drugs should not be taken for at least a year by patients who have their immune status tested. This is impossible!!! But I can say with conviction that men whose testosterone levels increased 2 fold during hCG therapy as well as those already experiencing androversion (for androversion see 3.3 LOH and androversion) claimed that they did not get colds and that

Now, the most widely practiced therapy for men with LOH are injections of testosterone esters or transcutaneous applications of 1% and 2% testosterone gel preparations. This is because many years ago it was erroneously believed that, with age, all men lose their ability

Another method known over the past 50 years, presently gaining "renaissance" of recognition, has been the stimulation of testosterone endo-synthesis by administering hCG (Gould, 1951, Gomula, 2001, Gomuła, 2002-a, Gomuła, 2002-b, Gomuła & Twarkowski, 2002,

The serum testosterone concentrations observed in patients treated with hCG increased by at least 150% and up to 200% (Gould, 1951, Janczewski, et al., 1966, Gomuła, 2001, Gomuła,

Yet, if applying hCG makes it possible to almost double testosterone concentration, why to inject testosterone esters to men when they still have the ability for endo-synthesis of their

The hCG therapy improves testicular function, while treatment with synthetic testosterone inhibits it. Years ago, there was a trend to produce oral contraceptives for men but this idea was soon abandoned. That oral contraceptive for men was supposed to be testosterone ester pill. The information that taking synthetic testosterone not only inhibits spermatogenesis, but also inhibits male's own testosterone endo-synthesis, is now almost labeled as "classified". In fact, after three months of taking synthetic testosterone, a man becomes

become popular as a treatment of choice for some cardiovascular diseases.

**3.10 Late - onset hypogonadism and immune resistance** 

they had no infections, which they had had quite often before.

2002-a, Liu et al., 2002, Gomuła, 2007, Gomuła & Rabijewski, 2010).

**4. The treatment of late - onset hypogonadism** 

indwelling catheter (Webb, C.M. et al., 1999).

of so called angina pectoris pain.

for endo-synthesis of testosterone.

Gomuła, 2006, Gomuła, 2007).

own, natural testosterone?

totally infertile (WHO, 1990, Wallace, et al., 1993, Anderson & Wu, 1996, Zhang et al., 1999, Baird, 2002, Anderson & Waites, 2003, Si-Tian, et al., 2004). Furthermore, long-time testosterone therapy may result in irreversible testicular atrophy. From this moment on, a man becomes dependent on taking testosterone. The discontinuation of testosterone intake leads to metabolic disorders, which may cause many other diseases to develop.

#### **4.1 Late - onset hypogonadism and hCG therapy**

The effect of the treatment of 908 patients taking hCG was described in 2010 (Gomula & Rabijewski, 2010). Below, I present a further study of 1200 men (age range 20-89 years; mean, 54). Mean follow-up period of the patients was over 37 months.

During the therapy with hCG (2 x 5000 i.u. per week) there was an average increase in serum concentrations of total testosterone from 18.4 nmol/L to 38.59 nmol/L. It was noted that during the hCG therapy, there was no increase of SHBG. To the contrary, the SHBG concentrations even showed a slight decrease. This meant that as the result of hCG treatment, free and bioavailable testosterone concentrations increased. The rise was proportional to that in total testosterone, as was reported (Fiers & Kaufman, 1999).

There was an average increase in free testosterone concentrations from 0.0829 ng/mL (1.98%) to 0.201 ng/mL (2.29%) The bio-available testosterone concentration also increased: − on average from 1.94 ng/mL (46.4%) to 4.71 ng /mL (53.6%). At the same time, there was a steady increase in the average concentration of estradiol, from 138.6 pmol/L to 280.9 pmol/L. In paralel, the average PSA level decreased by 40% (from 3.09 ng/mL to 1.83 ng/mL) after 37 months of therapy. These results are shown in Table 9, below.


T= total testosterone (in nmol/L), T-f = free testosterone (in ng/mL), T-ba = bioavailable testosterone (in ng/mL), E-2=estradiol (in pmol/L), SHBG=sex hormone binding globulin (in nmol/L), PSA-t = total Prostate-Specific Antigen (in ng/mL).

Table 9. Testosterone (total, free, bioavailable), estradiol, SHBG & PSA before and during/after 3 years of hCG therapy

During LOH treatment with testosterone there was also no significant PSA increase, which was regarded as evidence further supporting the safety of this therapy (Wang, et al., 2009). But achieving a 40% reduction in PSA during the hCG therapy, with a simultaneous increase of more than 2-fold in serum testosterone levels as reported above show for the first time that the hCG therapy is not only highly effective for all age groups, but quite safe as well.

There is clear evidence now that hCG therapy is safe and effective as a treatment of choice for men with late onset hypogonadism. During many years of continuous use of preparations, where hCG was the active ingredient, for LOH therapy, none of untoward side-effects were observed nor reported. It has also been confirmed by the files at the Division for Monitoring of Adverse Actions of the High Authority for Registration of Drugs and other Medical Products in Poland. The reports in these files have shown that in the last 14 years there has not been any case reporting a man showing the potential side effects of preparations used in the treatment, in which the hCG is the active ingredient (Reports from

Late - Onset Hypogonadism - New Point of View 151

The results of my follow-up of these 1200 patients show clearly that the decrease in the ability of testosterone endosynthesis is synonymous with increased risk of prostate cancer.

The effect of HRT with exo-testosterone is that after the treatment is discontinued, the patient's testosterone concentration level is lower than that which was when the treatment was initiated. In contrast to this, when hCG therapy is discontinued, the abilities for testosterone endo-synthesis are the same as they were before the treatment. Around 30% of patients developed a phenomenon, which can be called androversion – no need nor willingness to continue the hCG therapy, once an increase of 50–150% of testosterone levels

The long term hCG treatment (6-24 months) can cause permanent increase ( i.e. saturation) in testosterone endosynthesis, so the further induction by hCG administration will be not indicated. Is it possible to predict whether a man has the chance of achieving such a state of androversion? The answer is still uncertain. When a man, in his initial blood tests, has a very high concentration of LH and low testosterone levels, the prognosis for him to reach a state of androversion is negative. In such cases, the thorough prostate cancer detection tests are highly indicated, as such a patient belongs to the high-risk group. What is more, there is no increased PC risk associated with the continuing of hCG therapy for such a patient. Importantly, however, there may be an increased risk of prostate cancer, if and when the hCG therapy is discontinued. In case when there is a decrease in SHBG, this indicates that hCG therapy can be safely discontinued. The concentrations of LH, testosterone, SHGB, E-2, and PSA must be measured after one month and then again after two months. If for a period of two months, the concentrations of testosterone are high, in excess of those in the previous therapy, it is the proof of the onset of androversion. Furthermore, it is the sign of the end of indications for further hCG treatment. The aforementioned parameters should be monitored every 6 months. If, after 6 months, one notices a drop in serum testosterone, it is advisable to renew regular hCG treatment. If, however, a low concentration of testosterone, high LH levels, and elevated PSA reccur in the subsequent blood tests, this should be a signal to do further diagnostic tests, in order to be able to exclude or to undertake measures to treat

**4.4 Recomendation for Late - onset hypogonadism therapy – discussion** 

The problem with therapy of LOH in men, is the long-standing notion that andropause is like mirror image of menopause. Commonly, by the term « menopause » we understand the period when amenorrhea sets in for many years to follow, and women may even undergo HRT. A rather correct term for this period of women's life is « post-menopause ». The medically defined « menopause » lasts for only few months during which, the menstrual cycle gradually comes to a stop. This is due to the rapid decay and eventually total cessation

In men, such a phenomenon does not take place. A man loses his hormonal capacity, fertility and sexuality gradually over a period of many, many years. There are no such things in

The official recommendations of the National Health Service of Poland for treatments of men with LOH, recommend the use of both: either Testosterone or hCG therapies with no

men, like such rapid, almost dramatic drops in concentrations of gonadal hormones.

emphasis on either one. This well-balanced approach merits recognition.

**4.3 Late - onset hypogonadism and androversion** 

was reached by natural endosynthesis.

prostate cancer.

of synthesis capacity of hormones in ovaries.

the years 1996-2009). This applies to 1200 patients treated by my and to the thousands of other patients treated by other physicians across Europe.

#### **4.2 LOH and the possibility to restore testosterone endosynthesis**

Stimulation of hormonal balance with hCG in LOH men, in most cases does increase endosynthesis of natural testosterone. This is true also for all age groups. In young men the increase can be even as high as 300%. With age, the induction of endosynthesis with hCG therapy gets relatively weaker, but still effective enough to eradicate manifestations of the Testosterone Deficiency Syndrome (TDS). The analysis of the findings of the author's studies show that the opinions about the Testosterone Deficiency Syndrome etiology, so far reported in medical publications were erroneous. The author's studies on the material of 908 males treated with the hCG preparations for inducing testosterone endosynthesis, has proved that such induction is possible in any age group, but physiologically it diminishes with man's age. (Gomuła & Rabijewski, 2010). Yet, effective endosynthesis is maintained until late senility, which is in the contrast to the belief that Leydig's cells completely cease to function with age. In men aged 30–39 years, the mean increase of serum testosterone concentration resulting from hCG therapy was 128%, in those aged 40 – 49 years – 126%, in age group of 50 – 59 years – 129%, aged 60 – 69 years – 121%, aged 70 – 79 years – 99%, and in those aged 80 – 99 years it was 88%.

A statistical analysis has revealed that the effectiveness of hCG therapy depends mainly on the LH/F ratio, and to a lesser extent on the patient's age (where: LH = luteinizing hormone in the IU/L, and F = free PSA (expressed as free PSA/total PSA in %).

The data showing the relationship of the decrease in the ability of testosterone endosynthesis with the LH/F ratio, rather than age, are provided in Table 10, below.


Table 10. Ability of testosterone endosynthesis versus the LH/F ratio and age

Knowing the LH/F ratio helps to predict the outcome of the hCG treatment. When the LH/F ratio is lower than **0.15,** a patient is still able to rebuild the synthesis of endogenic testosterone. Once LH/F ratio increases above **0.30**, a patient is much less likely to be able to achieve that. Another very important correlation. The serum LH and free PSA levels are essential for the health of prostate. In the recent studies of drugs for treatment of benign prostatic hyperplasia (BPH), there is an emphasis on lowering LH. For many years the LHRH analogues have been used to treat prostate cancer.

When the LH/F index in a patient with late onset hypogonadism exceeds 0.5, there is an indication for particularly thorough detection of prostate cancer.

the years 1996-2009). This applies to 1200 patients treated by my and to the thousands of

Stimulation of hormonal balance with hCG in LOH men, in most cases does increase endosynthesis of natural testosterone. This is true also for all age groups. In young men the increase can be even as high as 300%. With age, the induction of endosynthesis with hCG therapy gets relatively weaker, but still effective enough to eradicate manifestations of the Testosterone Deficiency Syndrome (TDS). The analysis of the findings of the author's studies show that the opinions about the Testosterone Deficiency Syndrome etiology, so far reported in medical publications were erroneous. The author's studies on the material of 908 males treated with the hCG preparations for inducing testosterone endosynthesis, has proved that such induction is possible in any age group, but physiologically it diminishes with man's age. (Gomuła & Rabijewski, 2010). Yet, effective endosynthesis is maintained until late senility, which is in the contrast to the belief that Leydig's cells completely cease to function with age. In men aged 30–39 years, the mean increase of serum testosterone concentration resulting from hCG therapy was 128%, in those aged 40 – 49 years – 126%, in age group of 50 – 59 years – 129%, aged 60 – 69 years – 121%, aged 70 – 79 years – 99%, and

A statistical analysis has revealed that the effectiveness of hCG therapy depends mainly on the LH/F ratio, and to a lesser extent on the patient's age (where: LH = luteinizing hormone

The data showing the relationship of the decrease in the ability of testosterone

 LH:F= 0.1 LH:F= 0.15 LH:F= 0.20 LH:F= 0.25 LH:F= 0.3 LH:F= 0.35 30-40y 175% 160% 145% 130% 115% 100% 40-50y 164% 149% 134% 119% 104% 89% 50-60y 153% 138% 123% 108% 93% 78% 60-70y 142% 127% 112% 97% 82% 67% 70-80y 131% 110% 109% 89% 72% 52% 80-89y 119% 98% 84% 81% 60% 41%

Knowing the LH/F ratio helps to predict the outcome of the hCG treatment. When the LH/F ratio is lower than **0.15,** a patient is still able to rebuild the synthesis of endogenic testosterone. Once LH/F ratio increases above **0.30**, a patient is much less likely to be able to achieve that. Another very important correlation. The serum LH and free PSA levels are essential for the health of prostate. In the recent studies of drugs for treatment of benign prostatic hyperplasia (BPH), there is an emphasis on lowering LH. For many years the

When the LH/F index in a patient with late onset hypogonadism exceeds 0.5, there is an

endosynthesis with the LH/F ratio, rather than age, are provided in Table 10, below.

Table 10. Ability of testosterone endosynthesis versus the LH/F ratio and age

LHRH analogues have been used to treat prostate cancer.

indication for particularly thorough detection of prostate cancer.

other patients treated by other physicians across Europe.

in those aged 80 – 99 years it was 88%.

**4.2 LOH and the possibility to restore testosterone endosynthesis** 

in the IU/L, and F = free PSA (expressed as free PSA/total PSA in %).

The results of my follow-up of these 1200 patients show clearly that the decrease in the ability of testosterone endosynthesis is synonymous with increased risk of prostate cancer.

#### **4.3 Late - onset hypogonadism and androversion**

The effect of HRT with exo-testosterone is that after the treatment is discontinued, the patient's testosterone concentration level is lower than that which was when the treatment was initiated. In contrast to this, when hCG therapy is discontinued, the abilities for testosterone endo-synthesis are the same as they were before the treatment. Around 30% of patients developed a phenomenon, which can be called androversion – no need nor willingness to continue the hCG therapy, once an increase of 50–150% of testosterone levels was reached by natural endosynthesis.

The long term hCG treatment (6-24 months) can cause permanent increase ( i.e. saturation) in testosterone endosynthesis, so the further induction by hCG administration will be not indicated. Is it possible to predict whether a man has the chance of achieving such a state of androversion? The answer is still uncertain. When a man, in his initial blood tests, has a very high concentration of LH and low testosterone levels, the prognosis for him to reach a state of androversion is negative. In such cases, the thorough prostate cancer detection tests are highly indicated, as such a patient belongs to the high-risk group. What is more, there is no increased PC risk associated with the continuing of hCG therapy for such a patient. Importantly, however, there may be an increased risk of prostate cancer, if and when the hCG therapy is discontinued. In case when there is a decrease in SHBG, this indicates that hCG therapy can be safely discontinued. The concentrations of LH, testosterone, SHGB, E-2, and PSA must be measured after one month and then again after two months. If for a period of two months, the concentrations of testosterone are high, in excess of those in the previous therapy, it is the proof of the onset of androversion. Furthermore, it is the sign of the end of indications for further hCG treatment. The aforementioned parameters should be monitored every 6 months. If, after 6 months, one notices a drop in serum testosterone, it is advisable to renew regular hCG treatment. If, however, a low concentration of testosterone, high LH levels, and elevated PSA reccur in the subsequent blood tests, this should be a signal to do further diagnostic tests, in order to be able to exclude or to undertake measures to treat prostate cancer.

#### **4.4 Recomendation for Late - onset hypogonadism therapy – discussion**

The problem with therapy of LOH in men, is the long-standing notion that andropause is like mirror image of menopause. Commonly, by the term « menopause » we understand the period when amenorrhea sets in for many years to follow, and women may even undergo HRT. A rather correct term for this period of women's life is « post-menopause ». The medically defined « menopause » lasts for only few months during which, the menstrual cycle gradually comes to a stop. This is due to the rapid decay and eventually total cessation of synthesis capacity of hormones in ovaries.

In men, such a phenomenon does not take place. A man loses his hormonal capacity, fertility and sexuality gradually over a period of many, many years. There are no such things in men, like such rapid, almost dramatic drops in concentrations of gonadal hormones.

The official recommendations of the National Health Service of Poland for treatments of men with LOH, recommend the use of both: either Testosterone or hCG therapies with no emphasis on either one. This well-balanced approach merits recognition.

Late - Onset Hypogonadism - New Point of View 153

2. treatment with hCG improves functioning of testicles, with corresponding increase of secretion of « good », natural testosterone, the conclusion is simple: the treatment of LOH with naturally secreted testosterone is better, safer and more economical then it is

The superiority of the hCG treatment over the testosterone treatment has already been

This information, however, did not receive universal recognition. Yet hCG therapy has been proven to be effective and safe. Moreover, as a result of testosterone treatment, permanent sterilization of men follows. Other risks, which mainly occur with long-term use include irreversible testicular atrophy. And then only successful marketing of pharmaceutical companies remains. Patients seem condemned to a lifetime use of testosterone. Otherwise their cellular metabolism drops so low that they are vulnerable to many diseases and to

I want to apologize to the guideline authors – some of whom are my friends − but I cannot accept the fact that not having conducted any studies on hCG treatment they ruled that the method was uncertain. They claimed that perhaps it was even dangerous. Is the lack of experience concerning the effects of hCG in the LOH treatment a sufficient reason to reject

Among the authors of LOH therapy guidelines, which were established at international levels, there are my long-time friends. I do not want to upset them because I strongly believe that what they did was acting in good faith, but I cannot exclude the fact that they had been insidiously stimulated by the pharmaceutical industry. Yet, the ultimate aim of a physician's actions is acting for the good of a patient, so in such context, the friendship takes a second place. Consequently, the conclusions that are listed below are very delicate ethically but

1. There is no doubt now that testosterone deficit, which grows with age, negatively

2. The own material presented in the paper allows the author to conclude that the minimal reference values for testosterone concentration levels, at which testosterone therapy

4. Applying testosterone replacement therapy to a man with LOH makes sense only when it is impossible to provoke the man's own testosterone endosynthesis. If a man is still able to rebuild his own testosterone endosynthesis, the treatment with hCG is recommended, otherwise the testosterone replacement therapy will soon make him infertile. Moreover applying testosterone replacement therapy for a long time, will

5. Consequently, the guidelines and recommendations on LOH treatment should be

should be initiated, which are accepted universally so far, are definitely too low. 3. Introducing a therapy to increase testosterone levels only when for many years testosterone deficit has inflicted irreversible anatomical changes, seems to be clearly

a case synthetic exo-applied testosterone!

this alternative safe, effective and affordable therapy?

This has turned out to be the most troublesome section…

affects the man's biology in its broad meaning.

delayed, and is harmful for the man's health.

verified and modified as soon as possible.

Not because I had objective problems, no, but because of my ethical issues.

described (Gould, 1951).

early death.

**5. Conclusions** 

unambiguously to the point.

result in testicular atrophy.

Testosterone preparations − hormone replacement therapy (HRT) − are recommended in a series of guidelines for the LOH treatment established by the world renowned doctors and scientists. In their work, there is also reference to the HCG treatment: « Human chorionic gonadotropin (hCG) stimulates testosterone production of Leydig cells, albeit at a lower rate in older men, than in younger men. Since insufficient information exists about the therapeutic and adverse effects of hCG treatment in older men and its higher cost, this treatment cannot be recommended in LOH, except when fertility is an issue » (Wang, et al., 2009).

Over the last almost 50 years there have been many studies published on the effectiveness of hCG to induce endosynthesis of testosterone. They showed increases of testosterone concentrations from 150% to over 200% resulting from hCG therapy (Gould, 1951, Gomuła, 2001, Gomuła, 2002-a, Liu, et al., 2002, Gomuła, 2007, Gomuła & Rabijewski, 2010).

Own prospective study of 1200 patients aged from 20 to 89 y and monitored for >10 years undeniably showed hCG effectiveness in inducing endosynthesis of T. In effect, the hCGtherapy resulted in the increase of serum testosterone from a mean of 18.4 nmol/L initially, to mean 38.59 nmol/L during therapy (Gomuła & Rabijewski, 2010). There is clear evidence that hCG therapy is safe and effective as a treatment of choice for men with late onset hypogonadism. It has also been confirmed by the reports on file at the Division for Monitoring of Adverse Actions of the High Authority for Registration of Drugs and other Medical Products in Poland. The reports have shown that in the last 16 years there has not been any case reporting a man showing the potential side effects of preparations used in the treatment, in which the hCG is the active ingredient (Reports from the years 1996-2011).

This applies to 1200 patients treated by me and to the thousands of other patients treated by other doctors across Europe.

On the economic side, the treatment cost of LOH when using hCG is lower than the cost of the newly available T preparations. In the UE countries, it amounts to about 50 Euro/month. It is beyond any doubt that some authors of such consensus statement are aware of the negative impact of testosterone replacement therapy on fertility of men. They are the authors who published on the contraceptive action of **T** in men (Wallace, et al., 1993, Anderson & Wu, 1996, Weinbauer, et al, 2001).

Furthermore, many peer reviewed papers, including the report from World Health Organization, confirmed that after up to three months of HRT with testosterone, from 40% to 90% male patients developed azoo-spermy or significant oligo-spermy – clearly a contraceptive situation - leading to a permanent pharmacological sterilization of men! (WHO, 1990, Wallace, et al., 1993, Anderson & Wu, 1996, Zhang et al., 1999, Baird, 2002, Anderson & Waites, 2003, Si-Tian, et al., 2004).

These facts have been known for over 20 years, but are still suppressed from information available to the general public. Just to the contrary, the mandatory information for physicians and patients attached to each box of the products, does not even mention the infertility/sterilization as a possible side-effect action of HRT with testosterone! Even in the newest-generation testosterone product ANDROTOP, the attached leaflet lists only the following warning: « The Androtop medication is not intended for treatment of male infertility or impotence.. » .

When comparing the two facts:

1. treatment of men with exo-applied synthetic testosterone may cause not only infertility but also anatomical and functional destruction of testicles and eventually leads to their complete atrophy including disappearance;

Testosterone preparations − hormone replacement therapy (HRT) − are recommended in a series of guidelines for the LOH treatment established by the world renowned doctors and scientists. In their work, there is also reference to the HCG treatment: « Human chorionic gonadotropin (hCG) stimulates testosterone production of Leydig cells, albeit at a lower rate in older men, than in younger men. Since insufficient information exists about the therapeutic and adverse effects of hCG treatment in older men and its higher cost, this treatment cannot be

Over the last almost 50 years there have been many studies published on the effectiveness of hCG to induce endosynthesis of testosterone. They showed increases of testosterone concentrations from 150% to over 200% resulting from hCG therapy (Gould, 1951, Gomuła,

Own prospective study of 1200 patients aged from 20 to 89 y and monitored for >10 years undeniably showed hCG effectiveness in inducing endosynthesis of T. In effect, the hCGtherapy resulted in the increase of serum testosterone from a mean of 18.4 nmol/L initially, to mean 38.59 nmol/L during therapy (Gomuła & Rabijewski, 2010). There is clear evidence that hCG therapy is safe and effective as a treatment of choice for men with late onset hypogonadism. It has also been confirmed by the reports on file at the Division for Monitoring of Adverse Actions of the High Authority for Registration of Drugs and other Medical Products in Poland. The reports have shown that in the last 16 years there has not been any case reporting a man showing the potential side effects of preparations used in the treatment, in which the hCG is the active ingredient (Reports from the years 1996-2011). This applies to 1200 patients treated by me and to the thousands of other patients treated by

On the economic side, the treatment cost of LOH when using hCG is lower than the cost of the newly available T preparations. In the UE countries, it amounts to about 50 Euro/month. It is beyond any doubt that some authors of such consensus statement are aware of the negative impact of testosterone replacement therapy on fertility of men. They are the authors who published on the contraceptive action of **T** in men (Wallace, et al., 1993,

Furthermore, many peer reviewed papers, including the report from World Health Organization, confirmed that after up to three months of HRT with testosterone, from 40% to 90% male patients developed azoo-spermy or significant oligo-spermy – clearly a contraceptive situation - leading to a permanent pharmacological sterilization of men! (WHO, 1990, Wallace, et al., 1993, Anderson & Wu, 1996, Zhang et al., 1999, Baird, 2002,

These facts have been known for over 20 years, but are still suppressed from information available to the general public. Just to the contrary, the mandatory information for physicians and patients attached to each box of the products, does not even mention the infertility/sterilization as a possible side-effect action of HRT with testosterone! Even in the newest-generation testosterone product ANDROTOP, the attached leaflet lists only the following warning: « The Androtop medication is not intended for treatment of male

1. treatment of men with exo-applied synthetic testosterone may cause not only infertility but also anatomical and functional destruction of testicles and eventually leads to their

2001, Gomuła, 2002-a, Liu, et al., 2002, Gomuła, 2007, Gomuła & Rabijewski, 2010).

recommended in LOH, except when fertility is an issue » (Wang, et al., 2009).

other doctors across Europe.

infertility or impotence.. » . When comparing the two facts:

Anderson & Wu, 1996, Weinbauer, et al, 2001).

Anderson & Waites, 2003, Si-Tian, et al., 2004).

complete atrophy including disappearance;

2. treatment with hCG improves functioning of testicles, with corresponding increase of secretion of « good », natural testosterone, the conclusion is simple: the treatment of LOH with naturally secreted testosterone is better, safer and more economical then it is a case synthetic exo-applied testosterone!

The superiority of the hCG treatment over the testosterone treatment has already been described (Gould, 1951).

This information, however, did not receive universal recognition. Yet hCG therapy has been proven to be effective and safe. Moreover, as a result of testosterone treatment, permanent sterilization of men follows. Other risks, which mainly occur with long-term use include irreversible testicular atrophy. And then only successful marketing of pharmaceutical companies remains. Patients seem condemned to a lifetime use of testosterone. Otherwise their cellular metabolism drops so low that they are vulnerable to many diseases and to early death.

I want to apologize to the guideline authors – some of whom are my friends − but I cannot accept the fact that not having conducted any studies on hCG treatment they ruled that the method was uncertain. They claimed that perhaps it was even dangerous. Is the lack of experience concerning the effects of hCG in the LOH treatment a sufficient reason to reject this alternative safe, effective and affordable therapy?

#### **5. Conclusions**

This has turned out to be the most troublesome section…

Not because I had objective problems, no, but because of my ethical issues.

Among the authors of LOH therapy guidelines, which were established at international levels, there are my long-time friends. I do not want to upset them because I strongly believe that what they did was acting in good faith, but I cannot exclude the fact that they had been insidiously stimulated by the pharmaceutical industry. Yet, the ultimate aim of a physician's actions is acting for the good of a patient, so in such context, the friendship takes a second place. Consequently, the conclusions that are listed below are very delicate ethically but unambiguously to the point.


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#### **6. Acknowledgements**

I wish to express my sincere gratitude to my close friend – Dr Bruno Lunenfeld – for inspiring me to work in the field of the Aging Male for many years, as especially to conduct studies in the effectiveness of hCG therapy in inducing testosterone endosynthesis. I received a similar support from Prof. Alvaro Morales, for which I am very grateful too. During performing my studies, I felt a constant support from Dr. Malcolm Carruthers, Prof. Assam Yashin, Prof. Peter Sheplev, Prof. Nykola Boiko, Prof. Stefan Zgliczynski – thank you all, my friends. Walking the steep path of a professional career in science, where I have attempted to prove the safety and effectiveness of hCG therapy, I have encountered numerous enemies. I want to thank them too – their activities have made me fight for the scientific truth even more tenaciously.

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**7** 

**Hypogonadism After** 

*1Institute of Oncology,* 

*Slovenia* 

**Childhood Cancer Treatment** 

*2University of Ljubljana, Faculty of Administration*

Lorna Zadravec Zaletel1, Ljupčo Todorovski2 and Berta Jereb1

Long-term survival of children with cancer has greatly improved in the last decades due to effective treatment, especially multiagent chemotherapy (ChT). The chief concern is now being directed toward the late effects of treatment. Endocrine glands, gonads in particular, are very susceptible to damaging effects of anticancer therapy. The damaging effect of both ChT and radiotherapy (RT) on gonads is well known (Cohen 2003, Diamond et al. 2001, Spoudeas 2002). In a study of 2283 long-term survivors of childhood cancer Byrne and colleagues found that RT below the diaphragm depressed fertility in both sexes for about 25%, ChT with alkylating agents (AA) with or without RT below the diaphragm depressed fertility by 60% in men, in women, however, AA therapy administered alone had no apparent effect on fertility (Byrne et al. 1987). Hypogonadism is most often due to direct damage by ChT, RT and/or surgery, rarely is due to damage to the hypothalamus and/or

The gonads have two main functions, the production of sex hormones (estrogens and testosterone) and germ cells (ova and sperm). Both of them depend on a normal function of the hypothalamic-pituitary-gonadal axis. Long term survivors of childhood cancer are at risk of hypogonadism related to gonadotropin secretion, but more frequently hypogonadism is caused by direct damage of testes or ovaries. In human testis two functions are combined: sex steroid production and sperm production. Germ cells form sperm, Sertoli cells support and nurture the developing germ cells and Leydig cells produce testosterone. These three cell types are organized into two functional compartments: germ cells and Sertoli cells form the seminiferous tubules where spermatogenesis takes place, and the network of Leydig cells are responsible for the production of testosterone, which is necessary for normal spermatogenesis. These two compartments are under separate controls and affected in different ways by cancer treatments (Meistrich 2009, Shalet 2009, Sklar 1999). In the ovary, follicle is the site where the production of sex hormones and germ cells takes place. As a result, when ovarian failure occurs, both sex hormone production and fertility are disrupted. Germ cells in the ovary unlike spermatogonial cells lack the ability of repopulation. Preliminary stages of oogenesis are completed shortly after birth, the dominant part of the cell population in ovary being oocytes in the stationary stage of prophase. Older age is an important risk factor for ovarian failure following childhood

**1. Introduction** 

pituitary gland (Cicognani et al. 2003, Müller 2003).


### **Hypogonadism After Childhood Cancer Treatment**

Lorna Zadravec Zaletel1, Ljupčo Todorovski2 and Berta Jereb1 *1Institute of Oncology, 2University of Ljubljana, Faculty of Administration Slovenia* 

#### **1. Introduction**

160 Sex Hormones

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injectable testosterone undecanoate as a potential male contraceptive in normal

Long-term survival of children with cancer has greatly improved in the last decades due to effective treatment, especially multiagent chemotherapy (ChT). The chief concern is now being directed toward the late effects of treatment. Endocrine glands, gonads in particular, are very susceptible to damaging effects of anticancer therapy. The damaging effect of both ChT and radiotherapy (RT) on gonads is well known (Cohen 2003, Diamond et al. 2001, Spoudeas 2002). In a study of 2283 long-term survivors of childhood cancer Byrne and colleagues found that RT below the diaphragm depressed fertility in both sexes for about 25%, ChT with alkylating agents (AA) with or without RT below the diaphragm depressed fertility by 60% in men, in women, however, AA therapy administered alone had no apparent effect on fertility (Byrne et al. 1987). Hypogonadism is most often due to direct damage by ChT, RT and/or surgery, rarely is due to damage to the hypothalamus and/or pituitary gland (Cicognani et al. 2003, Müller 2003).

The gonads have two main functions, the production of sex hormones (estrogens and testosterone) and germ cells (ova and sperm). Both of them depend on a normal function of the hypothalamic-pituitary-gonadal axis. Long term survivors of childhood cancer are at risk of hypogonadism related to gonadotropin secretion, but more frequently hypogonadism is caused by direct damage of testes or ovaries. In human testis two functions are combined: sex steroid production and sperm production. Germ cells form sperm, Sertoli cells support and nurture the developing germ cells and Leydig cells produce testosterone. These three cell types are organized into two functional compartments: germ cells and Sertoli cells form the seminiferous tubules where spermatogenesis takes place, and the network of Leydig cells are responsible for the production of testosterone, which is necessary for normal spermatogenesis. These two compartments are under separate controls and affected in different ways by cancer treatments (Meistrich 2009, Shalet 2009, Sklar 1999). In the ovary, follicle is the site where the production of sex hormones and germ cells takes place. As a result, when ovarian failure occurs, both sex hormone production and fertility are disrupted. Germ cells in the ovary unlike spermatogonial cells lack the ability of repopulation. Preliminary stages of oogenesis are completed shortly after birth, the dominant part of the cell population in ovary being oocytes in the stationary stage of prophase. Older age is an important risk factor for ovarian failure following childhood

Hypogonadism After Childhood Cancer Treatment 163

infertility is often associated with reduced testicular volume, increased FSH concentrations,

Leydig cells are less sensitive for damaging effect of RT than germ cells, requiring higher dose of ionizing radiation (more than 1500 cGy) for failure, therefore only direct testicular irradiation can cause significant damage of LC. LC are the most sensitive for damaging effect of RT in prepubertal period (Castillo et al. 1990, Shalet et al. 1985). The probability of radiation induced Leydig cell failure is directly related to the dose delivered and inversely related to age at treatment (Leiper et al. 1986, Sarafoglou et al. 1997, Shalet et al. 1989). In the majority of males who receive 2000 cGy fractionated radiation to the testes there is no impairment of testosterone production, but after 2400 cGy of fractionated irradiation as therapy for young males with testicular relapse of ALL there is a very high risk for Leydig cell damage (Sklar 1999). The majority of boys who are prepubertal at the time of treatment, will develop Leydig cell failure after 2400 cGy testicular irradiation and require androgen replacement (Leiper et al. 1986, Shalet et al. 1985). Low doses of ionizing radiation but above 75 cGy can lead to dysfunction of the LC (compensated insufficiency of LC with normal

Unlike germinal epithelium LC impairment may develop several years after RT and is usually irreparable (Shalet et al. 1985). Treatment-induced Leydig cell failure and testosterone insufficiency following cancer tretament are relatively uncommon compared with germ cell dysfunction and infertility. Leydig cell failure results in delayed/arrested puberty and lack of secondary sexual characteristics if it occurs before onset of puberty. If it occurs following the completion of normal pubertal development, it can result in reduced libido, erectile dysfunction, decreased bone mineral density, decreased muscle mass, and other metabolic disturbances (Sklar 1999). Increased plasma concentrations of LH combined with low levels of testosterone are characteristic for Leydig cell dysfunction, but these changes may not become

The chemotherapeutic agents most commonly associated with impaired male fertility are alkylating agents (AA). These citostatic agents are used in the treatment of many types of childhood cancer. Agents in this group are: cyclophosphamide (CY), busulfan, melphalan, nitrogen mustard (NM), DTIC, nitrosoureas (CCNU, BCNU), procarbazine, chlorambucil, ifosfamide. Alkylating agents damage especially late (differentiating) spermatogonial cells and early spermatocytes, and less mature spermatozoa (Meistrich et al. 1982). In the treatment of childhood cancer many cytostatic agents are used at the same time, making it difficult to identify gonadotoxic effect of individual cytostatic. The toxic effect of CY has been studied most. After the cumulative dose of CY of less than 7.5 g/m2 males may retain normal sperm production, after a dose between 7.5 and 22.5 g/m2 oligo-and azoospermia are observed, but the dose greater than 25 g / m 2 causes azoospermia (Kenney et al. 2001). It seems that the threshold dose of CY for azoospermia is around 10 g / m2 (Aubier et al. 1989, Casteren et al. 2009, Relander et al. 2000). Patients treated in prepubertal period have a lower risk for germ cell damage than those treated in postpubertal period (Aubier et al. 1989, Brämswig et al. 1990, Pennisi et al. 1975). Procarbazine, another alkylating agent, commonly used in the treatment of

apparent until the individual has reached mid-adolescence (Shalet et al. 1985).

and reduced plasma concentrations of inhibin B.

levels of testosterone) (Rowley et al. 1974).

**1.2 Toxic effects of cytostatic agents on testes** 

**1.2.1 Germ cell epithelium** 

**1.1.2 Leydig cells** 

cancer and its treatment, given the progressive decline in oocyte reserve with increasing age (Johnston & Wallace 2009, Sklar 1999). If ovarian function is lost prior to the onset of puberty, it results in delayed puberty and primary amenorrhea. If ovarian function is lost during or after pubertal maturation, arrested puberty, secondary amenorrhea, and premature maenopause are observed. In the adolescent and young adults with ovarian failure increased plasma concentrations of gonadotropins and reduced levels of estradiol are typically found.

#### **1.1 Toxic effects of ionizing radiation on testes**

#### **1.1.1 Germ cell epithelium**

The sperm-producing cells are more vulnerable to cancer treatment than Leydig cells, and are frequently impaired by radiotherapy and different types of chemotherapy. Even small doses of ionizing radiation can damage germinal epithelium of testes. Among the germ cells, type A spermatogonial cells are the most sensitive (especially more differentiated stages A2-4, which are at the stage of mitosis) and type B spermatogonial cells (Ash 1980, Greiner 1985, Lu & Meistrich 1979, Meistrich et al. 1982, Rowley et al. 1974). These sensitive cells can be destroyed with a single dose of radiation as low as 15 cGy. Germ cells in later stages of spermatogenesis, eg. spermatocytes, and spermatides, are less sensitive to ionizing radiation (destroyed by a single dose of 200 cGy or more) (Ash 1980, Lushbaugh & Casarett 1976). After irradiation, the surviving germ cells (early type A spermatogonial cells) develop into more radiosensitive germ cells. Therefore, fractionated RT (administered in several small doses) may be more harmful because it empties the storage of germ cells (Ash 1980, Greiner 1982). Only type A spermatogonial cells can repopulate. If a sufficient number of these cells survive, there is recovery of spermatogenesis, even after several years (Hahn et al. 1982). The rate of damage of spermatogenesis and time in which there's a full recovery depends on the size of RT dose to the testes. Duration of azoospermia is likely to depend on the number of destroyed germ cells. After a single dose of less than 100 cGy to the testes, recovery of spermatogenesis occurs in 9-18 months, after a dose of 200 to 300 cGy in 30 months and after a dose of 400 to 600 cGy in more than 5-years (even after more than 10 years) (Lushbaugh & Casarett 1976, Sandeman 1966, Sanders et al. 1991, Rowley et al. 1974). Single doses greater than 600 cGy cause irreparable damage of spermatogenesis. After fractionated RT a total dose of more than 150 to 200 cGy cause irreversible azoospermia (Greiner 1982, Sandeman 1966). Oligospermia or azoospermia may occur during treatment with RT or mostly during the 2- 3-months from the start of RT (Sandeman 1966). During radiation treatment testes are rarely directly exposed to ionizing radiation, but they are exposed to indirect radiation (e.g. abdominal RT). Several studies reported radiation doses the testes receive at the spillage of ionizing radiation during RT of areas under the diaphragm. This dose to the testes following RT of abdominal areas may be as high as 7 to 13% of the total dose (i.e. the order of 100 to 300 cGy) (da Cunha et al. 1984, Kinsella et al. 1989, Lushbaugh & Casarett 1976, Whitehead et al. 1982). The dose of this size can cause irreversible azoospermia (Greiner 1982, Sandeman 1966). This dose to the testes can be reduced by an additional lead shielding of testis to the level bellow 50cGy, which is not harmful for spermatogenesis (Kovač et al. 1990, Whitehead et al. 1982). Germ cell dysfunction with azoospermia is present in essentially all males treated with TBI (Sanders et al. 1991). Recovery of germ cell function has occurred rarely and primarily following singledose irradiation (Sanders et al. 1991, Sklar et al. 1984). Germ cell dysfunction with resultant infertility is often associated with reduced testicular volume, increased FSH concentrations, and reduced plasma concentrations of inhibin B.

#### **1.1.2 Leydig cells**

162 Sex Hormones

cancer and its treatment, given the progressive decline in oocyte reserve with increasing age (Johnston & Wallace 2009, Sklar 1999). If ovarian function is lost prior to the onset of puberty, it results in delayed puberty and primary amenorrhea. If ovarian function is lost during or after pubertal maturation, arrested puberty, secondary amenorrhea, and premature maenopause are observed. In the adolescent and young adults with ovarian failure increased plasma concentrations of gonadotropins and reduced levels of estradiol are

The sperm-producing cells are more vulnerable to cancer treatment than Leydig cells, and are frequently impaired by radiotherapy and different types of chemotherapy. Even small doses of ionizing radiation can damage germinal epithelium of testes. Among the germ cells, type A spermatogonial cells are the most sensitive (especially more differentiated stages A2-4, which are at the stage of mitosis) and type B spermatogonial cells (Ash 1980, Greiner 1985, Lu & Meistrich 1979, Meistrich et al. 1982, Rowley et al. 1974). These sensitive cells can be destroyed with a single dose of radiation as low as 15 cGy. Germ cells in later stages of spermatogenesis, eg. spermatocytes, and spermatides, are less sensitive to ionizing radiation (destroyed by a single dose of 200 cGy or more) (Ash 1980, Lushbaugh & Casarett 1976). After irradiation, the surviving germ cells (early type A spermatogonial cells) develop into more radiosensitive germ cells. Therefore, fractionated RT (administered in several small doses) may be more harmful because it empties the storage of germ cells (Ash 1980, Greiner 1982). Only type A spermatogonial cells can repopulate. If a sufficient number of these cells survive, there is recovery of spermatogenesis, even after several years (Hahn et al. 1982). The rate of damage of spermatogenesis and time in which there's a full recovery depends on the size of RT dose to the testes. Duration of azoospermia is likely to depend on the number of destroyed germ cells. After a single dose of less than 100 cGy to the testes, recovery of spermatogenesis occurs in 9-18 months, after a dose of 200 to 300 cGy in 30 months and after a dose of 400 to 600 cGy in more than 5-years (even after more than 10 years) (Lushbaugh & Casarett 1976, Sandeman 1966, Sanders et al. 1991, Rowley et al. 1974). Single doses greater than 600 cGy cause irreparable damage of spermatogenesis. After fractionated RT a total dose of more than 150 to 200 cGy cause irreversible azoospermia (Greiner 1982, Sandeman 1966). Oligospermia or azoospermia may occur during treatment with RT or mostly during the 2- 3-months from the start of RT (Sandeman 1966). During radiation treatment testes are rarely directly exposed to ionizing radiation, but they are exposed to indirect radiation (e.g. abdominal RT). Several studies reported radiation doses the testes receive at the spillage of ionizing radiation during RT of areas under the diaphragm. This dose to the testes following RT of abdominal areas may be as high as 7 to 13% of the total dose (i.e. the order of 100 to 300 cGy) (da Cunha et al. 1984, Kinsella et al. 1989, Lushbaugh & Casarett 1976, Whitehead et al. 1982). The dose of this size can cause irreversible azoospermia (Greiner 1982, Sandeman 1966). This dose to the testes can be reduced by an additional lead shielding of testis to the level bellow 50cGy, which is not harmful for spermatogenesis (Kovač et al. 1990, Whitehead et al. 1982). Germ cell dysfunction with azoospermia is present in essentially all males treated with TBI (Sanders et al. 1991). Recovery of germ cell function has occurred rarely and primarily following singledose irradiation (Sanders et al. 1991, Sklar et al. 1984). Germ cell dysfunction with resultant

typically found.

**1.1.1 Germ cell epithelium** 

**1.1 Toxic effects of ionizing radiation on testes** 

Leydig cells are less sensitive for damaging effect of RT than germ cells, requiring higher dose of ionizing radiation (more than 1500 cGy) for failure, therefore only direct testicular irradiation can cause significant damage of LC. LC are the most sensitive for damaging effect of RT in prepubertal period (Castillo et al. 1990, Shalet et al. 1985). The probability of radiation induced Leydig cell failure is directly related to the dose delivered and inversely related to age at treatment (Leiper et al. 1986, Sarafoglou et al. 1997, Shalet et al. 1989). In the majority of males who receive 2000 cGy fractionated radiation to the testes there is no impairment of testosterone production, but after 2400 cGy of fractionated irradiation as therapy for young males with testicular relapse of ALL there is a very high risk for Leydig cell damage (Sklar 1999). The majority of boys who are prepubertal at the time of treatment, will develop Leydig cell failure after 2400 cGy testicular irradiation and require androgen replacement (Leiper et al. 1986, Shalet et al. 1985). Low doses of ionizing radiation but above 75 cGy can lead to dysfunction of the LC (compensated insufficiency of LC with normal levels of testosterone) (Rowley et al. 1974).

Unlike germinal epithelium LC impairment may develop several years after RT and is usually irreparable (Shalet et al. 1985). Treatment-induced Leydig cell failure and testosterone insufficiency following cancer tretament are relatively uncommon compared with germ cell dysfunction and infertility. Leydig cell failure results in delayed/arrested puberty and lack of secondary sexual characteristics if it occurs before onset of puberty. If it occurs following the completion of normal pubertal development, it can result in reduced libido, erectile dysfunction, decreased bone mineral density, decreased muscle mass, and other metabolic disturbances (Sklar 1999). Increased plasma concentrations of LH combined with low levels of testosterone are characteristic for Leydig cell dysfunction, but these changes may not become apparent until the individual has reached mid-adolescence (Shalet et al. 1985).

#### **1.2 Toxic effects of cytostatic agents on testes 1.2.1 Germ cell epithelium**

The chemotherapeutic agents most commonly associated with impaired male fertility are alkylating agents (AA). These citostatic agents are used in the treatment of many types of childhood cancer. Agents in this group are: cyclophosphamide (CY), busulfan, melphalan, nitrogen mustard (NM), DTIC, nitrosoureas (CCNU, BCNU), procarbazine, chlorambucil, ifosfamide. Alkylating agents damage especially late (differentiating) spermatogonial cells and early spermatocytes, and less mature spermatozoa (Meistrich et al. 1982). In the treatment of childhood cancer many cytostatic agents are used at the same time, making it difficult to identify gonadotoxic effect of individual cytostatic. The toxic effect of CY has been studied most. After the cumulative dose of CY of less than 7.5 g/m2 males may retain normal sperm production, after a dose between 7.5 and 22.5 g/m2 oligo-and azoospermia are observed, but the dose greater than 25 g / m 2 causes azoospermia (Kenney et al. 2001). It seems that the threshold dose of CY for azoospermia is around 10 g / m2 (Aubier et al. 1989, Casteren et al. 2009, Relander et al. 2000). Patients treated in prepubertal period have a lower risk for germ cell damage than those treated in postpubertal period (Aubier et al. 1989, Brämswig et al. 1990, Pennisi et al. 1975). Procarbazine, another alkylating agent, commonly used in the treatment of

Hypogonadism After Childhood Cancer Treatment 165

et al. 1992). In women over 40 years of age radiation dose of 400 to 700 cGy is sufficient for the sterilization, in younger women the dose from 1250 to 1500 cGy is necessary for sterilization (Ash 1980), and for those treated at the age of 10 years or less even dose of 2000 cGy is necessary for permanent ovarian damage (Lushbaugh & Casarett 1976, Sanders et al. 1991, Wallace et al. 2005). Nevertheless doses of less than 1000 cGy are capable of inducing ovarian damage in patients who have additional risk factors, such as concomitant exposure to alkylating agents and older age at diagnosis. In a report from the CCSS, doses of radiotherapy to the ovary of at least 2000 cGy were associated with the highest risk of ovarian failure; more than 70% of patients exposed to such doses developed ovarian failure, with higher rates in older individuals (13–20 years) when compared with those who were younger (0–12 years) at the time of treatment (Chemaitilly et al. 2006). Moreover, if radiation is given in association with alkylating agents, ovarian dysfunction may occur despite the use of lower doses. In the report from CCSS acute ovarian failure occurred in 6.3% of eligible survivors, exposure of the ovaries to high-dose radiation (especially over 1000 cGy), alkylating agents and older ages being significant risk factors for ovarian failure (Chemaitilly et al. 2006). Premature nonsurgical menopause occurred in 8% of participants versus 0.8% of siblings. Risk factors for premature menopause included attained age, exposure to increasing doses of radiation to the ovaries, increasing alkylating agent score, and the diagnosis of Hodgkin's lymphoma. The cumulative incidence of premature menopause in individuals treated with both alkylating agents and abdominal– pelvic radiation was in the range of 30% (Sklar et al. 2006). Offspring of women who received uterine radiation doses of more than 500 cGy were more likely to be small for gestational age, but there was no evidence for an increased risk of congenital malformations (Green et al. 2002). These studies demonstrated that women treated with pelvic irradiation and/or high alkylating agent doses were at risk for acute ovarian failure, premature menopause,

Ovaries are less sensitive to harmful effect of cytostatics than germ cells of testes. Biopsy of the ovary in girls after treatment with chemotherapy showed decreased number of primordial and antral follicles (even more pronounced after treatment with multiagent ChT and RT), decreased follicular maturation, cortical and stromal fibrosis, with / without proliferation and thickening of blood vessels (Chapman et al. 1979, Nicosia et al. 1985). Most sensitive for damaging effect of ChT are growing and preovulatory follicles, therefore ovaries of prepubertal girls are less sensitive to injury after ChT exposure (Stillman et al. 1982). Another reason for higher resistance of ovaries of prepubertal girls to ChT is their greater follicular reserve when compared with the ovaries of adults (Chemaitilly et al. 2006, Grigg et al. 2000). Among chemotherapeutic agents, alkylating agents, which prevent cell division by interacting with DNA, are known to be associated with the occurrence of ovarian failure (Brydøy et al. 2007, Chematilly et al. 2006, Green et al. 2009, Ortin et al. 1990, Zacharin et al. 2010), ovarian failure being dependent on the cumulative dose of cytostatic and age of patients during treatment. In CCSS-study, it was reported that alkylating agents cyclophosphamide and procarbazine were significant risk factors for ovarian failure (Chematilly et al. 2006). Although exposure to procarbazine was an independent risk factor for ovarian failure, regardless of age at treatment, cyclophosphamide significantly increased that risk only in subjects treated at an older age. As the number of oocytes declines with

and small-for-gestational-age offspring.

**1.4 Toxic effects of cytostatic agents on ovaries** 

Hodgkin's disease, can also induce impaired sperm production in a dose-dependent fashion. MOPP (mechlorethamine, vincristine, procarbazine, and prednisone) or MOPP-like combinations, such as MVPP (mechlorethamine, vinblastine, procarbazine and prednisone) induce azoospermia in 90-100 % of pts with a 10-20% chance of recovery even 10 years after treatment (Chapman et al. 1979, Diamond & Bercu 2001, Viviani et al. 1985, Whitehead et al. 1982). Recovery of spermatogenesis following MOPP therapy appears to be dose-related, 3 courses of MOPP representing a limiting gonadal exposure for recovery, suggesting only a partial killing of germinal stem cells (da Cunha et al. 1984). Patients with Hodgkin's disease who received three cycles of MOPP alternating with three cycles of ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine) suffer less testicular damage than patients who received 6 cycles of MOPP (Berg et al. 2004, Mackie et al. 1996). Chemotherapy with COPP (CY, VCR, PBZ, prednisone) or OPPA (VCR, prednisone, procarbazine and adriamycin) can cause azoospermia in 50% of patients. ABVD (Adriamycin, bleomycin, vinblastine,DTIC) protocol is less gonadotoxic, usually causing transient germ cell impairment with total recovery (Berg et al. 2004, Santoro et al. 1987, Viviani et al. 1985). CY and cytarabine were reported the most damaging antileukemic drugs for spermatogenesis (Lendon et al. 1978). There has been a report that cytarabine in cumulative doses greater than 1 g/m2 is correlated with a decreased tubular fertility index in boys (Lendon et al. 1978). There are reports that vincristine also might have important role in causing azoospermia, when administered in childhood or adolescence (Waxmann et al. 1982).

Chemotherapy regimens containing cisplatin or carboplatin can induce germ cell damage with a different rate of recovery of spermatogenesis (Lampe et al. 1997). Recovery of spermatogenesis may be lower in those who received ChT with vinca alkaloids (vincristine and vinblastine) as well. Antimetabolites usually do not cause irreversible damage to testes, but can cause temporary oligospermia. (Sussman & Leonard 1980).

#### **1.2.2 Leydig cells**

Leydig cells are less vulnerable to damage from cancer therapy than germ cells, and chemotherapy-induced dysfunction of Leydig cells requiring testosterone replacement therapy is rare (Blatt et al. 1981, Sklar 1999). Leydig cell dysfunction may be observed following treatment with alkylating agent regimens. Ten to 57% of male patients can develop elevated serum concentrations of LH following treatment, but chemotherapyinduced Leydig cell dysfunction is generally subclinical (Bramswig et al. 1990, Kenney et al. 2001, Mackie et al. 1996, Relander et al. 2000, Romerius et al. 2009, Sklar 1999).

#### **1.3 Toxic effects of ionizing radiation on ovaries**

Ionizing radiation causes ovarian function impairment as a function of cumulative dose and age. Ovaries are frequently inside RT field or in its immediate vicinity during pelvic or abdominal RT. Preservation of ovarian function depends on the ability of single oocyte to repair damage. That is why RT with multiple small fractions is less toxic for ovaries than RT with one larger fraction, due to greater potential of damage repair during two smaller fractions (Greiner 1985). Females receiving abdominal, pelvic, or spinal irradiation are at increased risk of ovarian failure, especially if both ovaries are within the treatment field (Hamre et al. 1987, Horning et al. 1981, Sklar et al. 2006, Thibaud et al. 1992, Wallace 2005). However, when ovarian transposition is performed prior to RT, ovarian function is retained in the majority of young girls and adolescent females (Ortin et al. 1990, Sklar 1999, Thibaud

Hodgkin's disease, can also induce impaired sperm production in a dose-dependent fashion. MOPP (mechlorethamine, vincristine, procarbazine, and prednisone) or MOPP-like combinations, such as MVPP (mechlorethamine, vinblastine, procarbazine and prednisone) induce azoospermia in 90-100 % of pts with a 10-20% chance of recovery even 10 years after treatment (Chapman et al. 1979, Diamond & Bercu 2001, Viviani et al. 1985, Whitehead et al. 1982). Recovery of spermatogenesis following MOPP therapy appears to be dose-related, 3 courses of MOPP representing a limiting gonadal exposure for recovery, suggesting only a partial killing of germinal stem cells (da Cunha et al. 1984). Patients with Hodgkin's disease who received three cycles of MOPP alternating with three cycles of ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine) suffer less testicular damage than patients who received 6 cycles of MOPP (Berg et al. 2004, Mackie et al. 1996). Chemotherapy with COPP (CY, VCR, PBZ, prednisone) or OPPA (VCR, prednisone, procarbazine and adriamycin) can cause azoospermia in 50% of patients. ABVD (Adriamycin, bleomycin, vinblastine,DTIC) protocol is less gonadotoxic, usually causing transient germ cell impairment with total recovery (Berg et al. 2004, Santoro et al. 1987, Viviani et al. 1985). CY and cytarabine were reported the most damaging antileukemic drugs for spermatogenesis (Lendon et al. 1978). There has been a report that cytarabine in cumulative doses greater than 1 g/m2 is correlated with a decreased tubular fertility index in boys (Lendon et al. 1978). There are reports that vincristine also might have important role in causing azoospermia, when administered in

Chemotherapy regimens containing cisplatin or carboplatin can induce germ cell damage with a different rate of recovery of spermatogenesis (Lampe et al. 1997). Recovery of spermatogenesis may be lower in those who received ChT with vinca alkaloids (vincristine and vinblastine) as well. Antimetabolites usually do not cause irreversible damage to testes,

Leydig cells are less vulnerable to damage from cancer therapy than germ cells, and chemotherapy-induced dysfunction of Leydig cells requiring testosterone replacement therapy is rare (Blatt et al. 1981, Sklar 1999). Leydig cell dysfunction may be observed following treatment with alkylating agent regimens. Ten to 57% of male patients can develop elevated serum concentrations of LH following treatment, but chemotherapyinduced Leydig cell dysfunction is generally subclinical (Bramswig et al. 1990, Kenney et al.

Ionizing radiation causes ovarian function impairment as a function of cumulative dose and age. Ovaries are frequently inside RT field or in its immediate vicinity during pelvic or abdominal RT. Preservation of ovarian function depends on the ability of single oocyte to repair damage. That is why RT with multiple small fractions is less toxic for ovaries than RT with one larger fraction, due to greater potential of damage repair during two smaller fractions (Greiner 1985). Females receiving abdominal, pelvic, or spinal irradiation are at increased risk of ovarian failure, especially if both ovaries are within the treatment field (Hamre et al. 1987, Horning et al. 1981, Sklar et al. 2006, Thibaud et al. 1992, Wallace 2005). However, when ovarian transposition is performed prior to RT, ovarian function is retained in the majority of young girls and adolescent females (Ortin et al. 1990, Sklar 1999, Thibaud

childhood or adolescence (Waxmann et al. 1982).

**1.3 Toxic effects of ionizing radiation on ovaries** 

**1.2.2 Leydig cells** 

but can cause temporary oligospermia. (Sussman & Leonard 1980).

2001, Mackie et al. 1996, Relander et al. 2000, Romerius et al. 2009, Sklar 1999).

et al. 1992). In women over 40 years of age radiation dose of 400 to 700 cGy is sufficient for the sterilization, in younger women the dose from 1250 to 1500 cGy is necessary for sterilization (Ash 1980), and for those treated at the age of 10 years or less even dose of 2000 cGy is necessary for permanent ovarian damage (Lushbaugh & Casarett 1976, Sanders et al. 1991, Wallace et al. 2005). Nevertheless doses of less than 1000 cGy are capable of inducing ovarian damage in patients who have additional risk factors, such as concomitant exposure to alkylating agents and older age at diagnosis. In a report from the CCSS, doses of radiotherapy to the ovary of at least 2000 cGy were associated with the highest risk of ovarian failure; more than 70% of patients exposed to such doses developed ovarian failure, with higher rates in older individuals (13–20 years) when compared with those who were younger (0–12 years) at the time of treatment (Chemaitilly et al. 2006). Moreover, if radiation is given in association with alkylating agents, ovarian dysfunction may occur despite the use of lower doses. In the report from CCSS acute ovarian failure occurred in 6.3% of eligible survivors, exposure of the ovaries to high-dose radiation (especially over 1000 cGy), alkylating agents and older ages being significant risk factors for ovarian failure (Chemaitilly et al. 2006). Premature nonsurgical menopause occurred in 8% of participants versus 0.8% of siblings. Risk factors for premature menopause included attained age, exposure to increasing doses of radiation to the ovaries, increasing alkylating agent score, and the diagnosis of Hodgkin's lymphoma. The cumulative incidence of premature menopause in individuals treated with both alkylating agents and abdominal– pelvic radiation was in the range of 30% (Sklar et al. 2006). Offspring of women who received uterine radiation doses of more than 500 cGy were more likely to be small for gestational age, but there was no evidence for an increased risk of congenital malformations (Green et al. 2002). These studies demonstrated that women treated with pelvic irradiation and/or high alkylating agent doses were at risk for acute ovarian failure, premature menopause, and small-for-gestational-age offspring.

#### **1.4 Toxic effects of cytostatic agents on ovaries**

Ovaries are less sensitive to harmful effect of cytostatics than germ cells of testes. Biopsy of the ovary in girls after treatment with chemotherapy showed decreased number of primordial and antral follicles (even more pronounced after treatment with multiagent ChT and RT), decreased follicular maturation, cortical and stromal fibrosis, with / without proliferation and thickening of blood vessels (Chapman et al. 1979, Nicosia et al. 1985). Most sensitive for damaging effect of ChT are growing and preovulatory follicles, therefore ovaries of prepubertal girls are less sensitive to injury after ChT exposure (Stillman et al. 1982). Another reason for higher resistance of ovaries of prepubertal girls to ChT is their greater follicular reserve when compared with the ovaries of adults (Chemaitilly et al. 2006, Grigg et al. 2000). Among chemotherapeutic agents, alkylating agents, which prevent cell division by interacting with DNA, are known to be associated with the occurrence of ovarian failure (Brydøy et al. 2007, Chematilly et al. 2006, Green et al. 2009, Ortin et al. 1990, Zacharin et al. 2010), ovarian failure being dependent on the cumulative dose of cytostatic and age of patients during treatment. In CCSS-study, it was reported that alkylating agents cyclophosphamide and procarbazine were significant risk factors for ovarian failure (Chematilly et al. 2006). Although exposure to procarbazine was an independent risk factor for ovarian failure, regardless of age at treatment, cyclophosphamide significantly increased that risk only in subjects treated at an older age. As the number of oocytes declines with

Hypogonadism After Childhood Cancer Treatment 167

Leukemia 30 37 67 (22.5) Hodgkin's disease 40 24 64 (21.5) Brain tumor 30 18 48 (16) NHL 35 3 38 (13) Soft tissue sarcoma 16 8 24 (8) Wilms' tumor 11 7 18 (6) Bone sarcoma 6 6 12 (4) Germ cell tumor 4 6 10 (3.5) Neuroblastoma 4 1 5 (1.5) Retinoblastoma 2 3 5 (1.5) Carcinoma of nasopharynx 2 1 3 (1) Other ♣ 2 1 3 (1) All 182 115 297 (100)

♣ retroperitoneal paraganglioma, hepatoblastoma, invasive adenoma of suprarenal gland, one each

The patient´s data regarding diagnosis and treatment were collected from medical files, information concerning quality of life including attained educational level, marital status, employment and social life, past and present menstrual histories, the course of puberty and fertility histories was ascertained by interview. General physical examination was performed in terms of recording height, weight, clinical abnormalities and Tanner stages of pubic hair and genital development were recorded. Each patient´s blood samples were analysed for basal concentrations of testosterone (RIA, IMUNOTECH), estradiol (DELFIA-LKB) and prolactin (DELFIA-LKB). Concentrations of LH (DELFIA-LKB) and FSH (DELFIA-LKB) were determined before and 10, 20, 30, 60 minutes after i.v. administration of gonadotropin releasing hormone (50 mcg/m2) (LH-RH). Primary hypogonadism (PH) was defined as basal serum FSH and/or LH level above the normal upper limit and exaggerated response after stimulation with LH-RH. In men, elevated basal serum FSH levels indicated germinal epithelium damage (GE-DA), while elevated LH levels (with/without reduced testosterone levels) indicated Leydig cells (LC) damage (LC-DA). Normal basal values of LH and/or FSH and exaggerated response after LH-RH stimulation were considered as subclinical impairment (SIG). Exaggerated response of FSH after LH-RH was considered as dysfunction of germinal epithelium (GE-dys), while exaggerated response of LH after LH-RH was considered as dysfunction of LC (LC-dys). PH and SIG together were named gonadal impairment (GI). Low serum basal FSH and LH levels with poor response after i.v.

Males Females All n n n (%)

DIAGNOSIS

Table 1. Diagnosis in 297 patients

**2.2.1 Assessment of gonadal function** 

bolus of LH-RH was considered as secondary hypogonadism.

**2.2 Methods** 

advancing age, the ovaries of older individuals become more vulnerable to gonadal toxins compared with that seen in younger subjects (Sarafoglou et al. 1997, Sklar 1999). In the study of Ortin and colleagues 10% of girls were amenorrhoic after receiving 6 or more cycles of MOPP and none of those who received other regimes of ChT (e.g. MOPP / ABVD, ABVD). After combined treatment with ChT (6 or more cycles of MOPP) and pelvic RT at a dose of 2000 to 4400 cGy (with or without ovaropeksy) incidence of ovarian failure was about 50%. None of the girls who received 3 or less cycles of MOPP had ovarian failure (Ortin et al. 1990). Females who received, both before and after pubertal development, high-dose myeloablative therapy with alkylating agents such as busulfan, melphalan, and thiotepa in preparation for bone marrow transplantation are at high risk of developing ovarian failure (Michel et al. 1997). Recovery of function has been recorded only rarely (Michel et al. 1997, Thibaud et al. 1998). However, the majority of girls receiving standard chemotherapy maintain or recover ovarian function during the immediate posttreatment period (Horning et al. 1981, Sklar 1999). Histologic examination of ovarian tissue in prepubertal and postpubertal girls treated for solid tumors or leukemia nevertheless revealed a decreased number of ovarian follicles and inhibition of follicular growth compared with age-matched controls (Himelstein- Braw et al. 1978, Larsen et al. 2003). Therefore, among women who retain or recover ovarian function following treatment with ChT, a subset will experience premature menopause when they reach their 20s and 30s (Byrne et al. 1992, Sklar et al. 2006). In a report from the CCSS, female survivors with a history of exposure to high doses of alkylating agents, to lomustine or to cyclophosphamide were less likely to become pregnant when compared with sibling controls (Green et al. 2009). No adverse pregnancy outcomes were identified, however in a large study conducted within the framework of the CCSS (Green et al. 2002).

The aim of our study was: to establish the incidence of hypogonadism and the risk factors for its developement in childhood-cancer survivors in Slovenia and define the highest respective the lowest risk groups.

#### **2. Patients and methods**

#### **2.1 Patients**

In Slovenia, 1474 children were treated for cancer under the age of 16 years from 1.1.1965 to 31.12.1995 at the University Clinical Hospital Ljubljana and/or Institute of Oncology Ljubljana. At the time of our study 712 patients were alive, 460 of them were more than 16 years of age and were at least 3 years off treatment. Of those patients 390 were regularly followed at the outpatient clinic at the Institute of Oncology in Ljubljana (Jereb 2000, Zaletel 2004). We included in our study 297 consecutive patients in whom endocrinological evaluation was performed until 1.1.2003. Ninety-three patients refused examinations. There were 115 females and 182 males. They were 0-16 (median 9 yrs) years of age at the diagnosis of malignancy and had endocrinological evaluation 3-32 (median 11,5) years after the end of treatment at age of 14-42 (median 20) years. All pts were pubertal or postpubertal when studied. Distribution of diagnosis among patients included in our study is shown in table 1. The majority (90%) of patients had combined treatment including 2-3 modalities, surgery (S), ChT, RT, a quarter of them had all 3 modalities, while 41% had combined ChT and RT. To evaluate the risk factors for hypogonadism after treatment for childhood cancer, we used a multivariate analysis method of the classification trees.


♣ retroperitoneal paraganglioma, hepatoblastoma, invasive adenoma of suprarenal gland, one each

Table 1. Diagnosis in 297 patients

#### **2.2 Methods**

166 Sex Hormones

advancing age, the ovaries of older individuals become more vulnerable to gonadal toxins compared with that seen in younger subjects (Sarafoglou et al. 1997, Sklar 1999). In the study of Ortin and colleagues 10% of girls were amenorrhoic after receiving 6 or more cycles of MOPP and none of those who received other regimes of ChT (e.g. MOPP / ABVD, ABVD). After combined treatment with ChT (6 or more cycles of MOPP) and pelvic RT at a dose of 2000 to 4400 cGy (with or without ovaropeksy) incidence of ovarian failure was about 50%. None of the girls who received 3 or less cycles of MOPP had ovarian failure (Ortin et al. 1990). Females who received, both before and after pubertal development, high-dose myeloablative therapy with alkylating agents such as busulfan, melphalan, and thiotepa in preparation for bone marrow transplantation are at high risk of developing ovarian failure (Michel et al. 1997). Recovery of function has been recorded only rarely (Michel et al. 1997, Thibaud et al. 1998). However, the majority of girls receiving standard chemotherapy maintain or recover ovarian function during the immediate posttreatment period (Horning et al. 1981, Sklar 1999). Histologic examination of ovarian tissue in prepubertal and postpubertal girls treated for solid tumors or leukemia nevertheless revealed a decreased number of ovarian follicles and inhibition of follicular growth compared with age-matched controls (Himelstein- Braw et al. 1978, Larsen et al. 2003). Therefore, among women who retain or recover ovarian function following treatment with ChT, a subset will experience premature menopause when they reach their 20s and 30s (Byrne et al. 1992, Sklar et al. 2006). In a report from the CCSS, female survivors with a history of exposure to high doses of alkylating agents, to lomustine or to cyclophosphamide were less likely to become pregnant when compared with sibling controls (Green et al. 2009). No adverse pregnancy outcomes were identified, however in a large study conducted within the framework of the CCSS

The aim of our study was: to establish the incidence of hypogonadism and the risk factors for its developement in childhood-cancer survivors in Slovenia and define the highest

In Slovenia, 1474 children were treated for cancer under the age of 16 years from 1.1.1965 to 31.12.1995 at the University Clinical Hospital Ljubljana and/or Institute of Oncology Ljubljana. At the time of our study 712 patients were alive, 460 of them were more than 16 years of age and were at least 3 years off treatment. Of those patients 390 were regularly followed at the outpatient clinic at the Institute of Oncology in Ljubljana (Jereb 2000, Zaletel 2004). We included in our study 297 consecutive patients in whom endocrinological evaluation was performed until 1.1.2003. Ninety-three patients refused examinations. There were 115 females and 182 males. They were 0-16 (median 9 yrs) years of age at the diagnosis of malignancy and had endocrinological evaluation 3-32 (median 11,5) years after the end of treatment at age of 14-42 (median 20) years. All pts were pubertal or postpubertal when studied. Distribution of diagnosis among patients included in our study is shown in table 1. The majority (90%) of patients had combined treatment including 2-3 modalities, surgery (S), ChT, RT, a quarter of them had all 3 modalities, while 41% had combined ChT and RT. To evaluate the risk factors for hypogonadism after treatment for childhood cancer, we used

(Green et al. 2002).

**2.1 Patients** 

respective the lowest risk groups.

a multivariate analysis method of the classification trees.

**2. Patients and methods** 

#### **2.2.1 Assessment of gonadal function**

The patient´s data regarding diagnosis and treatment were collected from medical files, information concerning quality of life including attained educational level, marital status, employment and social life, past and present menstrual histories, the course of puberty and fertility histories was ascertained by interview. General physical examination was performed in terms of recording height, weight, clinical abnormalities and Tanner stages of pubic hair and genital development were recorded. Each patient´s blood samples were analysed for basal concentrations of testosterone (RIA, IMUNOTECH), estradiol (DELFIA-LKB) and prolactin (DELFIA-LKB). Concentrations of LH (DELFIA-LKB) and FSH (DELFIA-LKB) were determined before and 10, 20, 30, 60 minutes after i.v. administration of gonadotropin releasing hormone (50 mcg/m2) (LH-RH). Primary hypogonadism (PH) was defined as basal serum FSH and/or LH level above the normal upper limit and exaggerated response after stimulation with LH-RH. In men, elevated basal serum FSH levels indicated germinal epithelium damage (GE-DA), while elevated LH levels (with/without reduced testosterone levels) indicated Leydig cells (LC) damage (LC-DA). Normal basal values of LH and/or FSH and exaggerated response after LH-RH stimulation were considered as subclinical impairment (SIG). Exaggerated response of FSH after LH-RH was considered as dysfunction of germinal epithelium (GE-dys), while exaggerated response of LH after LH-RH was considered as dysfunction of LC (LC-dys). PH and SIG together were named gonadal impairment (GI). Low serum basal FSH and LH levels with poor response after i.v. bolus of LH-RH was considered as secondary hypogonadism.

Hypogonadism After Childhood Cancer Treatment 169

In the second group of independent variables, variables from the first group were further broken down (type of surgery, parts of the body, which was irradiated, type of ChT), because we wanted to determine the effect of various treatments on the gonadal function. The observation time, i.e. the time from the end of treatment to the gonadal evaluation, was

 surgery (no, outside the abdomen, abdominal surgery, orchidectomy, ovariectomy), radiotherapy (no, brain RT, RT above the diaphragm except the brain, RT of the upper

We analyzed the influence of both groups of independent variables to each of the two dependent of PH and GI (i.e., PH and/or SIG) in three different groups of patients: all patients, females and males (impairment of LC, LC-DA and LC-dys as well as impairment of

Primary hypogonadism was found in 76 (26%) adolescents, in 62 (34%) males and 14 (12%) females. Gonadal impairment was found in 114 (38%) adolescents, in 89 (49%) males and 25

n (%) All (n) Pts with PH

n (%) All (n) Pts with PH

n (%)

Diagnosis Males Females All

Leukaemia 30 5 (16.5) 37 1 (2.7) 67 6 (9) Hodgkin's disease 40 26 (65) 24 6 (25) 64 32 (50) Brain tumor 30 3(10) 18 2 (11) 48 5 (10) NHL 35 12 (34) 3 0 38 12 (32) Soft tissue sarcoma 16 7 (44) 8 1 (12.5) 24 8 (33) Wilms' tumor 11 2 (18) 7 0 18 2 (11) Bone sarcoma 6 2 (33) 6 1 (16.5) 12 3 (25) Germ cell tumor 4 2 (50) 6 3 (50) 10 5 (50) Neuroblastoma 4 2 (50) 1 0 5 2 (40) Retinoblastoma 2 1 (50) 3 0 5 1 (20)

nasopharynx 2 0 1 0 3 0 Others ♣ 2 0 1 0 3 0 All 182 62 (34) 115 14 (22) 297 76 (26) ♣ retroperitoneal paraganglioma, hepatoblastoma, invasive adenoma of suprarenal gland, one each

Table 2. Primary hypogonadism versus diagnosis and gender in 297 patients.

All (n) Pts with PH

added as a new variable. Variables of the second groups were:

chemotherapy (no, ChT without AA, ChT with AA),

 gender (female, male) age at diagnosis (in years),

observation time.

(22%) females (Table 2).

**3. Results** 

Carcinoma of

PH - primary hypogonadism

type of malignancy (1-12, see Table 1),

abdomen, pelvic RT, testicular RT)

germinal epithelium, GE-DA and GE-dys).

#### **2.2.2 Classification tree analysis**

Classification tree analysis is a multivariate analysis method that allows for studying of simultaneous influence of a series of independent variables on a single dependent variable (Jereb 1973). The output of the analysis is a classification tree, read from the root node, through the internal nodes all the way to the leaves. In each internal node, a test on the value of a single independent variable for the given case is being performed. Based on the outcome of the test, we follow one of the branches originating from the node. Following the branches in that manner, we arrive in one of the leaf nodes of the tree that provides a classification, i.e., the predicted value of the dependent variable, of the case at hand. In addition to predicting the value of the dependent variable for a given case, the structure of the classification tree also reveals the influence and relative importance of the values of independent variables on the dependent one.

Classification tree is being constructed by successive divisions of the original group of cases into pairs of subgroups, where each division is based on the value of a single independent variable. For each division (often referred to as a split), the variable is being selected that produces "pure" subgroups; the purity being measured as a fraction of cases with the same value of the dependent variable. In ideal case, a completely "pure" group of cases that share the same value of the dependent variable is obtained. Each of the subgroups generated in the process becomes a parent group in the next step of the analysis and is further divided in the same way. The division of cases stops when the group of cases is completely pure or when it contains less than a user-defined minimal number of cases. In our study, the C4.5 (Quinlan 1993) program for constructing classification trees was used. C4.5 allows the setting of several parameters that influence branching and quality of the final classification tree: most notably there is one parameter that determines the smallest number of cases to be included in a single group, and another parameter that determines the degree of the tree post-pruning performed. For details please refer to the description in (Quinlan 1993). The optimal values of these parameters were determined using a standard cross-validation method (Jazbec et al. 2007, Macedoni-Lukšič et al. 2003, Velensek et al. 2008). The usual performance measure for classification trees is the accuracy of the tree when predicting the outcome (the value of the dependent variable) on samples not seen during the process of tree building.

Note finally, that since we use an alternative performance criterion, the classification tree obtained the cross-validation procedure outlined above is not expected to provide accurate classification of cases into hypogonadism and non-hypogonadism classes. Instead of using the tree as an accurate predictor, we were interested in analyzing the tree structure and identifying the risk group where incidence of hypogonadism is significantly higher than the one observed in the population of 297 patients included in the study. Multivariate analysis with classification tree was not done when specific abnormalities were found in less than ten percent of examinated childhood cancer survivors.

Multivariate statistical analysis with classification tree analysis was performed with two groups of independent variables and their values. The first group included six independent variables:


In the second group of independent variables, variables from the first group were further broken down (type of surgery, parts of the body, which was irradiated, type of ChT), because we wanted to determine the effect of various treatments on the gonadal function. The observation time, i.e. the time from the end of treatment to the gonadal evaluation, was added as a new variable. Variables of the second groups were:

gender (female, male)

168 Sex Hormones

Classification tree analysis is a multivariate analysis method that allows for studying of simultaneous influence of a series of independent variables on a single dependent variable (Jereb 1973). The output of the analysis is a classification tree, read from the root node, through the internal nodes all the way to the leaves. In each internal node, a test on the value of a single independent variable for the given case is being performed. Based on the outcome of the test, we follow one of the branches originating from the node. Following the branches in that manner, we arrive in one of the leaf nodes of the tree that provides a classification, i.e., the predicted value of the dependent variable, of the case at hand. In addition to predicting the value of the dependent variable for a given case, the structure of the classification tree also reveals the influence and relative importance of the values of

Classification tree is being constructed by successive divisions of the original group of cases into pairs of subgroups, where each division is based on the value of a single independent variable. For each division (often referred to as a split), the variable is being selected that produces "pure" subgroups; the purity being measured as a fraction of cases with the same value of the dependent variable. In ideal case, a completely "pure" group of cases that share the same value of the dependent variable is obtained. Each of the subgroups generated in the process becomes a parent group in the next step of the analysis and is further divided in the same way. The division of cases stops when the group of cases is completely pure or when it contains less than a user-defined minimal number of cases. In our study, the C4.5 (Quinlan 1993) program for constructing classification trees was used. C4.5 allows the setting of several parameters that influence branching and quality of the final classification tree: most notably there is one parameter that determines the smallest number of cases to be included in a single group, and another parameter that determines the degree of the tree post-pruning performed. For details please refer to the description in (Quinlan 1993). The optimal values of these parameters were determined using a standard cross-validation method (Jazbec et al. 2007, Macedoni-Lukšič et al. 2003, Velensek et al. 2008). The usual performance measure for classification trees is the accuracy of the tree when predicting the outcome (the value of the

dependent variable) on samples not seen during the process of tree building.

percent of examinated childhood cancer survivors.

type of malignancy (1-12, see Table 1),

variables:

 gender (female, male) age at diagnosis (in years),

 surgery (yes, no) radiotherapy (yes, no) chemotherapy (yes, no).

Note finally, that since we use an alternative performance criterion, the classification tree obtained the cross-validation procedure outlined above is not expected to provide accurate classification of cases into hypogonadism and non-hypogonadism classes. Instead of using the tree as an accurate predictor, we were interested in analyzing the tree structure and identifying the risk group where incidence of hypogonadism is significantly higher than the one observed in the population of 297 patients included in the study. Multivariate analysis with classification tree was not done when specific abnormalities were found in less than ten

Multivariate statistical analysis with classification tree analysis was performed with two groups of independent variables and their values. The first group included six independent

**2.2.2 Classification tree analysis** 

independent variables on the dependent one.


We analyzed the influence of both groups of independent variables to each of the two dependent of PH and GI (i.e., PH and/or SIG) in three different groups of patients: all patients, females and males (impairment of LC, LC-DA and LC-dys as well as impairment of germinal epithelium, GE-DA and GE-dys).

#### **3. Results**

Primary hypogonadism was found in 76 (26%) adolescents, in 62 (34%) males and 14 (12%) females. Gonadal impairment was found in 114 (38%) adolescents, in 89 (49%) males and 25 (22%) females (Table 2).


♣ retroperitoneal paraganglioma, hepatoblastoma, invasive adenoma of suprarenal gland, one each PH - primary hypogonadism

Table 2. Primary hypogonadism versus diagnosis and gender in 297 patients.

Hypogonadism After Childhood Cancer Treatment 171

most important factor which divided a group of adolescents, treated for HD, was gender, and the next still important independant variable was therapy with ChT. Other independent variables from the first group didn't emerged as important risk factors for PH. Therefore, with this analysis we defined a group of 33 (11% of all) patients with the highest (72%) risk of PH; these are men treated for HD with ChT. Two hundred thirty-three patienst, who did not have HD, had low (19%) risk of PH. Low risk (25%) of PH was found also in the group

PH – primary hypogonadism, ChT – chemotherapy, HD – Hodgkin's disease

dependent variable in 297 patients

Fig. 1. Classification tree analysis with first group of independent variables and PH as

treated for HD without ChT, only two had PH. Both were treated with pelvic RT.

Statistical significance of this analysis was borderline (p = 0,068). We performed the analysis with the same independent variables only for males and it confirmed the results of previous analysis with statistically significance p = 0.045 (Figure 2). Namely, once again in the group of patients with the highest risk of PH (72%) were those treated for HD with ChT. Patients treated for other types of cancer, had a risk of PH of only 25%. Of the seven male patients

of female patients, treated for HD.

All but one male subjects with PH had damage of germinal epithelium (15 of them at the same time damage of LC, 30 of them at the same time dysfunction of LC), with one failure, we found LC and DKE. In 12 of the 61 patients with germinal epithelium damage semen analyses was performed; in 11 patients azoospermia was found, one had normal spermiogram. Dysfunction of LC was detected in 54 patients (in 21 patients the only finding), dysfunction of germinal epithelium was found in 9 patients. All 14 female patients with PH had elevated basal FSH and FSH after stimulation, 5 of them had elevated basal LH and LH after stimulation, four had increased LH after stimulation, 6 had decreased levels of estradiol. Among 4 patients with PH who were treated in prepubertal period, two had delayed puberty. Ovarian dysfunction was detected in 11 patients, all had elevated levels of LH after stimulation, 6 had elevated FSH after stimulation as well. All had normal serum estradiol. The highest incidence (50%) of PH was found in those patients treated for Hodgkin's disease (HD) or germ cell tumor (GCT), the lowest incidence (10%) was found in those treated for brain tumors, leukemia and Wilms' tumor (Table 2). The incidence of PH depended on type of treatment as well. The highest proportion of PH (26 - 40%) was found in patients treated with combined treatment, t.i. ChT and RT with / without surgery (Table 3).


PH - primary hypogonadism, RT - radiotherapy, ChT- chemotherapy, S – surgery

Table 3. Primary hypogonadism versus type of treatment in 297 patients.

Secondary hypogonadism was found in 6 patients. Three of them had panhypopituitarism after treatment of hypothalamic tumor (2 patients) or orbital tumor (one patient) with surgery and RT (from 4400 to 5000 cGy), 2 patients were treated for brain tumors with surgery and RT (5500 or. 6500 cGy), 1 patient was treated for leukemia with ChT and brain RT (3000 cGy).

Sixty-seven patients had whole brain irradiation with a dose of 1200 - 4000 (median 2400) cGy in prepubertal period. Six patients had precocious puberty; 5 girls after treatment of leukemia and 1 boy after treatment of NHL. Those 6 patients were treated with ChT and brain RT at the age of 5 to 8 years. In all 6 the dose of ionizing radiation to the brain was equal to or greater than 2400 cGy (2400 - 3400, med. 2400 cGy).

#### **3.1 Results of classification tree analysis 3.1.1 First group of independent variables**

#### **3.1.1.1 Dependent variable – primary hypogonadism, all patients**

PH was found in 76 (26%) of 297 patients. The most important risk factor for PH, which divided the basic group into two subgroups, was diagnosis of HD (Figure 1). The second

All but one male subjects with PH had damage of germinal epithelium (15 of them at the same time damage of LC, 30 of them at the same time dysfunction of LC), with one failure, we found LC and DKE. In 12 of the 61 patients with germinal epithelium damage semen analyses was performed; in 11 patients azoospermia was found, one had normal spermiogram. Dysfunction of LC was detected in 54 patients (in 21 patients the only finding), dysfunction of germinal epithelium was found in 9 patients. All 14 female patients with PH had elevated basal FSH and FSH after stimulation, 5 of them had elevated basal LH and LH after stimulation, four had increased LH after stimulation, 6 had decreased levels of estradiol. Among 4 patients with PH who were treated in prepubertal period, two had delayed puberty. Ovarian dysfunction was detected in 11 patients, all had elevated levels of LH after stimulation, 6 had elevated FSH after stimulation as well. All had normal serum estradiol. The highest incidence (50%) of PH was found in those patients treated for Hodgkin's disease (HD) or germ cell tumor (GCT), the lowest incidence (10%) was found in those treated for brain tumors, leukemia and Wilms' tumor (Table 2). The incidence of PH depended on type of treatment as well. The highest proportion of PH (26 - 40%) was found in patients treated with

Type of treatment All Pts with PH n (%)

Secondary hypogonadism was found in 6 patients. Three of them had panhypopituitarism after treatment of hypothalamic tumor (2 patients) or orbital tumor (one patient) with surgery and RT (from 4400 to 5000 cGy), 2 patients were treated for brain tumors with surgery and RT (5500 or. 6500 cGy), 1 patient was treated for leukemia with ChT and brain RT (3000 cGy). Sixty-seven patients had whole brain irradiation with a dose of 1200 - 4000 (median 2400) cGy in prepubertal period. Six patients had precocious puberty; 5 girls after treatment of leukemia and 1 boy after treatment of NHL. Those 6 patients were treated with ChT and brain RT at the age of 5 to 8 years. In all 6 the dose of ionizing radiation to the brain was

PH was found in 76 (26%) of 297 patients. The most important risk factor for PH, which divided the basic group into two subgroups, was diagnosis of HD (Figure 1). The second

RT + ChT 121 31 (26) S + RT+ ChT 76 31 (40.5) OP + RT 38 4 (10.5) OP + ChT 31 5 (16) ChT 14 2 (14) S 9 0 RT 8 2 (22) All 297 76 (26)

combined treatment, t.i. ChT and RT with / without surgery (Table 3).

PH - primary hypogonadism, RT - radiotherapy, ChT- chemotherapy, S – surgery Table 3. Primary hypogonadism versus type of treatment in 297 patients.

equal to or greater than 2400 cGy (2400 - 3400, med. 2400 cGy).

**3.1.1.1 Dependent variable – primary hypogonadism, all patients** 

**3.1 Results of classification tree analysis 3.1.1 First group of independent variables**  most important factor which divided a group of adolescents, treated for HD, was gender, and the next still important independant variable was therapy with ChT. Other independent variables from the first group didn't emerged as important risk factors for PH. Therefore, with this analysis we defined a group of 33 (11% of all) patients with the highest (72%) risk of PH; these are men treated for HD with ChT. Two hundred thirty-three patienst, who did not have HD, had low (19%) risk of PH. Low risk (25%) of PH was found also in the group of female patients, treated for HD.

PH – primary hypogonadism, ChT – chemotherapy, HD – Hodgkin's disease

Fig. 1. Classification tree analysis with first group of independent variables and PH as dependent variable in 297 patients

Statistical significance of this analysis was borderline (p = 0,068). We performed the analysis with the same independent variables only for males and it confirmed the results of previous analysis with statistically significance p = 0.045 (Figure 2). Namely, once again in the group of patients with the highest risk of PH (72%) were those treated for HD with ChT. Patients treated for other types of cancer, had a risk of PH of only 25%. Of the seven male patients treated for HD without ChT, only two had PH. Both were treated with pelvic RT.

Hypogonadism After Childhood Cancer Treatment 173

GI – gonadal impairment, GCT – germ cell tumor, L – leukemia, ChT – chemotherapy, RT –

Fig. 3. Classification tree analysis with first group of independent variables and GI as

We looked more in detail at the group of 29 male patient, after the last division in the classification tree analysis, the risk factor being diagnosis: leukemia. Less than half of them received ChT with AA, two of them had testicular RT, none had pelvic RT. In 5 patients PH was diagnosed; 2 after testicular RT, 3 after ChT with AA - 2 received the highest cumulative dose of CY and cytarabine in their group (7 g/m2 and 9.5 g/m2). Two patients had subclinical gonadal impairment (SIG) after ChT with AA and/or cytarabine. On the contrary, in the other group of 92 patients, treated for other malignancies, with the highest proportion of GI, as many as 90% received ChT with AA and a quarter of them had pelvic RT. Thirty-one of 46 patients with PH received ChT with AA, 14 ChT with AA and pelvic RT (1500 to 4000 cGy), one was treated with ChT without AA and RT to the whole abdomen (1400 cGy). Fourteen out of 17 patients with SIG received ChT with AA (one pelvic RT as well), 3 patients received ChT without AA, but had RT of the whole abdomen. Therefore the most significant risk factors for GI in our patients were beside the diagnosis of GCT male gender and therapy with ChT and RT. In the group with the highest risk of GI among risk factors has stood out ChT with AA and pelvic RT (and gonadal surgery and ChT with AA in the group of patients with GCT). In the group with low risk of GI mainly ChT with AA and

radiotherapy

dependent variable in 297 patients

testicular RT emerged as risk factors.

PH – primary hypogonadism, ChT – chemotherapy, HD – Hodgkin's disease

Fig. 2. Classification tree analysis with first group of independent variables and PH as dependent variable in 182 male patients

#### **3.1.1.2 Dependent variable – gonadal impairment (GI, gonadal damage and subclinical impairment) in all patients**

GI was found in 114 (38%) adolescents. Independent variable type of diagnosis, germ cell tumor, turned out as the most important risk factor for GI. Namely, 9 out of 10 patients treated for GCT had GI. In the remaining 287 patients the most significant risk factor for GI stood gender (Fig. 3). Males had 48% and females had 18% risk of GI. The group of male patiens further divided by important risk factors for GI: ChT, RT and diagnosis of cancer other than leukemia. With this analysis, we therefore defined the group of patients at highest, 68%, risk of GI: 92 male patients who were treated with ChT and RT for cancer other than leukemia. The lowest, 18%, risk of GI, had two groups of patients; group of females (excluding GCT) and group of 34 male patients who did not receive ChT.

In this multivariate analysis the highest (90%) risk of GI had a group od 10 patients (4 males, 6 females) treated for GCT; 5 patients had PH, 3 females after bilateral ovariectomy abdominal RT (one) and 2 males, one after unilateral orchidectomy and ChT with AA for testicular GCT, and the second after ChT with AA for mediastinal GCT. Four patients (2 females and 2 males) had SIG – all having been treated by unilateral removal of the ovary or testis and ChT with AA. The only patient of this group with normal function of the gonads was a female treated for GCT by unilateral ovariectomy and ChT (including bleomycin, etoposide, cisplatin and ifosfamide) at the age 13. Cumulative doses of AA were comparable to those received by the girls with GI following unilateral ovariectomy (ages 9 and 14 years). So, in this group of patients at high risk of GI gonadal surgery and ChT with AA seems to be important risk factors for gonadal impairment.

PH – primary hypogonadism, ChT – chemotherapy, HD – Hodgkin's disease

dependent variable in 182 male patients

important risk factors for gonadal impairment.

**impairment) in all patients** 

Fig. 2. Classification tree analysis with first group of independent variables and PH as

females (excluding GCT) and group of 34 male patients who did not receive ChT.

**3.1.1.2 Dependent variable – gonadal impairment (GI, gonadal damage and subclinical** 

GI was found in 114 (38%) adolescents. Independent variable type of diagnosis, germ cell tumor, turned out as the most important risk factor for GI. Namely, 9 out of 10 patients treated for GCT had GI. In the remaining 287 patients the most significant risk factor for GI stood gender (Fig. 3). Males had 48% and females had 18% risk of GI. The group of male patiens further divided by important risk factors for GI: ChT, RT and diagnosis of cancer other than leukemia. With this analysis, we therefore defined the group of patients at highest, 68%, risk of GI: 92 male patients who were treated with ChT and RT for cancer other than leukemia. The lowest, 18%, risk of GI, had two groups of patients; group of

In this multivariate analysis the highest (90%) risk of GI had a group od 10 patients (4 males, 6 females) treated for GCT; 5 patients had PH, 3 females after bilateral ovariectomy abdominal RT (one) and 2 males, one after unilateral orchidectomy and ChT with AA for testicular GCT, and the second after ChT with AA for mediastinal GCT. Four patients (2 females and 2 males) had SIG – all having been treated by unilateral removal of the ovary or testis and ChT with AA. The only patient of this group with normal function of the gonads was a female treated for GCT by unilateral ovariectomy and ChT (including bleomycin, etoposide, cisplatin and ifosfamide) at the age 13. Cumulative doses of AA were comparable to those received by the girls with GI following unilateral ovariectomy (ages 9 and 14 years). So, in this group of patients at high risk of GI gonadal surgery and ChT with AA seems to be

GI – gonadal impairment, GCT – germ cell tumor, L – leukemia, ChT – chemotherapy, RT – radiotherapy

Fig. 3. Classification tree analysis with first group of independent variables and GI as dependent variable in 297 patients

We looked more in detail at the group of 29 male patient, after the last division in the classification tree analysis, the risk factor being diagnosis: leukemia. Less than half of them received ChT with AA, two of them had testicular RT, none had pelvic RT. In 5 patients PH was diagnosed; 2 after testicular RT, 3 after ChT with AA - 2 received the highest cumulative dose of CY and cytarabine in their group (7 g/m2 and 9.5 g/m2). Two patients had subclinical gonadal impairment (SIG) after ChT with AA and/or cytarabine. On the contrary, in the other group of 92 patients, treated for other malignancies, with the highest proportion of GI, as many as 90% received ChT with AA and a quarter of them had pelvic RT. Thirty-one of 46 patients with PH received ChT with AA, 14 ChT with AA and pelvic RT (1500 to 4000 cGy), one was treated with ChT without AA and RT to the whole abdomen (1400 cGy). Fourteen out of 17 patients with SIG received ChT with AA (one pelvic RT as well), 3 patients received ChT without AA, but had RT of the whole abdomen. Therefore the most significant risk factors for GI in our patients were beside the diagnosis of GCT male gender and therapy with ChT and RT. In the group with the highest risk of GI among risk factors has stood out ChT with AA and pelvic RT (and gonadal surgery and ChT with AA in the group of patients with GCT). In the group with low risk of GI mainly ChT with AA and testicular RT emerged as risk factors.

Hypogonadism After Childhood Cancer Treatment 175

Fig. 5. Classification tree analysis with first group of independent variables and GI as

Again, all patients were included in the analysis.. The most important risk factor for PH turned out to be pelvic RT (Fig. 6). Risk for PH in patients, treated with pelvic RT, was 55% and 19% only in the other group. The second most important factor which divided the group of 51 patients, who had pelvic RT, was type of diagnosis, and the third one treatment with ChT. We defined a group of 29 patients with the highest, 79%, risk of PH, namely those who had pelvic RT, didn't have diagnosis of brain tumor and were treated with ChT including AA or did not receive ChT at all. In the group of 246 patients who didn't have pelvic RT, ChT with AA emerged as the most important risk factor for PH, followed by male gender and diagnosis of HD. Similar to the analysis with the first group of independent variables (Fig.1) we identified a group of 24 patients with the highest risk of PH (66%) among those patients who didn't have pelvic RT; t.i. males treated for HD with ChT including AA. We defined a group of patients with the lowest, 1%, risk of PH. Those were 87 patients who had neither pelvic RT nor ChT with AA (PH was found in one patient only after being treated with testicular RT). Low risk of PH (7%) had as well a group of 56 females who received ChT with AA, but didn't have pelvic RT. Males treated with ChT with AA but without pelvic RT, had much higher risk of PH (42%), suggesting that ChT with AA presented greater risk

The highest risk for PH (79%) had the group of patients treated for cancer other than brain tumor, with pelvic RT and ChT with AA (23 patients) or without ChT (6 patients). Only 6 patients in this group didn't develop PH; 2 females treated with unilateral RT to iliacoinguinal region (2400 cGy) and ChT with AA for HD (one female with 2 relapses 6 cycles of LOPP (chlorambucil, vincristine, procarbazine and prednisone), 6 cycles of MOPP-ABV and 6 cycles of ABVD). Among 4 males without PH one received RT to both iliacal regions (3000 cGy) and 2 cycles of MOPP ChT, one received RT to the left femur and iliac bone (4800 cGy) for hondrosarcoma of the iliac bone, and 2 were treated for NHL of the caecum with

**3.1.2.1 Dependent variable – primary hypogonadism in all patients** 

GI – gonadal impairment, GCT-germ cell tumor

dependent variable in 115 female patients

factor of PH in males than in females.

**3.1.2 Second group of independent variables** 

#### **3.1.1.3 Dependent variable – gonadal impairment (GI) in male patients**

The analysis confirmed the results of the previous analysis. The largest, 68%, risk of GI, had a group of men, who were treated for cancer other than leukemia with ChT and RT (Fig. 4).

GI – gonadal impairment, L – leukemia, ChT – chemotherapy, RT – radiotherapy

Fig. 4. Classification tree analysis with first group of independent variables and GI as dependent variable in 182 male patients

We looked more in detail at the group of 34 males who did not receive ChT. Six patients had GI. Three of them had PH, all after pelvic RT (3000 - 4000 cGy). Three patients had SIG after being treated for brain tumors, 2 with surgery only, one with brain RT. Of the remaining 28 patients of this group (with normal gonadal function six had pelvic RT (2800 to 4800 cGy). Therefore, in the group of patients with low risk of GI (without therapy with ChT) mainly pelvic RT emerged as risk factor.

#### **3.1.1.4 Dependent variable – gonadal impairment (GI) in female patients**

Classification tree analysis with first group of independent variables and GI as dependent variable was performed for the cohort of 115 female patients as well. It had only one division, the only risk factor being diagnosis GCT. Six patients treated for GCT were at high risk (83%) for GI. The other group of 109 patients was not further divided (Fig. 5). This tree was not significantly different from random predictions, probably because of a very small number of positive outcomes and some other independent variables relevant for ovarian failure, which are not yet known.

The analysis confirmed the results of the previous analysis. The largest, 68%, risk of GI, had a group of men, who were treated for cancer other than leukemia with ChT and RT (Fig. 4).

**3.1.1.3 Dependent variable – gonadal impairment (GI) in male patients** 

GI – gonadal impairment, L – leukemia, ChT – chemotherapy, RT – radiotherapy

**3.1.1.4 Dependent variable – gonadal impairment (GI) in female patients** 

dependent variable in 182 male patients

pelvic RT emerged as risk factor.

failure, which are not yet known.

Fig. 4. Classification tree analysis with first group of independent variables and GI as

We looked more in detail at the group of 34 males who did not receive ChT. Six patients had GI. Three of them had PH, all after pelvic RT (3000 - 4000 cGy). Three patients had SIG after being treated for brain tumors, 2 with surgery only, one with brain RT. Of the remaining 28 patients of this group (with normal gonadal function six had pelvic RT (2800 to 4800 cGy). Therefore, in the group of patients with low risk of GI (without therapy with ChT) mainly

Classification tree analysis with first group of independent variables and GI as dependent variable was performed for the cohort of 115 female patients as well. It had only one division, the only risk factor being diagnosis GCT. Six patients treated for GCT were at high risk (83%) for GI. The other group of 109 patients was not further divided (Fig. 5). This tree was not significantly different from random predictions, probably because of a very small number of positive outcomes and some other independent variables relevant for ovarian

GI – gonadal impairment, GCT-germ cell tumor

Fig. 5. Classification tree analysis with first group of independent variables and GI as dependent variable in 115 female patients

#### **3.1.2 Second group of independent variables**

#### **3.1.2.1 Dependent variable – primary hypogonadism in all patients**

Again, all patients were included in the analysis.. The most important risk factor for PH turned out to be pelvic RT (Fig. 6). Risk for PH in patients, treated with pelvic RT, was 55% and 19% only in the other group. The second most important factor which divided the group of 51 patients, who had pelvic RT, was type of diagnosis, and the third one treatment with ChT.

We defined a group of 29 patients with the highest, 79%, risk of PH, namely those who had pelvic RT, didn't have diagnosis of brain tumor and were treated with ChT including AA or did not receive ChT at all. In the group of 246 patients who didn't have pelvic RT, ChT with AA emerged as the most important risk factor for PH, followed by male gender and diagnosis of HD. Similar to the analysis with the first group of independent variables (Fig.1) we identified a group of 24 patients with the highest risk of PH (66%) among those patients who didn't have pelvic RT; t.i. males treated for HD with ChT including AA. We defined a group of patients with the lowest, 1%, risk of PH. Those were 87 patients who had neither pelvic RT nor ChT with AA (PH was found in one patient only after being treated with testicular RT). Low risk of PH (7%) had as well a group of 56 females who received ChT with AA, but didn't have pelvic RT. Males treated with ChT with AA but without pelvic RT, had much higher risk of PH (42%), suggesting that ChT with AA presented greater risk factor of PH in males than in females.

The highest risk for PH (79%) had the group of patients treated for cancer other than brain tumor, with pelvic RT and ChT with AA (23 patients) or without ChT (6 patients). Only 6 patients in this group didn't develop PH; 2 females treated with unilateral RT to iliacoinguinal region (2400 cGy) and ChT with AA for HD (one female with 2 relapses 6 cycles of LOPP (chlorambucil, vincristine, procarbazine and prednisone), 6 cycles of MOPP-ABV and 6 cycles of ABVD). Among 4 males without PH one received RT to both iliacal regions (3000 cGy) and 2 cycles of MOPP ChT, one received RT to the left femur and iliac bone (4800 cGy) for hondrosarcoma of the iliac bone, and 2 were treated for NHL of the caecum with

Hypogonadism After Childhood Cancer Treatment 177

10%. With this statistical analysis we found that the significant risk factors for PH were pelvic RT, ChT with AA, male gender and diagnosis of HD. In the group with a lower risk

Gonadal impairment was found in 114 (38%) adolescents. The most significant risk factor for GI was RT (Fig. 7). The group of 100 patients who had brain RT only had the lowest (15%) risk of GI, in the group of the remaining 197 patients, who were irradiated to any other part of the body or had no RT, the risk of GI was 50%. Similar to the analysis of the first set of variables male patients treated with ChT and RT stood out as a group with the highest (75%) risk of GI.

GI – gonadal impairment, AA – alkylating agents, GCT – germ cell tumor, ChT – chemotherapy, RT –

Fig. 7. Classification tree analysis with second group of independent variables and GI as

of PH craniospinal RT and ChT with AA emerged as important risk factors for PH. **3.1.2.2 Dependent variable – gonadal impairment (GI) (t.i. gonadal damage and** 

**subclinical impairment)** 

radiotherapy

dependent variable in 297 patients

PH – primary hypogonadism, AA – alkylating agents, ChT – chemotherapy, RT – radiotherapy, HD – Hodgkin's disease

Fig. 6. Classification tree analysis with second group of independent variables and PH as dependent variable in 297 patients

surgery, ChT with AA and RT (one had 900 cGy to the whole central nervous system, the other had abdominal RT with 3000 cGy). Of the 6 patients in the group not receiving ChT with AA, as many as five had PH; 3 of them had whole abdominal RT, 2 RT of bilateral iliacoinguinal regions. Among patients who had pelvic RT, a group of 17 patients with low proportion of PH emerged. They were treated for brain tumor by craniospinal RT (600-4400 cGy), 7 of them also received ChT with AA. All 4 patients with PH were treated for medulloblastoma, 3 of them received, in addition to craniospinal RT, ChT with Procarbazine or CCNU. The risk of PH in patients treated with craniospinal RT and ChT with AA was therefore 43%, while in those treated with craniospinal RT without ChT with AA, was only

PH – primary hypogonadism, AA – alkylating agents, ChT – chemotherapy, RT – radiotherapy, HD –

Fig. 6. Classification tree analysis with second group of independent variables and PH as

surgery, ChT with AA and RT (one had 900 cGy to the whole central nervous system, the other had abdominal RT with 3000 cGy). Of the 6 patients in the group not receiving ChT with AA, as many as five had PH; 3 of them had whole abdominal RT, 2 RT of bilateral iliacoinguinal regions. Among patients who had pelvic RT, a group of 17 patients with low proportion of PH emerged. They were treated for brain tumor by craniospinal RT (600-4400 cGy), 7 of them also received ChT with AA. All 4 patients with PH were treated for medulloblastoma, 3 of them received, in addition to craniospinal RT, ChT with Procarbazine or CCNU. The risk of PH in patients treated with craniospinal RT and ChT with AA was therefore 43%, while in those treated with craniospinal RT without ChT with AA, was only

Hodgkin's disease

dependent variable in 297 patients

10%. With this statistical analysis we found that the significant risk factors for PH were pelvic RT, ChT with AA, male gender and diagnosis of HD. In the group with a lower risk of PH craniospinal RT and ChT with AA emerged as important risk factors for PH.

#### **3.1.2.2 Dependent variable – gonadal impairment (GI) (t.i. gonadal damage and subclinical impairment)**

Gonadal impairment was found in 114 (38%) adolescents. The most significant risk factor for GI was RT (Fig. 7). The group of 100 patients who had brain RT only had the lowest (15%) risk of GI, in the group of the remaining 197 patients, who were irradiated to any other part of the body or had no RT, the risk of GI was 50%. Similar to the analysis of the first set of variables male patients treated with ChT and RT stood out as a group with the highest (75%) risk of GI.

GI – gonadal impairment, AA – alkylating agents, GCT – germ cell tumor, ChT – chemotherapy, RT – radiotherapy

Fig. 7. Classification tree analysis with second group of independent variables and GI as dependent variable in 297 patients

Hypogonadism After Childhood Cancer Treatment 179

GEI - impairment of germinal epithelium, AA – alkylating agents, ChT – chemotherapy, RT –

Fig. 8. Classification tree analysis with second group of independent variables and GEI (impairment of germinal epithelium) as dependent variable in 182 male patients

**3.1.2.4 Dependent variable - impairment of Leydig cells (LCI), t.i. damage of LC (LC-DA)** 

In this analysis as the most important risk factor for LCI stood out treatment with ChT (Fig. 9). All but three patients with LCI received ChT. In the group of 148 patients who received ChT, 67 patients had LCI; all but the 6 of them received ChT with AA (13 of them also had pelvic RT, one testicular RT). Five of the 6 patients not receiving ChT with AA had

The highest, 83%, risk of LCI was in the group of patients, who received ChT and had orchidectomy. Similar to the above analysis (GI in all patients), we defined a group of 79 patients with high risk for LCI (61%); namely patients treated with ChT and RT of other regions than brain. Eleven patients in this group had both, damage of GE and damage of LC (2 after testicular RT, 3 after ChT with AA and pelvic RT, 6 after the ChT), 37 patients had dysfunction of LC; 24 of them with the damage of GE as well (15 after ChT with AA, 8 after ChT with AA and pelvic RT, one after pelvic RT), 13 patients had an isolated finding (10

In the group of 22 patients treated with ChT, but without RT (with 23% risk of LCI), all 5 patients with LCI had only dysfunction of LC and all received ChT with AA. In the group of 34 patients with the lowest, 9%, risk of LCI, patients didn't receive ChT; only 3 patients had

radiotherapy, HD – Hodgkin's disease

abdominal RT.

**and dysfunction of LC (LC-dys) in male patients** 

after ChT with AA, 3 following abdominal RT).

LCI (one patient following abdominal RT, 2 patients after brain RT).

We looked more in detail at the group of 100 (63 males, 37 females) patients with the lowest risk of GI who had only brain RT; 69% of them were treated for ALL, 27% for brain tumors, 9% for NHL, 4% for soft tissue sarcoma. Only 45% of this group of patients received ChT with AA. All 7 males who had PH, received ChT with AA, as did also 6 of 8 patients (3 females, 5 males) with SIG. In the second division of decision tree the subgroup of male patients had 61% risk of GI and the subgroup of females only 31%, although a similar proportion of patients in both subgroups received ChT with AA (72%: 75%) or did not receive ChT (28%: 25%) and the same (27%) proportion of patients had pelvic RT.

Therefore, in this statistical analysis the most important risk factors for GI turned out to be: male gender, treatment with ChT with AA and RT and orchidectomy.

#### **3.1.2.3 Dependent variable - impairment of germinal epithelium GEI, t.i. germinal epithelium damage (GE-DA) and germinal epithelium dysfunction (GE-dys) in male patients**

In this analysis as the most important risk factor for GEI emerged ChT with AA (Fig. 8). Only 10% of males who didn't receive ChT with AA had GEI (all 6 patients in this group who had GEI, had pelvic or testicular RT). The second most important factor that divided the group of 121 males who received ChT with AA, was pelvic RT. The risk of GEI in the group of 18 patients who had pelvic RT was as high as 89%. The next most important risk factor, which divided the group of patients who received ChT with AA and didn't have pelvic RT, was diagnosis HD. It is the same result as above when analyzing all patiens (with dependent variable PH), a group of 24 patients treated for HD with ChT with AA but without pelvic RT (71% risk of GEI). In the other group of patients treated for other malignancies than HD were at greater risk of GEI those who had RT above the diaphragm and were at the age of 10 years or less at the time of treatment (83% risk of GEI). In this analysis the most important risk factors for GEI turned out to be: ChT with AA and pelvic RT.

In the group of 18 patients who received ChT with AA and had pelvic RT, only 2 patients had normal gonadal function; one patient received 2 cycles of MOPP and RT of iliac regions with a dose of 3000 cGy for HD, the other had craniospinal RT with 900 cGy and ChT following BFM protocol (including 7 g/ m 2 of CY and 3, 2 g/m2 of cytarabine) for NHL. High, 71% the risk of GEI, had a group of 24 patients with HD treated with ChT with AA without pelvic RT. Seven patients of this group didn't have GEI. They received ChT following protocol LOPP (6 cycles), MOPP (1 to 4 cycles) or OPPA (2 cycles).

At the last division of this tree patients age at treatment emerged as risk factor for GEI. The group of 25 patients who were treated for HD with ChT with AA and RT above the diaphragm, was divided into those who were 10 or less years of age at diagnosis (83% risk of GEI ), and into the group of older patients (31% risk of GEI). The younger patients received a slightly higher cumulative dose of CY (2.8 - 40, med. 10 g/m2) than the group of older patients (1.4 - 16, med. 5 g/m2), doses of cytarabine were approximately equal in both groups. Among patients without GEI in the group of younger patients there was one who received CY 15 g/m2. Taking in account that the two age groups have different cumulative doses of AA, we can not consider patient's age at treatment as an important risk factor for GEI.

Therefore, in this analysis the most important risk factors for damage or dysfunction of germinal epithelium were ChT with AA, pelvic RT, diagnosis HD and orchidectomy.

We looked more in detail at the group of 100 (63 males, 37 females) patients with the lowest risk of GI who had only brain RT; 69% of them were treated for ALL, 27% for brain tumors, 9% for NHL, 4% for soft tissue sarcoma. Only 45% of this group of patients received ChT with AA. All 7 males who had PH, received ChT with AA, as did also 6 of 8 patients (3 females, 5 males) with SIG. In the second division of decision tree the subgroup of male patients had 61% risk of GI and the subgroup of females only 31%, although a similar proportion of patients in both subgroups received ChT with AA (72%: 75%) or did not

Therefore, in this statistical analysis the most important risk factors for GI turned out to be:

In this analysis as the most important risk factor for GEI emerged ChT with AA (Fig. 8). Only 10% of males who didn't receive ChT with AA had GEI (all 6 patients in this group who had GEI, had pelvic or testicular RT). The second most important factor that divided the group of 121 males who received ChT with AA, was pelvic RT. The risk of GEI in the group of 18 patients who had pelvic RT was as high as 89%. The next most important risk factor, which divided the group of patients who received ChT with AA and didn't have pelvic RT, was diagnosis HD. It is the same result as above when analyzing all patiens (with dependent variable PH), a group of 24 patients treated for HD with ChT with AA but without pelvic RT (71% risk of GEI). In the other group of patients treated for other malignancies than HD were at greater risk of GEI those who had RT above the diaphragm and were at the age of 10 years or less at the time of treatment (83% risk of GEI). In this analysis the most important risk factors for GEI turned out to be: ChT with AA and

In the group of 18 patients who received ChT with AA and had pelvic RT, only 2 patients had normal gonadal function; one patient received 2 cycles of MOPP and RT of iliac regions with a dose of 3000 cGy for HD, the other had craniospinal RT with 900 cGy and ChT following BFM protocol (including 7 g/ m 2 of CY and 3, 2 g/m2 of cytarabine) for NHL. High, 71% the risk of GEI, had a group of 24 patients with HD treated with ChT with AA without pelvic RT. Seven patients of this group didn't have GEI. They received ChT

At the last division of this tree patients age at treatment emerged as risk factor for GEI. The group of 25 patients who were treated for HD with ChT with AA and RT above the diaphragm, was divided into those who were 10 or less years of age at diagnosis (83% risk of GEI ), and into the group of older patients (31% risk of GEI). The younger patients received a slightly higher cumulative dose of CY (2.8 - 40, med. 10 g/m2) than the group of older patients (1.4 - 16, med. 5 g/m2), doses of cytarabine were approximately equal in both groups. Among patients without GEI in the group of younger patients there was one who received CY 15 g/m2. Taking in account that the two age groups have different cumulative doses of AA, we can not consider patient's age at treatment as an important

Therefore, in this analysis the most important risk factors for damage or dysfunction of

germinal epithelium were ChT with AA, pelvic RT, diagnosis HD and orchidectomy.

following protocol LOPP (6 cycles), MOPP (1 to 4 cycles) or OPPA (2 cycles).

receive ChT (28%: 25%) and the same (27%) proportion of patients had pelvic RT.

**3.1.2.3 Dependent variable - impairment of germinal epithelium GEI, t.i. germinal epithelium damage (GE-DA) and germinal epithelium dysfunction (GE-dys) in male** 

male gender, treatment with ChT with AA and RT and orchidectomy.

**patients** 

pelvic RT.

risk factor for GEI.

GEI - impairment of germinal epithelium, AA – alkylating agents, ChT – chemotherapy, RT – radiotherapy, HD – Hodgkin's disease

Fig. 8. Classification tree analysis with second group of independent variables and GEI (impairment of germinal epithelium) as dependent variable in 182 male patients

#### **3.1.2.4 Dependent variable - impairment of Leydig cells (LCI), t.i. damage of LC (LC-DA) and dysfunction of LC (LC-dys) in male patients**

In this analysis as the most important risk factor for LCI stood out treatment with ChT (Fig. 9). All but three patients with LCI received ChT. In the group of 148 patients who received ChT, 67 patients had LCI; all but the 6 of them received ChT with AA (13 of them also had pelvic RT, one testicular RT). Five of the 6 patients not receiving ChT with AA had abdominal RT.

The highest, 83%, risk of LCI was in the group of patients, who received ChT and had orchidectomy. Similar to the above analysis (GI in all patients), we defined a group of 79 patients with high risk for LCI (61%); namely patients treated with ChT and RT of other regions than brain. Eleven patients in this group had both, damage of GE and damage of LC (2 after testicular RT, 3 after ChT with AA and pelvic RT, 6 after the ChT), 37 patients had dysfunction of LC; 24 of them with the damage of GE as well (15 after ChT with AA, 8 after ChT with AA and pelvic RT, one after pelvic RT), 13 patients had an isolated finding (10 after ChT with AA, 3 following abdominal RT).

In the group of 22 patients treated with ChT, but without RT (with 23% risk of LCI), all 5 patients with LCI had only dysfunction of LC and all received ChT with AA. In the group of 34 patients with the lowest, 9%, risk of LCI, patients didn't receive ChT; only 3 patients had LCI (one patient following abdominal RT, 2 patients after brain RT).

Hypogonadism After Childhood Cancer Treatment 181

GI in the subgroup of 93 female patients without pelvic RT was as low as 13%. Only 4 out of 12 patients with GI in this group had damage of ovarian function (all after ChT with AA), 8

In the group of 6 females treated for GCT at the age of 9 to 15 (med. 13) years, 5 had GI; 2 female patients had bilateral ovariectomy, one had ovarian damage after unilateral ovariectomy and abdominal RT, two females had dysfunction of ovarian function after

In females, therefore, the most important risk factors for ovarian failure turned out to be

Fig. 10. Classification tree analysis with second group of independent variables and gonadal

Forty-six patients (21 males, 25 females) in our study had no PH, despite treatment with either ChT including AA or pelvic or testicular RT. Thirty-four (19 males, 15 females) of them were treated with ChT, which contained the antimetabolites methotrexate and 6 mercaptopurine, VCR, corticosteroids with or without L-asparaginase and adriamycin. Eleven patients (2 males and 9 females) received ChT containing actinomycin D (AMD) and vincristine (VCR) with or without adriamycin. One girl received ChT with vinblastine. Six patients (3 males and 3 females) out of 46 in this group of patients had subclinical impairment of the gonads (males had dysfunction of LC only) and as many as 4 patients (2 males and 2 females) had ChT with AMD and VCR. The impact of antimetabolite cytarabine on gonadal function could not be assessed because this cytostatic was administered to patients as part of ChT protocols, which contained AA (especially CY). So, we can conclude on the basis of our cohort of patients that ChT with antimetabolites (other

patients had only ovarian dysfunction (4 following ChT with AA).

GI – gonadal impairment, GCT – germ cell tumor, RT - radiotherapy

impairment as dependent variable in 115 female patients

*General observations* 

unilateral removal of the ovary and ChT with AA.

pelvic RT, ChT with AA and ovariectomy.

Therefore, in this analysis orchidectomy and therapy with ChT and RT emerged as the most important risk factors for LCI.

Fig. 9. Classification tree analysis with second group of independent variables and impairment of Leydig cells (LCI) as dependent variable in 182 male patients

#### **3.1.2.5 Dependent variable –gonadal impairment (GI) in female patients, t.i. damage of ovarian function and dysfunction of ovarian function in female patients**

The decision tree with the second group of independent variables (Fig. 10) was more branched than the one using the first group of independent variables (Fig. 5), although this tree was not significantly different from statistical random predictions. Similar to the previous tree the highest risk for GI was observed in the group of females treated for GCT (GI in 5 out of the 6 patients). The remaining group of 109 patients with 18% risk of GI, treated for other malignancies, was further divided by the most important risk factor pelvic RT. In the group of 16 patients treated with pelvic RT risk of GI was 50%. As many as 7 out of the 8 patients with GI had damage of ovarian function, t.i.PH (one following RT of the whole abdomen (2000 cGy) and ChT with AA for leukemia, 2 after craniospinal RT (3400 and 3600 cGy) and ChT with AA (only one) for medulloblastoma, 2 after RT of unilateral iliac region (3000 and 2400 cGy) and ChT with AA for HD, 2 following pelvic RT (3000 and 4000 cGy) and ChT with AA for sarcoma. One of the patients in this group had only ovarian dysfunction after craniospinal RT (2400 cGy) and ChT without AA for leukemia. The risk of

Therefore, in this analysis orchidectomy and therapy with ChT and RT emerged as the most

LCI - impairment of Leydig cells, ChT – chemotherapy, RT♣ – radiotherapy to other region than brain

**3.1.2.5 Dependent variable –gonadal impairment (GI) in female patients, t.i. damage of** 

The decision tree with the second group of independent variables (Fig. 10) was more branched than the one using the first group of independent variables (Fig. 5), although this tree was not significantly different from statistical random predictions. Similar to the previous tree the highest risk for GI was observed in the group of females treated for GCT (GI in 5 out of the 6 patients). The remaining group of 109 patients with 18% risk of GI, treated for other malignancies, was further divided by the most important risk factor pelvic RT. In the group of 16 patients treated with pelvic RT risk of GI was 50%. As many as 7 out of the 8 patients with GI had damage of ovarian function, t.i.PH (one following RT of the whole abdomen (2000 cGy) and ChT with AA for leukemia, 2 after craniospinal RT (3400 and 3600 cGy) and ChT with AA (only one) for medulloblastoma, 2 after RT of unilateral iliac region (3000 and 2400 cGy) and ChT with AA for HD, 2 following pelvic RT (3000 and 4000 cGy) and ChT with AA for sarcoma. One of the patients in this group had only ovarian dysfunction after craniospinal RT (2400 cGy) and ChT without AA for leukemia. The risk of

Fig. 9. Classification tree analysis with second group of independent variables and impairment of Leydig cells (LCI) as dependent variable in 182 male patients

**ovarian function and dysfunction of ovarian function in female patients** 

important risk factors for LCI.

GI in the subgroup of 93 female patients without pelvic RT was as low as 13%. Only 4 out of 12 patients with GI in this group had damage of ovarian function (all after ChT with AA), 8 patients had only ovarian dysfunction (4 following ChT with AA).

In the group of 6 females treated for GCT at the age of 9 to 15 (med. 13) years, 5 had GI; 2 female patients had bilateral ovariectomy, one had ovarian damage after unilateral ovariectomy and abdominal RT, two females had dysfunction of ovarian function after unilateral removal of the ovary and ChT with AA.

In females, therefore, the most important risk factors for ovarian failure turned out to be pelvic RT, ChT with AA and ovariectomy.

GI – gonadal impairment, GCT – germ cell tumor, RT - radiotherapy

Fig. 10. Classification tree analysis with second group of independent variables and gonadal impairment as dependent variable in 115 female patients

#### *General observations*

Forty-six patients (21 males, 25 females) in our study had no PH, despite treatment with either ChT including AA or pelvic or testicular RT. Thirty-four (19 males, 15 females) of them were treated with ChT, which contained the antimetabolites methotrexate and 6 mercaptopurine, VCR, corticosteroids with or without L-asparaginase and adriamycin. Eleven patients (2 males and 9 females) received ChT containing actinomycin D (AMD) and vincristine (VCR) with or without adriamycin. One girl received ChT with vinblastine. Six patients (3 males and 3 females) out of 46 in this group of patients had subclinical impairment of the gonads (males had dysfunction of LC only) and as many as 4 patients (2 males and 2 females) had ChT with AMD and VCR. The impact of antimetabolite cytarabine on gonadal function could not be assessed because this cytostatic was administered to patients as part of ChT protocols, which contained AA (especially CY). So, we can conclude on the basis of our cohort of patients that ChT with antimetabolites (other

Hypogonadism After Childhood Cancer Treatment 183

In our study, therefore, we didn't observe the impact of age at diagnosis or pubertal status of patient during treatment on severity of gonadal damage. The results of various studies regarding age of male patients at diagnosis as independent variable are contradictory. Some authors reported lesser degree of testicular damage in those patients treated in prepubertal period in comparison with those treated in postpubertal period (Rivkees & Crawford 1988, Waxman et al. 1982), while others didn't confirm that observation (Aubier et al. 1989, Casteren et al. 2009, Hoorweg -Nijman et al. 1992, Lendon et al. 1978, Mustieles et al. 1995). However, in very few studies took into account the size of cumulative doses of AA, that patients in different age groups received (Lendon et al. 1978, Rivkees & Crawford 1988). On the other hand there are numerous studies reporting reduced susceptibility of ovaries to deleterious effects of cancer therapy in prepubertal period, when the number of oocytes is larger and they are in the "peaceful" phase (Chapman et al. 1979, Chemaitilly at al. 2006, Lushbaugh & Casarett 1976, Sanders et al. 1991, Wallace et al. 2005, Waxman et al. 1982).

The largest, 50%, incidence of PH was observed in patients treated for HD and germ cell tumors and the lowest, 9-11 %, in those treated for leukemia, brain tumors and Wilms' tumor (Table 2). In multivariate analysis diagnosis of GCT and HD repeatedly turned out to be important risk factors for PH and GI (Fig. 1,2,3,5,8,10). This is related to the nature of the disease (germ cell tumors of the gonads) and the type of treatment. Majority of patients with HD were treated with combination therapy (ChT and RT with / without surgery), in which the largest, 26 to 40%, proportion of patients with PH was observed (Table 3). In addition, ChT for HD usually contains more than one gonadotoxic AA simultaneously in the same

Another reason for high incidence of PH in males treated for HD is preexistent impairment of spermatogenesis before treatment. Indeed, some authors have observed reduced number and / or reduced sperm motility in the ejaculate in as many as one third of adult males with HD prior to treatment (Vigersky et al. 1982, Whitehead et al. 1982). On the contrary, in adult women ovarian biopsy did not reveal any abnormalities prior to treatment of HD (Chapman

As for surgery in the multivariate analysis only orchidectomy stood as important risk factor for GI as well as germinal epithelium impairment (GEI) and LC impairment (LCI) (Fig. 7-9). In 6 of our male patients unilateral orchidectomy was performed during treatment (3 had testicular GCT, 2 paratesticular rhabdomyosarcoma, one leukemia). In all 6 gonadal impairment was observed, namely 4 had damage of germ cell epithelium and LC , one had dysfunction of germ cell epithelium and LC and one had dysfunction of LC only. It is true that all those 6 patients received ChT with AA as well but probably surgery itself also contributed to the testicular damage. Nijman et al. (1987) observed elevated levels of FSH, LH, and decreased levels of testosterone in adult patients undergoing unilateral orchidectomy. These findings were attributed to the LC insufficiency in the remaining testis. Unilateral ovariectomy as independent variable itself didn't stand as an important risk factor in the analysis, but the diagnosis GCT was the most important risk factor for ovarian damage and dysfunction (Fig. 5, 10). In all 6 females with GCT ovariectomy was performed, in 3 bilateral, in 3 unilateral. All 3 girls with unilateral ovariectomy received also ChT with

protocol. As many as 65% of male subjects with diagnosis of HD had PH (Fig. 1,2).

**4.3 Type of malignancy** 

et al. 1979).

**4.4 Surgery** 

than cytarabine), antibiotics and vinca alkaloids wasn't toxic to germinal epithelium of testes, but primarily AMD and VCR may cause mild failure of ovaries and LC.

Chemotherapy regimens usually include several cytostatics, to study the toxic effect of each one on the gonads was therefore not possible. In our study, one male patient received the largest cumulative dose of cyclofosfamide 24 g/m2 at the age of 5 for retinoblastoma by ChT protocol containing vincristine and Adriamycin as well. In this patient we discovered damage of GE and LC-dysfunction. On the other hand male patient treated at the age of 9 years with ChT containing cumulative dose of CY 20 g/m2 for bone sarcoma had normal gonadal function. Normal functioning of the testes after treatment with combined ChT was found in another male patients after receiving a cumulative dose of CY of 15 g/m2, and other 3 males after receiving 11 -12 g/m2.

One male patient received the highest cumulative dose of cisplatinum (1 g/m2) at the age of 16 years for nasopharyngeal cancer with local RT and ChT, containing platinol, vinblastine, methotrexate and bleomycin. He maintained normal gonadal function.

#### **4. Discussion**

We found primary hypogonadism (PH) in 76 (26%) long-term survivors, 62 (34%) males and 14 (12%) females. High incidence (50%) of PH was found in those treated for Hodgkin's disease (HD) or germ cell tumor (GCT). A group of males, treated for HD with ChT was at high risk (72%) for PH as was a group treated with RT to the pelvis and ChT with AA, 79% (Fig. 1). At high risk of damage to and dysfunction of the germ epithelium of the testes were those treated with pelvic RT and ChT with AA. At high risk for damage to and dysfunction of ovaries were those treated for GCT (89%) and those treated with pelvic RT (50%).

#### **4.1 Gender**

Primary hypogonadism was detected in one third of males and in 12% females, indicating a greater susceptibility of male gonads for the deleterious effects of cancer treatment in childhood. In the multivariate analysis, 3 trees showed gender as the second most important risk factor for PH or GI (Fig.1,3,7); in the analysis of risk factors for PH in patients treated for HD the risk for PH in males was 65%, in females 25% (Fig. 1), in the analysis of risk factors for GI after exclusion of patients treated for GCT, the risk for GI in males was 48%, in females 18% (Fig. 3) and in the analysis of risk factors for GI (the second group of independent variables) in patients who had brain RT the risk of GI was 61% in males and 31% in females (Fig. 7). This is consistent with observations of other authors (Byrne et al. 1987, Kinsella et al. 1989, Rivkees & Crawford 1988).

#### **4.2 Age at diagnosis**

In multivariate analysis, age at treatment did not turned out as a significant risk factor for hypogonadism. Only in the analysis of risk factors for germ epithelium impairment at the last division patients age at diagnosis emerged as a risk factor for GEI. Namely, the group of 25 patients who were treated for HD with ChT with AA and RT above the diaphragm, was divided into those who were 10 or less years of age at diagnosis (83% risk of GEI ), and into the group of older patients (31% risk of GEI). But the cumulative doses of AA were different in the two age groups and we can not consider patient's age at treatment as an important risk factor for GEI.

In our study, therefore, we didn't observe the impact of age at diagnosis or pubertal status of patient during treatment on severity of gonadal damage. The results of various studies regarding age of male patients at diagnosis as independent variable are contradictory. Some authors reported lesser degree of testicular damage in those patients treated in prepubertal period in comparison with those treated in postpubertal period (Rivkees & Crawford 1988, Waxman et al. 1982), while others didn't confirm that observation (Aubier et al. 1989, Casteren et al. 2009, Hoorweg -Nijman et al. 1992, Lendon et al. 1978, Mustieles et al. 1995). However, in very few studies took into account the size of cumulative doses of AA, that patients in different age groups received (Lendon et al. 1978, Rivkees & Crawford 1988). On the other hand there are numerous studies reporting reduced susceptibility of ovaries to deleterious effects of cancer therapy in prepubertal period, when the number of oocytes is larger and they are in the "peaceful" phase (Chapman et al. 1979, Chemaitilly at al. 2006, Lushbaugh & Casarett 1976, Sanders et al. 1991, Wallace et al. 2005, Waxman et al. 1982).

#### **4.3 Type of malignancy**

182 Sex Hormones

than cytarabine), antibiotics and vinca alkaloids wasn't toxic to germinal epithelium of

Chemotherapy regimens usually include several cytostatics, to study the toxic effect of each one on the gonads was therefore not possible. In our study, one male patient received the largest cumulative dose of cyclofosfamide 24 g/m2 at the age of 5 for retinoblastoma by ChT protocol containing vincristine and Adriamycin as well. In this patient we discovered damage of GE and LC-dysfunction. On the other hand male patient treated at the age of 9 years with ChT containing cumulative dose of CY 20 g/m2 for bone sarcoma had normal gonadal function. Normal functioning of the testes after treatment with combined ChT was found in another male patients after receiving a cumulative dose of CY of 15 g/m2, and

One male patient received the highest cumulative dose of cisplatinum (1 g/m2) at the age of 16 years for nasopharyngeal cancer with local RT and ChT, containing platinol, vinblastine,

We found primary hypogonadism (PH) in 76 (26%) long-term survivors, 62 (34%) males and 14 (12%) females. High incidence (50%) of PH was found in those treated for Hodgkin's disease (HD) or germ cell tumor (GCT). A group of males, treated for HD with ChT was at high risk (72%) for PH as was a group treated with RT to the pelvis and ChT with AA, 79% (Fig. 1). At high risk of damage to and dysfunction of the germ epithelium of the testes were those treated with pelvic RT and ChT with AA. At high risk for damage to and dysfunction

Primary hypogonadism was detected in one third of males and in 12% females, indicating a greater susceptibility of male gonads for the deleterious effects of cancer treatment in childhood. In the multivariate analysis, 3 trees showed gender as the second most important risk factor for PH or GI (Fig.1,3,7); in the analysis of risk factors for PH in patients treated for HD the risk for PH in males was 65%, in females 25% (Fig. 1), in the analysis of risk factors for GI after exclusion of patients treated for GCT, the risk for GI in males was 48%, in females 18% (Fig. 3) and in the analysis of risk factors for GI (the second group of independent variables) in patients who had brain RT the risk of GI was 61% in males and 31% in females (Fig. 7). This is consistent with observations of other authors (Byrne et al.

In multivariate analysis, age at treatment did not turned out as a significant risk factor for hypogonadism. Only in the analysis of risk factors for germ epithelium impairment at the last division patients age at diagnosis emerged as a risk factor for GEI. Namely, the group of 25 patients who were treated for HD with ChT with AA and RT above the diaphragm, was divided into those who were 10 or less years of age at diagnosis (83% risk of GEI ), and into the group of older patients (31% risk of GEI). But the cumulative doses of AA were different in the two age groups and we can not consider patient's age at treatment as an important

of ovaries were those treated for GCT (89%) and those treated with pelvic RT (50%).

testes, but primarily AMD and VCR may cause mild failure of ovaries and LC.

methotrexate and bleomycin. He maintained normal gonadal function.

other 3 males after receiving 11 -12 g/m2.

1987, Kinsella et al. 1989, Rivkees & Crawford 1988).

**4. Discussion** 

**4.1 Gender** 

**4.2 Age at diagnosis** 

risk factor for GEI.

The largest, 50%, incidence of PH was observed in patients treated for HD and germ cell tumors and the lowest, 9-11 %, in those treated for leukemia, brain tumors and Wilms' tumor (Table 2). In multivariate analysis diagnosis of GCT and HD repeatedly turned out to be important risk factors for PH and GI (Fig. 1,2,3,5,8,10). This is related to the nature of the disease (germ cell tumors of the gonads) and the type of treatment. Majority of patients with HD were treated with combination therapy (ChT and RT with / without surgery), in which the largest, 26 to 40%, proportion of patients with PH was observed (Table 3). In addition, ChT for HD usually contains more than one gonadotoxic AA simultaneously in the same protocol. As many as 65% of male subjects with diagnosis of HD had PH (Fig. 1,2).

Another reason for high incidence of PH in males treated for HD is preexistent impairment of spermatogenesis before treatment. Indeed, some authors have observed reduced number and / or reduced sperm motility in the ejaculate in as many as one third of adult males with HD prior to treatment (Vigersky et al. 1982, Whitehead et al. 1982). On the contrary, in adult women ovarian biopsy did not reveal any abnormalities prior to treatment of HD (Chapman et al. 1979).

#### **4.4 Surgery**

As for surgery in the multivariate analysis only orchidectomy stood as important risk factor for GI as well as germinal epithelium impairment (GEI) and LC impairment (LCI) (Fig. 7-9). In 6 of our male patients unilateral orchidectomy was performed during treatment (3 had testicular GCT, 2 paratesticular rhabdomyosarcoma, one leukemia). In all 6 gonadal impairment was observed, namely 4 had damage of germ cell epithelium and LC , one had dysfunction of germ cell epithelium and LC and one had dysfunction of LC only. It is true that all those 6 patients received ChT with AA as well but probably surgery itself also contributed to the testicular damage. Nijman et al. (1987) observed elevated levels of FSH, LH, and decreased levels of testosterone in adult patients undergoing unilateral orchidectomy. These findings were attributed to the LC insufficiency in the remaining testis. Unilateral ovariectomy as independent variable itself didn't stand as an important risk factor in the analysis, but the diagnosis GCT was the most important risk factor for ovarian damage and dysfunction (Fig. 5, 10). In all 6 females with GCT ovariectomy was performed, in 3 bilateral, in 3 unilateral. All 3 girls with unilateral ovariectomy received also ChT with

Hypogonadism After Childhood Cancer Treatment 185

This observation is consistent with reports of other authors (Byrne et al.1987, Casteren et al. 2009). In the group of 61 male patients who didn't receive ChT with AA, all 6 patients with

By the last division in the analysis of risk factors for PH (Fig. 1), a group of 7 male patients, treated for HD without ChT, was identified. PH was observed in 2 patients only, in both following pelvic RT with 4000 cGy. Only one of our male patients had additional shielding of testes during pelvic RT (3600 cGy to bilateral iliacoinguinal regions) by a lead capsule, he received 6 cycles of LOPP. In spite laboratory testing showing damage of germinal epithelium and LC dysfunction he fathered 2 children, suggesting only a partial impairment of spermatogenesis. This is consistent with the study of Kovač et al. (1990) in which authors reported about significant reduction of testicular dose by an additional lead shielding of

ChT emerged as an important risk factor in all multivariate analysis using the first set of variables. In the analysis of risk factors for PH as well as GI in male patients ChT turned out to be very important risk factor, immediately after the type of malignancy (GCT or HD) (Fig. 2,4). In the analysis of risk factors for PH using a second set of independent variables (Fig. 8), a group of 246 patients who didn't have pelvic RT divided further by therapy with ChT. So, we identified a group of 87 patients neither receiving ChT with AA nor pelvic RT, with the lowest, 1%, risk of PH. The only one patient with PH in this group had testicular RT. Using multivariate analysis we, therefore, identified a group of childhood cancer survivors in

We observed greater impact of ChT with AA on gonadal function in boys than girls, as 42% males and only 7% females treated with ChT with AA without pelvic RT, had PH (Fig. 6). Similar conclusions were drawn in the study of Byrne et al. (1987) analyzing 2283 long-term survivors of childhood cancer and showed that RT under the diaphragm decreased fertility in both sexes for 25%, ChT with AA (with or without abdominal RT) decreased fertility only

This reflects in the analysis of risk factors for impairment of germ cell epithelium (Fig. 8) where the most important risk factor for GEI was ChT with AA, followed by pelvic RT. Males who received ChT with AA for HD without having pelvic RT, had incidence of GEI as high as 71%. Only 7 of 24 patients in this group of patients had normal testicular function, 4 patients after 6 cycles of LOPP cycles, 2 patients after 1 to 4 cycles of MOPP, one after 2 cycles of OPPA. On the other hand none of the male subjects after 6 or more cycles of MOPP ChT had normal function of germ cell epithelium. Our findings are in concordance with data from other studies establishing that MOPP or MOPP-like combinations, such as MVPP (mechlorethamine, vinblastine, procarbazine and prednisone) and COPP induce azoospermia in 90-100 % of pts with a 10-20% chance of recovery even 10 years after treatment (Chapman et al. 1979, Diamond& Bercu 2001, Viviani et al. 1985, Waxmann et al. 1982, Whitehead et al. 1982, Zaletel 2010). Recovery of spermatogenesis following MOPP therapy appears to be dose-related with 3 courses of MOPP representing a limiting gonadal exposure for the recovery, suggesting only partial killing of germinal stem cells (da Cunha et al. 1984). Indeed, in our study we found normal gonadal function in two males after having received 1 and 2 cycles of MOPP ChT. We found ChT according to the protocol LOPP less damaging for testicular function than MOPP, causing GEI in 5 of 10 males having

GEI had pelvic or testicular RT (Fig.8).

testis (Kovač et al. 1990).

**4.6 Chemotherapy (ChT)** 

whom gonadal testing could be omitted.

in boys for 60%, but not in girls.

AA without pelvic RT; in 2 ovarian dysfunction was observed, the third one had normal gonadal function. Unilateral ovariectomy is therefore compatible with normal gonadal function, which was also observed by other authors (Perrin et al. 1999).

In 12 patients within diagnostic procedures for HD staging laparotomy with transposition of ovaries to the posterior wall of the uterus was performed. Only 2 of these patients had pelvic RT (unilateral iliac region), all but one received ChT with AA. Ovarian damage was observed in 3 of those 12 females, namely in one following unilateral iliac RT with 2400 cGy and 6 cycles of LOPP at the age of 6 years (primary amenorrhea), the other 2 after RT of the upper abdomen with 3000 and 3600 cGy and 6 cycles of MOPP at the age of 11 and 14 years (both delivered healthy children). The remaining 9 patients had no signs of ovarian failure, six of them gave birth to healthy babies. Therefore, it seems that transposition of ovaries itself does not reduce the fertility. Indeed Thomas et al. (1976) didn't observe neither worsening of ovarian function nor interrupted gamets transfer after ovarian transposition in their patients with HD. Nevertheless, possible complications of this surgery, among others, are tubal obstruction and ovarian failure due to vascular damage (Thibaud et al. 1992).

#### **4.5 Radiotherapy (RT)**

RT has emerged as an important risk factor in the analysis of GI in males (Fig. 3,4) and pelvic RT was a major risk factor for PH in all subjects (Fig. 6). Risk for PH after pelvic RT was as high as 55%. With another division we identified a group of 17 patients treated with craniospinal RT for brain tumors, with low, 24%, incidence of PH. Addition of ChT with AA (mostly CCNU and PBZ) to RT, increased incidence of PH to 43% (being only 10% in those without ChT including AA). So, ChT with CCNU or PBZ in our subjects markedly increased risk of PH, especially in men, as 2 of the 3 males after craniospinal RT and ChT with AA had PH and none of the 6 males after only craniospinal RT had PH. One of 4 females had PH after RT alone. Higher gonadotoxicity of craniospinal RT in girls than in boys is in concordance with reports of other authors (Hamre et al. 1987, Sklar et al. 1990).

In the analysis of risk factors for ovarian impairment (Fig. 10) as the most important risk factor emerged GCT and in the second division pelvic RT. Similarly, other authors observed that pelvic RT is the most important risk factor for ovarian failure (Hamre et al. 1987, Stillman et al. 1981) observed that the only risk factor for ovarian failure in 182 long-term survivors of childhood cancer was ovarian position relative to the RT field and not ChT. Of course, ovarian failure may be caused by ChT with AA (Chapman et al. 1979, Nicosia et al. 1985, Ortin et al. 1990, Siris et al. 1976), what was observed in our study as well, namely all females with PH without receiving pelvic RT had ChT with AA.

Four females in our series had unilateral pelvic RT. Two had evidence of PH, but all received ChT with AA and PBZ as well. After RT of paraaortic lymph nodes the estimated ovarian dose is about 6 % of the prescribed dose (in the range of 100 cGy) and this dose of radiation can cause transient disturbances of menstrual cycle. Haie-Mader and colleagues (1993) analyzed ovarian function in 134 females treated for HD or gynecological cancer and showed that the age over 25 years, MOPP ChT and total dose to the ovaries higher than 500 cGy are important risk factors for ovarian castration.

In the analysis of risk factors for impairment of germ cell epithelium (GEI) the highest, 89%, risk of GEI, was observed in the group of males who received ChT with AA and had pelvic RT (Fig. 8). Only two of the 18 patients in this group did not have GEI, namely one patient after RT of bilateral iliac regions (3000 cGy) and two cycles of MOPP and the other following craniospinal RT (900 cGy) and ChT with AA (incl. 7 g/m2 CY).

This observation is consistent with reports of other authors (Byrne et al.1987, Casteren et al. 2009). In the group of 61 male patients who didn't receive ChT with AA, all 6 patients with GEI had pelvic or testicular RT (Fig.8).

By the last division in the analysis of risk factors for PH (Fig. 1), a group of 7 male patients, treated for HD without ChT, was identified. PH was observed in 2 patients only, in both following pelvic RT with 4000 cGy. Only one of our male patients had additional shielding of testes during pelvic RT (3600 cGy to bilateral iliacoinguinal regions) by a lead capsule, he received 6 cycles of LOPP. In spite laboratory testing showing damage of germinal epithelium and LC dysfunction he fathered 2 children, suggesting only a partial impairment of spermatogenesis. This is consistent with the study of Kovač et al. (1990) in which authors reported about significant reduction of testicular dose by an additional lead shielding of testis (Kovač et al. 1990).

#### **4.6 Chemotherapy (ChT)**

184 Sex Hormones

AA without pelvic RT; in 2 ovarian dysfunction was observed, the third one had normal gonadal function. Unilateral ovariectomy is therefore compatible with normal gonadal

In 12 patients within diagnostic procedures for HD staging laparotomy with transposition of ovaries to the posterior wall of the uterus was performed. Only 2 of these patients had pelvic RT (unilateral iliac region), all but one received ChT with AA. Ovarian damage was observed in 3 of those 12 females, namely in one following unilateral iliac RT with 2400 cGy and 6 cycles of LOPP at the age of 6 years (primary amenorrhea), the other 2 after RT of the upper abdomen with 3000 and 3600 cGy and 6 cycles of MOPP at the age of 11 and 14 years (both delivered healthy children). The remaining 9 patients had no signs of ovarian failure, six of them gave birth to healthy babies. Therefore, it seems that transposition of ovaries itself does not reduce the fertility. Indeed Thomas et al. (1976) didn't observe neither worsening of ovarian function nor interrupted gamets transfer after ovarian transposition in their patients with HD. Nevertheless, possible complications of this surgery, among others, are tubal obstruction and ovarian failure due to vascular damage (Thibaud et al. 1992).

RT has emerged as an important risk factor in the analysis of GI in males (Fig. 3,4) and pelvic RT was a major risk factor for PH in all subjects (Fig. 6). Risk for PH after pelvic RT was as high as 55%. With another division we identified a group of 17 patients treated with craniospinal RT for brain tumors, with low, 24%, incidence of PH. Addition of ChT with AA (mostly CCNU and PBZ) to RT, increased incidence of PH to 43% (being only 10% in those without ChT including AA). So, ChT with CCNU or PBZ in our subjects markedly increased risk of PH, especially in men, as 2 of the 3 males after craniospinal RT and ChT with AA had PH and none of the 6 males after only craniospinal RT had PH. One of 4 females had PH after RT alone. Higher gonadotoxicity of craniospinal RT in girls than in boys is in

In the analysis of risk factors for ovarian impairment (Fig. 10) as the most important risk factor emerged GCT and in the second division pelvic RT. Similarly, other authors observed that pelvic RT is the most important risk factor for ovarian failure (Hamre et al. 1987, Stillman et al. 1981) observed that the only risk factor for ovarian failure in 182 long-term survivors of childhood cancer was ovarian position relative to the RT field and not ChT. Of course, ovarian failure may be caused by ChT with AA (Chapman et al. 1979, Nicosia et al. 1985, Ortin et al. 1990, Siris et al. 1976), what was observed in our study as well, namely all

Four females in our series had unilateral pelvic RT. Two had evidence of PH, but all received ChT with AA and PBZ as well. After RT of paraaortic lymph nodes the estimated ovarian dose is about 6 % of the prescribed dose (in the range of 100 cGy) and this dose of radiation can cause transient disturbances of menstrual cycle. Haie-Mader and colleagues (1993) analyzed ovarian function in 134 females treated for HD or gynecological cancer and showed that the age over 25 years, MOPP ChT and total dose to the ovaries higher than 500

In the analysis of risk factors for impairment of germ cell epithelium (GEI) the highest, 89%, risk of GEI, was observed in the group of males who received ChT with AA and had pelvic RT (Fig. 8). Only two of the 18 patients in this group did not have GEI, namely one patient after RT of bilateral iliac regions (3000 cGy) and two cycles of MOPP and the other following

concordance with reports of other authors (Hamre et al. 1987, Sklar et al. 1990).

females with PH without receiving pelvic RT had ChT with AA.

craniospinal RT (900 cGy) and ChT with AA (incl. 7 g/m2 CY).

cGy are important risk factors for ovarian castration.

function, which was also observed by other authors (Perrin et al. 1999).

**4.5 Radiotherapy (RT)** 

ChT emerged as an important risk factor in all multivariate analysis using the first set of variables. In the analysis of risk factors for PH as well as GI in male patients ChT turned out to be very important risk factor, immediately after the type of malignancy (GCT or HD) (Fig. 2,4). In the analysis of risk factors for PH using a second set of independent variables (Fig. 8), a group of 246 patients who didn't have pelvic RT divided further by therapy with ChT. So, we identified a group of 87 patients neither receiving ChT with AA nor pelvic RT, with the lowest, 1%, risk of PH. The only one patient with PH in this group had testicular RT. Using multivariate analysis we, therefore, identified a group of childhood cancer survivors in whom gonadal testing could be omitted.

We observed greater impact of ChT with AA on gonadal function in boys than girls, as 42% males and only 7% females treated with ChT with AA without pelvic RT, had PH (Fig. 6). Similar conclusions were drawn in the study of Byrne et al. (1987) analyzing 2283 long-term survivors of childhood cancer and showed that RT under the diaphragm decreased fertility in both sexes for 25%, ChT with AA (with or without abdominal RT) decreased fertility only in boys for 60%, but not in girls.

This reflects in the analysis of risk factors for impairment of germ cell epithelium (Fig. 8) where the most important risk factor for GEI was ChT with AA, followed by pelvic RT. Males who received ChT with AA for HD without having pelvic RT, had incidence of GEI as high as 71%. Only 7 of 24 patients in this group of patients had normal testicular function, 4 patients after 6 cycles of LOPP cycles, 2 patients after 1 to 4 cycles of MOPP, one after 2 cycles of OPPA. On the other hand none of the male subjects after 6 or more cycles of MOPP ChT had normal function of germ cell epithelium. Our findings are in concordance with data from other studies establishing that MOPP or MOPP-like combinations, such as MVPP (mechlorethamine, vinblastine, procarbazine and prednisone) and COPP induce azoospermia in 90-100 % of pts with a 10-20% chance of recovery even 10 years after treatment (Chapman et al. 1979, Diamond& Bercu 2001, Viviani et al. 1985, Waxmann et al. 1982, Whitehead et al. 1982, Zaletel 2010). Recovery of spermatogenesis following MOPP therapy appears to be dose-related with 3 courses of MOPP representing a limiting gonadal exposure for the recovery, suggesting only partial killing of germinal stem cells (da Cunha et al. 1984). Indeed, in our study we found normal gonadal function in two males after having received 1 and 2 cycles of MOPP ChT. We found ChT according to the protocol LOPP less damaging for testicular function than MOPP, causing GEI in 5 of 10 males having

Hypogonadism After Childhood Cancer Treatment 187

within a few years after treatment, and usually there is no correction of LC damage (Shalet et al. 1985). Indeed, in 4 male patients in our cohort we found LC impairment at the second testing 3 to 10 years after their first testing, when they had normal function or only

LC damage was detected in 16 adolescents. All who were on treatment in prepubertal period (10 patients) had normal pubertal development. Lowered testosterone level was observed only in one patient, who received ChT with AA and RT above the diaphragm at the age of 10 years for HD. At relapse, 5 years later, he had additional ChT with AA and RT to the upper abdomen (3400 cGy). So, the majority of these 16 patients had clinically insignificant impairment of LC function. Two of these patients had testicular RT (1200 cGy in 4 fractions). Other authors did neither observe clinically significant impairment of LC after the testicular dose of this size (Brauner et al. 1983, Castillo et al. 1999). Of the remaining 14 patients with LC damage, only 4 had pelvic RT with / without ChT with AA, 10 patients had ChT with AA without pelvic RT. So, failure of LC in these adolescents was not simply a consequence of RT, but was mainly caused by ChT with AA. Most studies after ChT with AA observed compensatory insufficiency of LC (normal testosterone level and elevated basal level of LH and / or elevated level of LH after stimulation) (Brämswig et al . 1990, Kenney et al. 2001, Meistrich 2009, Romerius at al. 2009, Sherins et al. 1978, Whitehead et al. 1982). However, in two studies LC dysfunction following ChT with AA was not identified (Pennisi et al. 1975, Shalet et al. 1981). The likely cause of this discrepancy lies in the fact that in one of these studies LC function was evaluated only by basal LH levels without GnRHtest, but LC dysfunction can reflect in increased LH response to GnRH (Pennisi et al. 1975).

All our 16 patients with LC damage had damage of germinal epithelium as well.

to reports of others (Constine et al. 1993, Gleeson & Shalet 2004).

pathogenesis of compensatory LC damage.

**4.9 Secondary hypogonadism** 

**4.10 Classification tree analysis** 

is discovered.

LC dysfunction was observed in 54 patients. All but 6 received ChT with AA (9 of them had pelvic RT as well). Again, this result confirms that ChT with AA contributes in the

Incidence of LC damage is increasing in the years after treatment, therefore, patients with elevated basal or stimulated LH require annual monitoring of LH and testosterone and the timely introduction of hormone replacement therapy when reduced secretion of testosterone

Secondary hypogonadism was detected in two female patients with panhipopituitarism after combination therapy of GCT of hypothalamus, in three patients after treatment of brain tumors located outside the hypothalamus or pituitary gland with surgery and postoperative RT (5000 - 6500 cGy) and in one female after whole brain RT with 3000 cGy for leukemia. All patients with secondary hypogonadism had hiposomatotropism as well, which corresponds

We analyzed our data by multivariate analysis method, classification tree model, which allows for studying of simultaneous influence of a series of independent variables on a single dependent variable. The main advantage of this method is its ability to detect the mutual effect of independent variables. The decision tree determines groups of subjects with

dysfunction of LC.

**4.8 Leydig cell damage** 

received 6 cycles, a finding not published elsewhere to our knowledge. It seems that chlorambucil inside LOPP protocol is more gonadotoxic than nitrogen mustard in MOPP protocol. Our subjects received many different chemotherapy regimens, so the comparison with the results of other studies is difficult.

Among patients who didn't receive pelvic RT, the highest incidence of PH was observed in the group of males treated for HD with ChT including AA (66%) (Fig. 6). Those males who were treated with ChT including AA for other malignancies, had much lower incidence of PH (34%). This finding is consistent with other studies reporting that ChT used in treatment of HD is more gonadotoxic from other ChT regimens (Müller 2003).

In women, in the analysis of risk factors for ovarian impairment pelvic RT emerged as the most important risk factor, immediately after diagnosis GCT (Fig. 10). But 6 of the 8 patients who had pelvic RT received ChT with AA as well. On the other hand, all females with ovarian impairment, who didn't receive pelvic RT, were treated with ChT with AA. So, we can conclude that ChT with AA contributed to development of ovarian impairment. ChT according to MOPP protocol is more toxic to the ovaries than other types of ChT. There are data of adverse effects of ChT that is in use for HD, on ovarian function in adult females, but very little on ovarian function in girls. In the study of Ortin and colleagues 2 of 18 girls were amenorrhoic after having received 6 or more cycles of MOPP (Ortin et al. 1990). In our study none of the 6 females was amenorrhoic after 6 or more cycles of MOPP, but 2 had evidence of ovarian damage while retaining fertility Probably in girls younger than 16 years ChT is less gonadotoxic because relative quiescence of stromal cells and oocytes in prepubertal period protects ovaries from cell cycle specific cytotoxic agents (Siris et al. 1976, Stillman et al. 1981).

Forty-six patients didn't receive neither testicular or pelvic RT nor ChT with AA. They were treated with ChT containing antimetabolites (except cytarabine), antibiotics and vinca alkaloids. None of them had PH. Other authors as well, didn't observe important role of this type of ChT in pathogenesis of gonadal failure (Blatt et al. 1981, Müller 2003), although they reported on transient oligospermia in adult patients following treatment with methotrexate (Sussman & Leonard 1980) and on possible role of VCR in pathogenesis of germ cell epithelium damage in childhood and adolescence (Rautonen et al. 1992). In the group of patients treated with ChT containing AMD and VCR, 2 female patients had ovarian dysfunction and 2 males had LC dysfunction, suggesting that therapy with these 2 chemotherapy agents can cause mild degree of ovarian or LC damage. To our knowledge there is no article reporting on the potential gonadotoxicity of AMD.

#### **4.7 Observation time**

In none of the multivariate analysis observation time emerged as a significant risk factor for gonadal impairment. This is in accordance with expectations, as normally gonadal failure develops during or shortly after administration of toxic therapy and correction of gonadal damage eventually takes place within the first decade thereafter or so (Mustieles et al. 1995, Rowley et al. 1974, Viviani et al. 1985, Waxman et al. 1982). In 73 patients, we performed gonadal testing twice. Indeed, in 3 males the second testing showed normal functioning of the germ cell epithelium, after first testing showing damage of germ cell epithelium (4 to 16 years earlier). In female subjects as well the correction of the ovarian function was found. One of them had secondary amenorrhea after treatment and after 5 years menstrual cycle restored. The second one had primary amenorrhea till 16 years of age and then restored normal menstrual cycle. In contrast to the germ cell failure, damage of LC can develop within a few years after treatment, and usually there is no correction of LC damage (Shalet et al. 1985). Indeed, in 4 male patients in our cohort we found LC impairment at the second testing 3 to 10 years after their first testing, when they had normal function or only dysfunction of LC.

#### **4.8 Leydig cell damage**

186 Sex Hormones

received 6 cycles, a finding not published elsewhere to our knowledge. It seems that chlorambucil inside LOPP protocol is more gonadotoxic than nitrogen mustard in MOPP protocol. Our subjects received many different chemotherapy regimens, so the comparison

Among patients who didn't receive pelvic RT, the highest incidence of PH was observed in the group of males treated for HD with ChT including AA (66%) (Fig. 6). Those males who were treated with ChT including AA for other malignancies, had much lower incidence of PH (34%). This finding is consistent with other studies reporting that ChT used in treatment

In women, in the analysis of risk factors for ovarian impairment pelvic RT emerged as the most important risk factor, immediately after diagnosis GCT (Fig. 10). But 6 of the 8 patients who had pelvic RT received ChT with AA as well. On the other hand, all females with ovarian impairment, who didn't receive pelvic RT, were treated with ChT with AA. So, we can conclude that ChT with AA contributed to development of ovarian impairment. ChT according to MOPP protocol is more toxic to the ovaries than other types of ChT. There are data of adverse effects of ChT that is in use for HD, on ovarian function in adult females, but very little on ovarian function in girls. In the study of Ortin and colleagues 2 of 18 girls were amenorrhoic after having received 6 or more cycles of MOPP (Ortin et al. 1990). In our study none of the 6 females was amenorrhoic after 6 or more cycles of MOPP, but 2 had evidence of ovarian damage while retaining fertility Probably in girls younger than 16 years ChT is less gonadotoxic because relative quiescence of stromal cells and oocytes in prepubertal period protects ovaries from cell cycle specific cytotoxic agents (Siris et al. 1976, Stillman et al. 1981). Forty-six patients didn't receive neither testicular or pelvic RT nor ChT with AA. They were treated with ChT containing antimetabolites (except cytarabine), antibiotics and vinca alkaloids. None of them had PH. Other authors as well, didn't observe important role of this type of ChT in pathogenesis of gonadal failure (Blatt et al. 1981, Müller 2003), although they reported on transient oligospermia in adult patients following treatment with methotrexate (Sussman & Leonard 1980) and on possible role of VCR in pathogenesis of germ cell epithelium damage in childhood and adolescence (Rautonen et al. 1992). In the group of patients treated with ChT containing AMD and VCR, 2 female patients had ovarian dysfunction and 2 males had LC dysfunction, suggesting that therapy with these 2 chemotherapy agents can cause mild degree of ovarian or LC damage. To our knowledge

of HD is more gonadotoxic from other ChT regimens (Müller 2003).

there is no article reporting on the potential gonadotoxicity of AMD.

In none of the multivariate analysis observation time emerged as a significant risk factor for gonadal impairment. This is in accordance with expectations, as normally gonadal failure develops during or shortly after administration of toxic therapy and correction of gonadal damage eventually takes place within the first decade thereafter or so (Mustieles et al. 1995, Rowley et al. 1974, Viviani et al. 1985, Waxman et al. 1982). In 73 patients, we performed gonadal testing twice. Indeed, in 3 males the second testing showed normal functioning of the germ cell epithelium, after first testing showing damage of germ cell epithelium (4 to 16 years earlier). In female subjects as well the correction of the ovarian function was found. One of them had secondary amenorrhea after treatment and after 5 years menstrual cycle restored. The second one had primary amenorrhea till 16 years of age and then restored normal menstrual cycle. In contrast to the germ cell failure, damage of LC can develop

**4.7 Observation time** 

with the results of other studies is difficult.

LC damage was detected in 16 adolescents. All who were on treatment in prepubertal period (10 patients) had normal pubertal development. Lowered testosterone level was observed only in one patient, who received ChT with AA and RT above the diaphragm at the age of 10 years for HD. At relapse, 5 years later, he had additional ChT with AA and RT to the upper abdomen (3400 cGy). So, the majority of these 16 patients had clinically insignificant impairment of LC function. Two of these patients had testicular RT (1200 cGy in 4 fractions). Other authors did neither observe clinically significant impairment of LC after the testicular dose of this size (Brauner et al. 1983, Castillo et al. 1999). Of the remaining 14 patients with LC damage, only 4 had pelvic RT with / without ChT with AA, 10 patients had ChT with AA without pelvic RT. So, failure of LC in these adolescents was not simply a consequence of RT, but was mainly caused by ChT with AA. Most studies after ChT with AA observed compensatory insufficiency of LC (normal testosterone level and elevated basal level of LH and / or elevated level of LH after stimulation) (Brämswig et al . 1990, Kenney et al. 2001, Meistrich 2009, Romerius at al. 2009, Sherins et al. 1978, Whitehead et al. 1982). However, in two studies LC dysfunction following ChT with AA was not identified (Pennisi et al. 1975, Shalet et al. 1981). The likely cause of this discrepancy lies in the fact that in one of these studies LC function was evaluated only by basal LH levels without GnRHtest, but LC dysfunction can reflect in increased LH response to GnRH (Pennisi et al. 1975). All our 16 patients with LC damage had damage of germinal epithelium as well.

LC dysfunction was observed in 54 patients. All but 6 received ChT with AA (9 of them had pelvic RT as well). Again, this result confirms that ChT with AA contributes in the pathogenesis of compensatory LC damage.

Incidence of LC damage is increasing in the years after treatment, therefore, patients with elevated basal or stimulated LH require annual monitoring of LH and testosterone and the timely introduction of hormone replacement therapy when reduced secretion of testosterone is discovered.

#### **4.9 Secondary hypogonadism**

Secondary hypogonadism was detected in two female patients with panhipopituitarism after combination therapy of GCT of hypothalamus, in three patients after treatment of brain tumors located outside the hypothalamus or pituitary gland with surgery and postoperative RT (5000 - 6500 cGy) and in one female after whole brain RT with 3000 cGy for leukemia. All patients with secondary hypogonadism had hiposomatotropism as well, which corresponds to reports of others (Constine et al. 1993, Gleeson & Shalet 2004).

#### **4.10 Classification tree analysis**

We analyzed our data by multivariate analysis method, classification tree model, which allows for studying of simultaneous influence of a series of independent variables on a single dependent variable. The main advantage of this method is its ability to detect the mutual effect of independent variables. The decision tree determines groups of subjects with

2009).

**5. Conclusions** 

other studies, such as :

childhood,

our subjects were tested in postpubertal period.

semen analysis is confirmatory assessment of male gonadal function.

cancer treatment in childhood authors have not reported on hyperexcitability.

With the presented population based study we confirmed several already known results of

a greater susceptibility of male gonads for the deleterious effects of cancer treatment in

Hypogonadism After Childhood Cancer Treatment 189

possibility of impaired spermatogenesis (Aubier et al. 1989, Kenney et al. 2001). We couldn't confirm that observation because none of our male patients with normal laboratory findings performed spermanalysis. But none of them had problems with fertile capability. We didn't use testicular volume for evaluation of gonadal function in male subjects. Indeed, some studies reported that testicular volume is not a reliable indicator of spermatogenesis (Kenney et al. 2001, Relander et al. 2000). There are reports on Inhibin B as a good serum marker which correlate well with sperm concentration (Beek at al. 2007, Casteren et al.

GnRH-test served us for the evaluation of LC function as well. Good test for the evaluation of LC function is HCG test, which measures testosterone levels after repeated administration of chorionic gonadotropin (Brauner et al. 1983). However, this test is difficult to implement as it lasts for several days. Anyway, Brauner (1983) found good correlation between GnRH-test and HCG-test in males if performed in postpubertal period. Actually,

Five of 24 males with germ cell epithelium damage fathered children indicating that they are not azoospermic but possibly oligospermic and fertile. Hoorweg-Nijman and colleagues found elevated levels of FSH compatible with normospermia (Hoorweg-Nijman et al. 1992). FSH levels may provide an estimate of possible impaired spermatogenesis, however only

We used GnRH-test for evaluation of ovarian function as well. Primary hypogonadism was detected in 14 females, but only 6 of them are amenorrhoic, 3 after bilateral ovariectomy for GCT. Interestingly, one of our patients in spite of being amenorrhoic and having levels of gonadotropins in menopausal range, gave birth to a healthy boy. Of the remaining 8 female patients with PH, one had transient, secondary amenorrhea lasting for 5 years, one is in early menopause (at 38 years of age) after 2 deliveries, 6 of them have irregular periods, 2 after 1 to 2 deliveries. Indeed, in most studies the term ovarian failure was used in patients with amenorrhea, elevated levels of gonadotropins and lower levels of estradiol (Stillman et al. 1981, Chapman et al. 1979). Thus, ovarian failure, defined in such a way, was diagnosed in 6 of our female patients only. So we, maybe, slightly overestimated the rate of PH in female patients (as well as in male patients) taking under the cover of ovarian damage more subtile, clinically insignificant gonadal damage as well. But it is likely that these patients are at risk of early menopause which already happened in one of our female patients. After cancer therapy, indeed, the number of primordial follicles decreases further, increasing the "age" of ovaries and shortening fertile period (Larsen et al. 2003). Hyperexcitability of gonadal axis (elevated LH / FSH after stimulation with GnRH), t.i. ovarian dysfunction, was detected in 11 of our female patients. All have regular menstrual cycles and five of them gave birth to healthy children. However, ovarian hyperexcitability may indicate mild impairment of ovarian tissue and higher risk of early menopause in most subjects but does not appear to be clinically significant. Other comparable studies of ovarian function after

a set of specific values of independent variables, in which the risk of gonadal damage is the highest or the lowest. So, in analysis where we took damage and dysfunction of germinal epithelium of testes as dependent variable we identified a group of patients at highest risk of this outcome, namely males who were treated with ChT with AA and pelvic RT. In analysis of risk factors for primary hypogonadism (PH), we identified a group of patients with very low risk, 1%, for this outcome, namely, males who had neither ChT with AA nor pelvic or testicular RT as their treatment.

Classification trees in our study were mostly significantly different from random prediction, with the exception of those trees constructed on data from female patients, due to the low number of patients with "positive" outcomes (e.g. PH). Predictive accuracy of the trees was high and it would be even higher if the analysis included other independent variables that might further explain the difference between groups of subjects with different outcome regarding dependent variable. In our analysis, we wanted to include more independent variables, or several different values for independent variables (e.g. cumulative doses of various cytostatics), but, despite the relatively large number of subjects, we could not do it. Each multivariate analysis restricts the number of independent variables and their values. On the other hand, other risk factors for gonadal damage can exist which we haven't identified yet.

Maybe, a limitation of this method is that in some analysis, a larger group of patients is not further divided, e.g. a group of 233 patients who were treated for cancer other than HD (Fig. 1) and a group of 109 female patients who was not treated for GCT (Fig. 5). The reason for this was the small number of subjects with observed outcome (e.g. PH) in these groups of patient. So we could loose some information on potential risk factors for impaired gonadal function in these patients. Decision trees, which we got using the second group of independent variables, were more diversified and gave also more information (Fig. 6-10).

In the published articles studying gonadal function after childhood cancer treatment different multivariate analysis for analyzing risk factors were used, mainly logistic regression (Chematilly et al. 2006, Haie-Mader et al. 1993, Rautonen et al. 1992, Romerius et al. 2009, Stillman et al. 1981), linear regression (Siimes & Rautonen 1990), Cox regression analysis (Byrne et al. 1987). But no one used the decision tree classification model, therefore, not been able to identify links between risk factors for impaired gonadal function. But there are a number of articles published in medicine, in which a classification tree method was used (Jazbec et al. 2004, Jereb & Eklund 1973, Macedoni-Lukšič et al. 2003, Velensek et al. 2008).

#### **4.11 Assessment of gonadal function**

For assessment of gonadal function in long-term survivors of childhood leukemia we used, beside clinical evaluation, hormonal testing, which is an indirect measure of testicular and ovarian function. Several studies showed that in men basal FSH level and FSH response to GnRH correlate well with sperm production (Aubier et al. 1989, Hoorweg-Nijman et al. 1992, Kinsella et al.1989, Kirkland et al. 1976, Mustieles et al. 1995, Siimes & Rautonen 1990). An increased FSH response to GnRH can be the first manifestation of testicular damage, although normal FSH levels do not rule out the possibility of azoospermia (Aubier et al. 1989, Kenney et al. 2001). All our male patients with PH were advised to perform analysis of spermiogram, but only 12 of them decided to do so. In 11 of them azoospermia was found, confirming that GnRH-testing offers a good estimate of spermatogenesis. On the other hand 6 male patients with documented PH became fathers, suggesting that elevated levels of FSH do not rule out fertile ability. But on the other hand normal FSH levels do not exclude the

a set of specific values of independent variables, in which the risk of gonadal damage is the highest or the lowest. So, in analysis where we took damage and dysfunction of germinal epithelium of testes as dependent variable we identified a group of patients at highest risk of this outcome, namely males who were treated with ChT with AA and pelvic RT. In analysis of risk factors for primary hypogonadism (PH), we identified a group of patients with very low risk, 1%, for this outcome, namely, males who had neither ChT with AA nor

Classification trees in our study were mostly significantly different from random prediction, with the exception of those trees constructed on data from female patients, due to the low number of patients with "positive" outcomes (e.g. PH). Predictive accuracy of the trees was high and it would be even higher if the analysis included other independent variables that might further explain the difference between groups of subjects with different outcome regarding dependent variable. In our analysis, we wanted to include more independent variables, or several different values for independent variables (e.g. cumulative doses of various cytostatics), but, despite the relatively large number of subjects, we could not do it. Each multivariate analysis restricts the number of independent variables and their values. On the other hand, other risk factors for gonadal damage can exist which we haven't

Maybe, a limitation of this method is that in some analysis, a larger group of patients is not further divided, e.g. a group of 233 patients who were treated for cancer other than HD (Fig. 1) and a group of 109 female patients who was not treated for GCT (Fig. 5). The reason for this was the small number of subjects with observed outcome (e.g. PH) in these groups of patient. So we could loose some information on potential risk factors for impaired gonadal function in these patients. Decision trees, which we got using the second group of independent variables, were more diversified and gave also more information (Fig. 6-10). In the published articles studying gonadal function after childhood cancer treatment different multivariate analysis for analyzing risk factors were used, mainly logistic regression (Chematilly et al. 2006, Haie-Mader et al. 1993, Rautonen et al. 1992, Romerius et al. 2009, Stillman et al. 1981), linear regression (Siimes & Rautonen 1990), Cox regression analysis (Byrne et al. 1987). But no one used the decision tree classification model, therefore, not been able to identify links between risk factors for impaired gonadal function. But there are a number of articles published in medicine, in which a classification tree method was used (Jazbec et al. 2004, Jereb & Eklund 1973, Macedoni-Lukšič et al. 2003, Velensek et al. 2008).

For assessment of gonadal function in long-term survivors of childhood leukemia we used, beside clinical evaluation, hormonal testing, which is an indirect measure of testicular and ovarian function. Several studies showed that in men basal FSH level and FSH response to GnRH correlate well with sperm production (Aubier et al. 1989, Hoorweg-Nijman et al. 1992, Kinsella et al.1989, Kirkland et al. 1976, Mustieles et al. 1995, Siimes & Rautonen 1990). An increased FSH response to GnRH can be the first manifestation of testicular damage, although normal FSH levels do not rule out the possibility of azoospermia (Aubier et al. 1989, Kenney et al. 2001). All our male patients with PH were advised to perform analysis of spermiogram, but only 12 of them decided to do so. In 11 of them azoospermia was found, confirming that GnRH-testing offers a good estimate of spermatogenesis. On the other hand 6 male patients with documented PH became fathers, suggesting that elevated levels of FSH do not rule out fertile ability. But on the other hand normal FSH levels do not exclude the

pelvic or testicular RT as their treatment.

**4.11 Assessment of gonadal function** 

identified yet.

possibility of impaired spermatogenesis (Aubier et al. 1989, Kenney et al. 2001). We couldn't confirm that observation because none of our male patients with normal laboratory findings performed spermanalysis. But none of them had problems with fertile capability. We didn't use testicular volume for evaluation of gonadal function in male subjects. Indeed, some studies reported that testicular volume is not a reliable indicator of spermatogenesis (Kenney et al. 2001, Relander et al. 2000). There are reports on Inhibin B as a good serum marker which correlate well with sperm concentration (Beek at al. 2007, Casteren et al. 2009).

GnRH-test served us for the evaluation of LC function as well. Good test for the evaluation of LC function is HCG test, which measures testosterone levels after repeated administration of chorionic gonadotropin (Brauner et al. 1983). However, this test is difficult to implement as it lasts for several days. Anyway, Brauner (1983) found good correlation between GnRH-test and HCG-test in males if performed in postpubertal period. Actually, our subjects were tested in postpubertal period.

Five of 24 males with germ cell epithelium damage fathered children indicating that they are not azoospermic but possibly oligospermic and fertile. Hoorweg-Nijman and colleagues found elevated levels of FSH compatible with normospermia (Hoorweg-Nijman et al. 1992). FSH levels may provide an estimate of possible impaired spermatogenesis, however only semen analysis is confirmatory assessment of male gonadal function.

We used GnRH-test for evaluation of ovarian function as well. Primary hypogonadism was detected in 14 females, but only 6 of them are amenorrhoic, 3 after bilateral ovariectomy for GCT. Interestingly, one of our patients in spite of being amenorrhoic and having levels of gonadotropins in menopausal range, gave birth to a healthy boy. Of the remaining 8 female patients with PH, one had transient, secondary amenorrhea lasting for 5 years, one is in early menopause (at 38 years of age) after 2 deliveries, 6 of them have irregular periods, 2 after 1 to 2 deliveries. Indeed, in most studies the term ovarian failure was used in patients with amenorrhea, elevated levels of gonadotropins and lower levels of estradiol (Stillman et al. 1981, Chapman et al. 1979). Thus, ovarian failure, defined in such a way, was diagnosed in 6 of our female patients only. So we, maybe, slightly overestimated the rate of PH in female patients (as well as in male patients) taking under the cover of ovarian damage more subtile, clinically insignificant gonadal damage as well. But it is likely that these patients are at risk of early menopause which already happened in one of our female patients. After cancer therapy, indeed, the number of primordial follicles decreases further, increasing the "age" of ovaries and shortening fertile period (Larsen et al. 2003). Hyperexcitability of gonadal axis (elevated LH / FSH after stimulation with GnRH), t.i. ovarian dysfunction, was detected in 11 of our female patients. All have regular menstrual cycles and five of them gave birth to healthy children. However, ovarian hyperexcitability may indicate mild impairment of ovarian tissue and higher risk of early menopause in most subjects but does not appear to be clinically significant. Other comparable studies of ovarian function after cancer treatment in childhood authors have not reported on hyperexcitability.

#### **5. Conclusions**

With the presented population based study we confirmed several already known results of other studies, such as :

 a greater susceptibility of male gonads for the deleterious effects of cancer treatment in childhood,

Hypogonadism After Childhood Cancer Treatment 191

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But there was no multivariate analysis using the decision tree classification model, which is able to identify links between risk factors for impaired gonadal function. With this model we could also identify a group of patients with the lowest risk of gonadal impairment those who had neither pelvic or testicular RT nor ChT including AA. In those hormonal testing could be omitted.

#### **6. Acknowledgements**

The research was supported by Ministry of science and Ministry of health in Slovenia.

#### **7. References**


RT is an important risk factor for GI in males and pelvic RT as a major risk factor for PH

 unilateral orchidectomy is an important risk factor for germinal epithelium impairment as well as LC impairment , which is attributed to the LC insufficiency in the remaining

 gonadal failure develops during or shortly after administration of toxic therapy and correction of gonadal damage eventually takes place in male as well as in female survivors. In contrast to the germ cell failure, damage of LC can develop within a few years after treatment, and usually there is no correction of LC damage therefore. Therefore, patients with elevated basal or stimulated LH levels require annual monitoring of LH and testosterone and the timely introduction of hormone replacement

But there was no multivariate analysis using the decision tree classification model, which is able to identify links between risk factors for impaired gonadal function. With this model we could also identify a group of patients with the lowest risk of gonadal impairment those who had neither pelvic or testicular RT nor ChT including AA. In those hormonal testing

The research was supported by Ministry of science and Ministry of health in Slovenia.

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**1. Introduction** 

androgen levels imply adequate estrogen levels).

**2. Qualities of bone in men** 

**8** 

*Poland* 

**Osteoporosis in Men - A Crucial** 

**Role of Sex Hormones** 

*Department of Endocrinology,* 

Michał Rabijewski and Lucyna Papierska

*Medical Centre for Postgraduate Education, Warsaw,* 

Osteoporosis is a disorder characterized by reduced bone mass, impaired bone quality and a propensity to fracture. Traditionally, osteoporosis has been viewed as a syndrome characterized by back pain and vertebral fractures. Osteoporotic fractures have long been regarded as a female ailment, but with the increasing longevity of men, it appears that the incidence and prevalence of osteoporotic fractures in men is not very different from the rate in women although it occurs approximately 10 years later in the lives of men. Morbidity and mortality associated with fractures and their (surgical) treatment is considerably greater than in women. A multitude of factors determine bone strength: genetic, nutritional (calcium), vitamin D, physical activity, and hormonal factors. Hormonal factors are significant throughout life, from puberty onwards. In adolescence they are indispensable for the formation of peak bone mass. Throughout life, sex steroids maintain bone formation. Surprisingly, in men estrogens appear to be more significant for the development of peak bone mass and the maintenance of bone mineral density than androgens. In men estrogens are derived from androgens and levels of both of them are strongly interrelated (adequate

Fracture is the result of failure of the material composition and the structural design of bone to tolerate the loads imposed upon it. These properties, or bone 'qualities', are compromised with the emergence of age-related abnormalities in bone modelling and remodelling, the cellular machinery responsible for the attainment of peak bone strength during growth and its maintenance during adulthood. The abnormalities contributing to material and structural decay are: a negative bone balance produced by each basic multicellular unit (BMU), a sustained increase in remodelling intensity in midlife in women but not in men unless frankly hypogonadal, reduced periosteal apposition after completion of longitudinal growth and secondary hyperparathyroidism (Khosla et al. 2006). However, the most important cause of bone loss is the increase in the intensity of bone remodelling on the trabecular, intracortical and endocortical bone surfaces (Amin & Felson 2001; Benito et al. 2004). The trabecular bone loss proceeds mainly by thinning in men - see Figure 1. Remodelling on intracortical surfaces results in intracortical porosity, particularly in cortex adjacent to the


### **Osteoporosis in Men - A Crucial Role of Sex Hormones**

Michał Rabijewski and Lucyna Papierska *Department of Endocrinology, Medical Centre for Postgraduate Education, Warsaw, Poland* 

#### **1. Introduction**

196 Sex Hormones

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Osteoporosis is a disorder characterized by reduced bone mass, impaired bone quality and a propensity to fracture. Traditionally, osteoporosis has been viewed as a syndrome characterized by back pain and vertebral fractures. Osteoporotic fractures have long been regarded as a female ailment, but with the increasing longevity of men, it appears that the incidence and prevalence of osteoporotic fractures in men is not very different from the rate in women although it occurs approximately 10 years later in the lives of men. Morbidity and mortality associated with fractures and their (surgical) treatment is considerably greater than in women. A multitude of factors determine bone strength: genetic, nutritional (calcium), vitamin D, physical activity, and hormonal factors. Hormonal factors are significant throughout life, from puberty onwards. In adolescence they are indispensable for the formation of peak bone mass. Throughout life, sex steroids maintain bone formation. Surprisingly, in men estrogens appear to be more significant for the development of peak bone mass and the maintenance of bone mineral density than androgens. In men estrogens are derived from androgens and levels of both of them are strongly interrelated (adequate androgen levels imply adequate estrogen levels).

#### **2. Qualities of bone in men**

Fracture is the result of failure of the material composition and the structural design of bone to tolerate the loads imposed upon it. These properties, or bone 'qualities', are compromised with the emergence of age-related abnormalities in bone modelling and remodelling, the cellular machinery responsible for the attainment of peak bone strength during growth and its maintenance during adulthood. The abnormalities contributing to material and structural decay are: a negative bone balance produced by each basic multicellular unit (BMU), a sustained increase in remodelling intensity in midlife in women but not in men unless frankly hypogonadal, reduced periosteal apposition after completion of longitudinal growth and secondary hyperparathyroidism (Khosla et al. 2006). However, the most important cause of bone loss is the increase in the intensity of bone remodelling on the trabecular, intracortical and endocortical bone surfaces (Amin & Felson 2001; Benito et al. 2004). The trabecular bone loss proceeds mainly by thinning in men - see Figure 1. Remodelling on intracortical surfaces results in intracortical porosity, particularly in cortex adjacent to the

al. 2006).

**3.2 Estrogens and bone** 

loss (Barrett-Connor et al. 2000).

also normalizes plasma estradiol levels.

**3.3 Current model of influence of sex hormones on bone in men** 

essential for the process of periosteal bone expansion in men.

Osteoporosis in Men - A Crucial Role of Sex Hormones 199

total testosterone and estradiol are more likely to be osteoporotic and at greater risk of hip fracture. Men with a deficiency of total testosterone were more likely to have rapid bone loss from the hip or to suffer from hip fractures following minimal trauma. Also men with osteoporosis were more likely to have a deficiency of testosterone and estradiol (Khosla et

Estrogens play also an essential role in maintaining bone mass in men.. Men with estrogens deficiency or impaired estrogens action have delayed epiphyseal closure and osteopenia (Monshima et al. 1991). It was shown that in men with aromatase deficiency the administration of estrogen had a significant beneficial effect on skeletal growth and bone maturation (Rochira 2000). In elderly men, estrogen seems to play a more dominant role than testosterone in regulating bone resorption. In elderly men, lower than normal levels of estrogen appeared to be associated with vertebral fractures (Rochira et al. 2006). Age-related decreases of estradiol, especially levels below 40 pmol/l, may be the major cause of bone

Estrogens in men are predominately the product of peripheral aromatization of androgens. Androgens (androstenedione, dehydroepiandrosterone produced by the adrenal gland, and testosterone produced by the testis) serve as precursors for chemical conversion to estrone and estradiol via the enzyme aromatase. The testis itself produce approximately 20% of the total estradiol. Adipose tissue is the most important source of estrogens in men. Plasma testosterone levels show an age-related decline while plasma estrogen levels in men remain relatively constant with aging resulting in an increased estrogen/androgen ratio. Estrogen deficiency in elderly men is tightly coupled to androgen deficiency since all estrogens are derived from androgens through aromatization. Restoring plasma testosterone to normal

Recently data have challenged the traditional concept of stimulatory *vs.* inhibitory effects of androgens and estrogens. The role of androgens as the main determinant of male bone acquisition has been challenged by observations in men with aromatase deficiency (Carani et al. 1997; Rochira et al. 2000). These men, who have normal androgen concentrations, but undetectable levels of endogenous estrogen, have surprisingly low bone mass and areal density and respond very well to estrogen therapy. Reduced bone mass in these subjects is not due to reduced volumetric bone density, but reflects a deficit in bone size and the increase in bone mass during estrogen therapy was found to be driven primarily by an increase in bone size, without treatment effect on volumetric density. The enlargement of cortical bone reflected *periosteal apposition*. As evidenced in the aromatase-deficient adolescent androgens alone may not be sufficient to drive periosteal expansion. Moreover, the periosteal expansion observed in response to estrogen therapy demonstrates that estrogens stimulate rather than inhibit periosteal apposition. While, exposure to estrogens is

Thus, the interaction of estrogen with the periosteum has been studied. Animal data suggest that estrogens may decrease the set point of the mechanostat and thereby increase the sensitivity of bone for mechanical stimuli (Lee 2003). This may indirectly impact on the response of the bone to androgens, because androgens increase lean body mass and the

marrow cavity, trabecularisation of the endosteal cortex and a decrease in cortical width. The effect is likely greater in women than in men. Periosteal apposition slows after completion of growth. Some studies suggest that men have greater periosteal apposition than women (Vanderschueren et al. 2004).

Fig. 1. Gender differences in pattern of trabecular bone loss resulting in trabecular thinning in men and increased cortical porosity and trabecular perforation in women.

Compared with women, men have greater bone strength not because of increased bone mineral density, but because of greater bone size, resulting from bone acquired at the periosteal surface. Periosteal bone formation is a result of two endocrine effects: androgenmediated stimulatory effects on periosteal bone formation and estrogen induced inhibitory effects on periosteal expansion. So, greater periosteal bone expansion in men has been traditional assumed to result from exposure to higher levels of androgens and/or lower levels of estrogens (Vanderschueren et al. 2006).

#### **3. Hormonal determinants of bone mass**

#### **3.1 Androgens and bone**

Androgens are pivotal for the acquisition of bone mass in adolescence and the maintenance of bone mass in adulthood (Finkelstein et al. 1996). In men, chronically low androgen levels are associated with low bone mass, and testosterone replacement can enhance BMD. However, it is not yet precisely clear what role androgens play in the maintenance of bone mass in men. Peak bone mass is acquired between the ages of 12–16 years, and even beyond. It represents the sum of several processes including a marked increase in bone formation. Boys tend to reach peak 2 years later than girls and their BMD is higher than that in women at all skeletal sites. In part this relates to a greater cross-sectional bone area in males. The timing of gonadal steroid surges are critical for bone acquisition since there is a relatively short window of time in which bone formation is favored and matrix synthesis is markedly enhanced (Rochira et al. 2006). In adulthood normal testosterone levels are required for the maintenance of BMD. Hypogonadal men suffer from osteoporosis, and bone fractures in men in their forties or fifties may well be the first manifestation of undiagnosed hypogonadism (Vanderschueren et al. 2004). Variations of free testosterone within the normal range are an independent predictor of cortical bone density, and also of previous osteoporosis-related fractures. Elderly men and older men with a deficiency in total testosterone and estradiol are more likely to be osteoporotic and at greater risk of hip fracture. Men with a deficiency of total testosterone were more likely to have rapid bone loss from the hip or to suffer from hip fractures following minimal trauma. Also men with osteoporosis were more likely to have a deficiency of testosterone and estradiol (Khosla et al. 2006).

#### **3.2 Estrogens and bone**

198 Sex Hormones

marrow cavity, trabecularisation of the endosteal cortex and a decrease in cortical width. The effect is likely greater in women than in men. Periosteal apposition slows after completion of growth. Some studies suggest that men have greater periosteal apposition

Fig. 1. Gender differences in pattern of trabecular bone loss resulting in trabecular thinning

Compared with women, men have greater bone strength not because of increased bone mineral density, but because of greater bone size, resulting from bone acquired at the periosteal surface. Periosteal bone formation is a result of two endocrine effects: androgenmediated stimulatory effects on periosteal bone formation and estrogen induced inhibitory effects on periosteal expansion. So, greater periosteal bone expansion in men has been traditional assumed to result from exposure to higher levels of androgens and/or lower

Androgens are pivotal for the acquisition of bone mass in adolescence and the maintenance of bone mass in adulthood (Finkelstein et al. 1996). In men, chronically low androgen levels are associated with low bone mass, and testosterone replacement can enhance BMD. However, it is not yet precisely clear what role androgens play in the maintenance of bone mass in men. Peak bone mass is acquired between the ages of 12–16 years, and even beyond. It represents the sum of several processes including a marked increase in bone formation. Boys tend to reach peak 2 years later than girls and their BMD is higher than that in women at all skeletal sites. In part this relates to a greater cross-sectional bone area in males. The timing of gonadal steroid surges are critical for bone acquisition since there is a relatively short window of time in which bone formation is favored and matrix synthesis is markedly enhanced (Rochira et al. 2006). In adulthood normal testosterone levels are required for the maintenance of BMD. Hypogonadal men suffer from osteoporosis, and bone fractures in men in their forties or fifties may well be the first manifestation of undiagnosed hypogonadism (Vanderschueren et al. 2004). Variations of free testosterone within the normal range are an independent predictor of cortical bone density, and also of previous osteoporosis-related fractures. Elderly men and older men with a deficiency in

in men and increased cortical porosity and trabecular perforation in women.

than women (Vanderschueren et al. 2004).

levels of estrogens (Vanderschueren et al. 2006).

**3. Hormonal determinants of bone mass** 

**3.1 Androgens and bone** 

Estrogens play also an essential role in maintaining bone mass in men.. Men with estrogens deficiency or impaired estrogens action have delayed epiphyseal closure and osteopenia (Monshima et al. 1991). It was shown that in men with aromatase deficiency the administration of estrogen had a significant beneficial effect on skeletal growth and bone maturation (Rochira 2000). In elderly men, estrogen seems to play a more dominant role than testosterone in regulating bone resorption. In elderly men, lower than normal levels of estrogen appeared to be associated with vertebral fractures (Rochira et al. 2006). Age-related decreases of estradiol, especially levels below 40 pmol/l, may be the major cause of bone loss (Barrett-Connor et al. 2000).

Estrogens in men are predominately the product of peripheral aromatization of androgens. Androgens (androstenedione, dehydroepiandrosterone produced by the adrenal gland, and testosterone produced by the testis) serve as precursors for chemical conversion to estrone and estradiol via the enzyme aromatase. The testis itself produce approximately 20% of the total estradiol. Adipose tissue is the most important source of estrogens in men. Plasma testosterone levels show an age-related decline while plasma estrogen levels in men remain relatively constant with aging resulting in an increased estrogen/androgen ratio. Estrogen deficiency in elderly men is tightly coupled to androgen deficiency since all estrogens are derived from androgens through aromatization. Restoring plasma testosterone to normal also normalizes plasma estradiol levels.

#### **3.3 Current model of influence of sex hormones on bone in men**

Recently data have challenged the traditional concept of stimulatory *vs.* inhibitory effects of androgens and estrogens. The role of androgens as the main determinant of male bone acquisition has been challenged by observations in men with aromatase deficiency (Carani et al. 1997; Rochira et al. 2000). These men, who have normal androgen concentrations, but undetectable levels of endogenous estrogen, have surprisingly low bone mass and areal density and respond very well to estrogen therapy. Reduced bone mass in these subjects is not due to reduced volumetric bone density, but reflects a deficit in bone size and the increase in bone mass during estrogen therapy was found to be driven primarily by an increase in bone size, without treatment effect on volumetric density. The enlargement of cortical bone reflected *periosteal apposition*. As evidenced in the aromatase-deficient adolescent androgens alone may not be sufficient to drive periosteal expansion. Moreover, the periosteal expansion observed in response to estrogen therapy demonstrates that estrogens stimulate rather than inhibit periosteal apposition. While, exposure to estrogens is essential for the process of periosteal bone expansion in men.

Thus, the interaction of estrogen with the periosteum has been studied. Animal data suggest that estrogens may decrease the set point of the mechanostat and thereby increase the sensitivity of bone for mechanical stimuli (Lee 2003). This may indirectly impact on the response of the bone to androgens, because androgens increase lean body mass and the

Osteoporosis in Men - A Crucial Role of Sex Hormones 201

to perforation. Reasons for sex differences in bone fragility are multifaceted: sex differences in trabecular morphology (thinning in men and perforation in women), less cortical porosity, endocortical remodelling and thinning in men, particularly and possibly greater periosteal apposition in men. Other factors that may contribute to sex differences in bone fragility include differences in osteonal morphology, tissue mineralisation and matrix composition (Amin et al. 2001; Seeman et al. 2002;Vanderschueren et al. 2004). Model of sex

Fig. 2. Model of sex hormones action on bone: testosterone stimulates periosteal expansion, whereas estradiol has a double action on periosteal bone apposition. Estrogen action on the

It is agreed that the life-time risk of osteoporotic fracture in men is around one-third of that in women About 4 to 6 percent of men older than 50 have osteoporosis, and 33 to 47 percent have osteopenia. The prevalence of osteoporosis is 7 percent in white men, 5 percent in black men, and about 3 percent in Hispanic-American men. Because men have greater bone mass, they present with osteoporotic fracture about 10 years later than women. Thus, starting at about age 75, the incidence of hip fracture increases rapidly. The life-time risk of a hip fracture in Caucasian men is 13 to 25% (Cooper et al.1992; Johnell et al. 2005). Because of the predicted growth in the number of elderly persons, the number of men with osteoporotic

Fractures represent the primary clinical consequence of osteoporosis. The difference in fracture incidence observed between men and women is due not only to a difference in their bone strength but also to the type and frequency of trauma experienced by men over life. Fractures of the hip, vertebrae and forearm are more likely to occur after minimal trauma in aging men. Of these fractures, those involving the hip and vertebrae are associated with some of the greatest morbidity and mortality for men (Anderson et al.1999). The fracture incidence in men follows a bimodal distribution tending to peak in adolescence and with advanced age. Although women have a greater incidence of fractures with aging, men are

hormones action on bone is shown on Figure 2.

periosteum may be also result of indirectly changes in IGF-I.

**4. Epidemiology of osteoporosis in men** 

fracture is expected to increase.

**5. Fractures in men** 

mechanical loading of the male skeleton and this mechanical loading constitutes one of the main triggers for androgen induced periosteal apposition. Androgens indirectly induce mechanical loading through their anabolic action on muscle, and there is growing evidence that estrogens interact with this process. Exposure to estrogen may be critical to allow the increased loading to be translated into periosteal bone formation and radial expansion (Vanderschueren 2003).

Estrogen action on bone may reflect an interaction with GH and/or IGF-I, two main determinants of cortical bone growth (Bateman et al. 1998). Estrogens are known to have a biphasic effect on pubertal skeletal growth. During early puberty, low estrogen concentrations increase the secretion of GH and IGF-I synthesis. As a consequence, estrogens stimulate skeletal growth. Hence, sex steroid-related changes in GH and IGF-I secretion may impact on bone size and cross-sectional area. By the end of puberty, elevated concentrations of estrogen limit skeletal longitudinal growth through a direct effect on growth plate closure. By the end of puberty, estrogens may inhibit radial growth in men (Juul 2001). Estrogen-related changes in serum IGF-I may be more important than direct estrogen-mediated stimulation of the periosteal surfaces. Estradiol stimulates radial bone growth as a result of an up-regulation of hepatic IGF-I synthesis and secretion, but only ER activation results in changes in serum IGF-I, indicating that AR-mediated androgen action, is independent of GH and/or IGF-I.

Effects of estrogen on periosteal bone are dose dependent. Low levels of estrogen might increase the mechanical sensitivity of the periosteum and/or affect circulating IGF-I levels, while higher concentrations of estrogen might inhibit periosteal bone apposition and its interaction with mechanical loading, possibly through an ERβ effect (Moverare et al. 2003). The inhibitory effect of estrogen is not observed in men, because men are exposed to low endogenous estrogen concentrations. Also disruption of ERβ does not affect the male cortical phenotype. Men exhibit more periosteal expansion because they are more exposed to the stimulatory effects of androgens and less exposed to the inhibitory effects of estrogens. Androgens may primarily affect lean body mass and the loading of the male skeleton; exposure to low-dose estrogen may allow this loading to induce bone expansion.

In addition to the changes in trabecular and cortical morphology, abnormalities in bone remodelling produce changes in bone 'qualities' at higher levels of resolution. Advancing age is associated with a reduction in osteonal size, accompanied by an increase in haversian canal diameter due to the reduced bone formation by each BMU. Smaller osteons result in less resistance to crack propagation through interstitial bone (Khosla et al. 2006). Also, smaller osteons give rise to a greater proportion interstitial (rather than osteonal) bone which has a higher tissue mineralization density, fewer osteocytes and is liable to accumulate microdamage and offer less resistance to crack propagation. These phenomena are likely to occur in both sexes but in women, osteonal density (the number per unit volume) may increase because of the high intracortical remodelling; the osteons are smaller but there are more of them (Amin et al. 2001).

Sex differences in bone size are contributing to the lower incidence of fractures in men. Men also have larger muscle mass and higher body weight so that compressive stress (load/area) is similar in young adult men and women. Men and women have similar cortical thickness which confers a greater cortical area in men (because their bones have a larger perimeter). Peak trabecular number and thickness is similar in men and women at the iliac crest and vertebral bone. Men have thicker trabeculae at the distal radius which may be more resistant

mechanical loading of the male skeleton and this mechanical loading constitutes one of the main triggers for androgen induced periosteal apposition. Androgens indirectly induce mechanical loading through their anabolic action on muscle, and there is growing evidence that estrogens interact with this process. Exposure to estrogen may be critical to allow the increased loading to be translated into periosteal bone formation and radial expansion

Estrogen action on bone may reflect an interaction with GH and/or IGF-I, two main determinants of cortical bone growth (Bateman et al. 1998). Estrogens are known to have a biphasic effect on pubertal skeletal growth. During early puberty, low estrogen concentrations increase the secretion of GH and IGF-I synthesis. As a consequence, estrogens stimulate skeletal growth. Hence, sex steroid-related changes in GH and IGF-I secretion may impact on bone size and cross-sectional area. By the end of puberty, elevated concentrations of estrogen limit skeletal longitudinal growth through a direct effect on growth plate closure. By the end of puberty, estrogens may inhibit radial growth in men (Juul 2001). Estrogen-related changes in serum IGF-I may be more important than direct estrogen-mediated stimulation of the periosteal surfaces. Estradiol stimulates radial bone growth as a result of an up-regulation of hepatic IGF-I synthesis and secretion, but only ER activation results in changes in serum IGF-I, indicating that AR-mediated androgen action,

Effects of estrogen on periosteal bone are dose dependent. Low levels of estrogen might increase the mechanical sensitivity of the periosteum and/or affect circulating IGF-I levels, while higher concentrations of estrogen might inhibit periosteal bone apposition and its interaction with mechanical loading, possibly through an ERβ effect (Moverare et al. 2003). The inhibitory effect of estrogen is not observed in men, because men are exposed to low endogenous estrogen concentrations. Also disruption of ERβ does not affect the male cortical phenotype. Men exhibit more periosteal expansion because they are more exposed to the stimulatory effects of androgens and less exposed to the inhibitory effects of estrogens. Androgens may primarily affect lean body mass and the loading of the male skeleton; exposure to low-dose estrogen may allow this loading to induce bone expansion. In addition to the changes in trabecular and cortical morphology, abnormalities in bone remodelling produce changes in bone 'qualities' at higher levels of resolution. Advancing age is associated with a reduction in osteonal size, accompanied by an increase in haversian canal diameter due to the reduced bone formation by each BMU. Smaller osteons result in less resistance to crack propagation through interstitial bone (Khosla et al. 2006). Also, smaller osteons give rise to a greater proportion interstitial (rather than osteonal) bone which has a higher tissue mineralization density, fewer osteocytes and is liable to accumulate microdamage and offer less resistance to crack propagation. These phenomena are likely to occur in both sexes but in women, osteonal density (the number per unit volume) may increase because of the high intracortical remodelling; the osteons are smaller

Sex differences in bone size are contributing to the lower incidence of fractures in men. Men also have larger muscle mass and higher body weight so that compressive stress (load/area) is similar in young adult men and women. Men and women have similar cortical thickness which confers a greater cortical area in men (because their bones have a larger perimeter). Peak trabecular number and thickness is similar in men and women at the iliac crest and vertebral bone. Men have thicker trabeculae at the distal radius which may be more resistant

(Vanderschueren 2003).

is independent of GH and/or IGF-I.

but there are more of them (Amin et al. 2001).

to perforation. Reasons for sex differences in bone fragility are multifaceted: sex differences in trabecular morphology (thinning in men and perforation in women), less cortical porosity, endocortical remodelling and thinning in men, particularly and possibly greater periosteal apposition in men. Other factors that may contribute to sex differences in bone fragility include differences in osteonal morphology, tissue mineralisation and matrix composition (Amin et al. 2001; Seeman et al. 2002;Vanderschueren et al. 2004). Model of sex hormones action on bone is shown on Figure 2.

Fig. 2. Model of sex hormones action on bone: testosterone stimulates periosteal expansion, whereas estradiol has a double action on periosteal bone apposition. Estrogen action on the periosteum may be also result of indirectly changes in IGF-I.

#### **4. Epidemiology of osteoporosis in men**

It is agreed that the life-time risk of osteoporotic fracture in men is around one-third of that in women About 4 to 6 percent of men older than 50 have osteoporosis, and 33 to 47 percent have osteopenia. The prevalence of osteoporosis is 7 percent in white men, 5 percent in black men, and about 3 percent in Hispanic-American men. Because men have greater bone mass, they present with osteoporotic fracture about 10 years later than women. Thus, starting at about age 75, the incidence of hip fracture increases rapidly. The life-time risk of a hip fracture in Caucasian men is 13 to 25% (Cooper et al.1992; Johnell et al. 2005). Because of the predicted growth in the number of elderly persons, the number of men with osteoporotic fracture is expected to increase.

#### **5. Fractures in men**

Fractures represent the primary clinical consequence of osteoporosis. The difference in fracture incidence observed between men and women is due not only to a difference in their bone strength but also to the type and frequency of trauma experienced by men over life. Fractures of the hip, vertebrae and forearm are more likely to occur after minimal trauma in aging men. Of these fractures, those involving the hip and vertebrae are associated with some of the greatest morbidity and mortality for men (Anderson et al.1999). The fracture incidence in men follows a bimodal distribution tending to peak in adolescence and with advanced age. Although women have a greater incidence of fractures with aging, men are

Osteoporosis in Men - A Crucial Role of Sex Hormones 203

Development of osteoporosis in men is primarily related to aging and genetic factors but30 to 60 percent of cases of osteoporosis are associated with one or more secondary risk factors. The three major causes of secondary osteoporosis in men are alcohol abuse, glucocorticoid glucocorticoid therapy and hypogonadism. Of these, glucocorticoid-induced osteoporosis is

Long-term oral glucocorticoid therapy accounts for nearly one in six cases of male osteoporosis. The extent of bone loss is related to the duration of therapy and the dosage of the steroid. Because of the high risk of bone loss, treatment of osteoporosis is recommended for any patient taking 5 mg or more of steroids per day for longer than six months. The recommended treatment is a bisphosphonate supplemented with calcium and vitamin D

Tobacco use and excessive alcohol consumption are more prevalent in men than in women, and both are independently associated with an increased incidence of osteoporotic fractures (Anderson et al.1997). Tobacco-related bone loss is linked to smoking duration and quantity. The mechanism may be a combination of decreased body weight, decreased calcium absorption, decreased estradiol levels, and a direct toxic effect on bone metabolism. Alcohol in modest amounts may have a protective effect on bone density, but sustained high consumption causes bone loss. It is likely that alcohol has a direct toxic effect on osteoblastic function. Excessive alcohol consumption is also often associated with poor nutrition and decreased physical activity, both of which are associated with bone loss (Seeman et al. 1983). Further studies are needed to determine the quantity of alcohol above which the protective

Hypogonadism induces a state of high bone turnover with accelerated bone loss and increased fracture risk which results from combined deficiency of testosterone and estradiol (Goderie-Plomp et al. 2004). Symptomatic hypogonadism is an indication for testosterone substitution in all causes (primary and secondary hypogonadism). .Also in men with history of delayed poberty peak bone mass in significant lower than in healthy men, what can be the reason of greater risk of fracture and high incidence of osteoporosis in this population

Aging is accompanied by progressive moderate decrease in the population mean serum concentration of total testosterone. A marked age-related increase of serum sex hormonebinding globulin (SHBG) levels is also found which results in a decrease in the non-SHBGbound fractions of testosterone available for biological action, i.e. decreased free- and bioavailable testosterone, as well as a moderate decrease of free- and bioavailable estradiol (Araujo 2004)/ . Aging in men is also accompanied by increasing prevalence of signs and symptoms, including osteoporosis, that are reminiscent of those observed in young hypogonadal men, but which in the elderly are at most only in part related to the decline of testosterone production. With age, both low serum testosterone and symptoms and signs

**6. Risk factors for osteoporosis in men** 

(Recommendation of American Collefe of Rheumatology 2001).

the most common.

**6.1 Glucocorticoid therapy** 

**6.2 Tobacco and alcohol use** 

effect ceases and bone loss occurs.

**6.3 Hypogonadism** 

(Finkelstein et al. 1996).

actually more likely than women to sustain a fracture at younger ages, what is related, in part, to the greater frequency of severe trauma associated with their fractures. The incidence rate for a work-related fracture for male employees is more than twice that for female employees, These fractures appear to be related to high-energy trauma events (Anderson & Cooper 1999). After the age of 50 yr, the trend reverses, with women tending to have a higher incidence of overall fractures than men. In both men and women, there is an exponential rise in fracture incidence after age 75 yr, particularly for hip fractures; however, the absolute incidence tends to be lower in men. Osteoporotic fractures in men appear to involve fractures of the hip, vertebrae, forearm, and humerus, although fragility fractures at other sites, including the pelvis, ribs, and clavicle, also occur in aging men. Of all the hip, forearm and clinical vertebral fractures, approximately 30, 20, and 40% occur in men, respectively. The lower absolute incidence in osteoporotic fractures in older men may be due to the an increased frequency of falls in women (Bilezikian 1999; Campion & Maricic 2003).

#### **5.1 Hip fractures**

Among all osteoporotic fractures, hip fractures account for the greatest morbidity and mortality for men. Overall, the incidence of hip fractures in men is uncommon until after the age of 75 yr, when the risk increases exponentially. The age-adjusted female to male ratio for hip fractures has been observed to be highest for whites, with a ratio up to 3–4:1 (Maggi et al.1991). With the improving longevity of men and the increasing size of the population, the number of men with hip fracture worldwide is estimated to reach 1.8 million in 2050 (Khosla et al. 2006). The mortality and morbidity associated with hip fractures are greater for men than women (Forsens et al. 1999). Men are twice as likely to die in hospital after a hip fracture as women. Estimates for the 1-yr mortality rate after hip fracture ranges from 31– 35% in men compared with 17–22% in women (Bass et al. 2007). Greater number of comorbid conditions at the time of fracture contribute to mortality risk and up to 50% of men may need institutionalized care after hip fracture. Men are less likely to return to autonomous living circumstances than women at 1 yr after hip fracture. In men ages 60–69 yr, the decrease in life expectancy after a hip fracture is 11.5 yr, compared with men ages 70– 79 yr and age 80 yr or older, where the decrease in life expectancy is 5 and 1.5 yr, respectively (Center et al.1999). Despite these facts, men are less likely to be investigated or treated for osteoporosis after hip fracture.

#### **5.2 Vertebral fractures**

Vertebral fractures are not always associated with pain that would bring them to clinical attention. The true incidence of these fractures is often underestimated. The agestandardized prevalence of vertebral deformity in Europe is estimated to be the same for both men and women, either 12 or 20%, depending on the criteria used to define vertebral deformity (O'Neill et al.1996). Below age 65 yr, men had a higher prevalence of vertebral deformity than women, whereas after this age the trend was reversed. The prevalence of vertebral deformity increased with age. The age-adjusted incidence for radiographically defined vertebral fractures in men is half the rate of women. In a 10-yr study prevalent vertebral deformity was a predictor of mortality in men during the forthcoming decade (age-adjusted hazard ratio, 2.4) (Hasserius rt al. 2003). Severe vertebral deformity is related to functional impairment and the association between vertebral deformity and negative health outcomes appears to be even stronger in men than in women.

#### **6. Risk factors for osteoporosis in men**

Development of osteoporosis in men is primarily related to aging and genetic factors but30 to 60 percent of cases of osteoporosis are associated with one or more secondary risk factors. The three major causes of secondary osteoporosis in men are alcohol abuse, glucocorticoid glucocorticoid therapy and hypogonadism. Of these, glucocorticoid-induced osteoporosis is the most common.

#### **6.1 Glucocorticoid therapy**

202 Sex Hormones

actually more likely than women to sustain a fracture at younger ages, what is related, in part, to the greater frequency of severe trauma associated with their fractures. The incidence rate for a work-related fracture for male employees is more than twice that for female employees, These fractures appear to be related to high-energy trauma events (Anderson & Cooper 1999). After the age of 50 yr, the trend reverses, with women tending to have a higher incidence of overall fractures than men. In both men and women, there is an exponential rise in fracture incidence after age 75 yr, particularly for hip fractures; however, the absolute incidence tends to be lower in men. Osteoporotic fractures in men appear to involve fractures of the hip, vertebrae, forearm, and humerus, although fragility fractures at other sites, including the pelvis, ribs, and clavicle, also occur in aging men. Of all the hip, forearm and clinical vertebral fractures, approximately 30, 20, and 40% occur in men, respectively. The lower absolute incidence in osteoporotic fractures in older men may be due to the an increased frequency of

Among all osteoporotic fractures, hip fractures account for the greatest morbidity and mortality for men. Overall, the incidence of hip fractures in men is uncommon until after the age of 75 yr, when the risk increases exponentially. The age-adjusted female to male ratio for hip fractures has been observed to be highest for whites, with a ratio up to 3–4:1 (Maggi et al.1991). With the improving longevity of men and the increasing size of the population, the number of men with hip fracture worldwide is estimated to reach 1.8 million in 2050 (Khosla et al. 2006). The mortality and morbidity associated with hip fractures are greater for men than women (Forsens et al. 1999). Men are twice as likely to die in hospital after a hip fracture as women. Estimates for the 1-yr mortality rate after hip fracture ranges from 31– 35% in men compared with 17–22% in women (Bass et al. 2007). Greater number of comorbid conditions at the time of fracture contribute to mortality risk and up to 50% of men may need institutionalized care after hip fracture. Men are less likely to return to autonomous living circumstances than women at 1 yr after hip fracture. In men ages 60–69 yr, the decrease in life expectancy after a hip fracture is 11.5 yr, compared with men ages 70– 79 yr and age 80 yr or older, where the decrease in life expectancy is 5 and 1.5 yr, respectively (Center et al.1999). Despite these facts, men are less likely to be investigated or

Vertebral fractures are not always associated with pain that would bring them to clinical attention. The true incidence of these fractures is often underestimated. The agestandardized prevalence of vertebral deformity in Europe is estimated to be the same for both men and women, either 12 or 20%, depending on the criteria used to define vertebral deformity (O'Neill et al.1996). Below age 65 yr, men had a higher prevalence of vertebral deformity than women, whereas after this age the trend was reversed. The prevalence of vertebral deformity increased with age. The age-adjusted incidence for radiographically defined vertebral fractures in men is half the rate of women. In a 10-yr study prevalent vertebral deformity was a predictor of mortality in men during the forthcoming decade (age-adjusted hazard ratio, 2.4) (Hasserius rt al. 2003). Severe vertebral deformity is related to functional impairment and the association between vertebral deformity and negative

health outcomes appears to be even stronger in men than in women.

falls in women (Bilezikian 1999; Campion & Maricic 2003).

treated for osteoporosis after hip fracture.

**5.2 Vertebral fractures** 

**5.1 Hip fractures** 

Long-term oral glucocorticoid therapy accounts for nearly one in six cases of male osteoporosis. The extent of bone loss is related to the duration of therapy and the dosage of the steroid. Because of the high risk of bone loss, treatment of osteoporosis is recommended for any patient taking 5 mg or more of steroids per day for longer than six months. The recommended treatment is a bisphosphonate supplemented with calcium and vitamin D (Recommendation of American Collefe of Rheumatology 2001).

#### **6.2 Tobacco and alcohol use**

Tobacco use and excessive alcohol consumption are more prevalent in men than in women, and both are independently associated with an increased incidence of osteoporotic fractures (Anderson et al.1997). Tobacco-related bone loss is linked to smoking duration and quantity. The mechanism may be a combination of decreased body weight, decreased calcium absorption, decreased estradiol levels, and a direct toxic effect on bone metabolism. Alcohol in modest amounts may have a protective effect on bone density, but sustained high consumption causes bone loss. It is likely that alcohol has a direct toxic effect on osteoblastic function. Excessive alcohol consumption is also often associated with poor nutrition and decreased physical activity, both of which are associated with bone loss (Seeman et al. 1983). Further studies are needed to determine the quantity of alcohol above which the protective effect ceases and bone loss occurs.

#### **6.3 Hypogonadism**

Hypogonadism induces a state of high bone turnover with accelerated bone loss and increased fracture risk which results from combined deficiency of testosterone and estradiol (Goderie-Plomp et al. 2004). Symptomatic hypogonadism is an indication for testosterone substitution in all causes (primary and secondary hypogonadism). .Also in men with history of delayed poberty peak bone mass in significant lower than in healthy men, what can be the reason of greater risk of fracture and high incidence of osteoporosis in this population (Finkelstein et al. 1996).

Aging is accompanied by progressive moderate decrease in the population mean serum concentration of total testosterone. A marked age-related increase of serum sex hormonebinding globulin (SHBG) levels is also found which results in a decrease in the non-SHBGbound fractions of testosterone available for biological action, i.e. decreased free- and bioavailable testosterone, as well as a moderate decrease of free- and bioavailable estradiol (Araujo 2004)/ . Aging in men is also accompanied by increasing prevalence of signs and symptoms, including osteoporosis, that are reminiscent of those observed in young hypogonadal men, but which in the elderly are at most only in part related to the decline of testosterone production. With age, both low serum testosterone and symptoms and signs

Osteoporosis in Men - A Crucial Role of Sex Hormones 205

History of nontraumatic fracture (hip, vertebrae or wrist)

Glucocorticoid use of 5mg of more for longer than six months

Osteopenia seen on plane radiograph

Excess alcohol consumption and tobacco use Rheumatoid and other inflammatory arthritis

Prolonged immobilization, lack of physical activity Conditions associated with increased risk of falling

Osteoporosis can be defined as ''a skeletal disease'' characterized by low bone mass and micro-architectural deterioration of bone tissue, leading to enhanced bone fragility and a consequent increase in fracture risk. The World Health Organization has definitions for osteopenia and osteoporosis in women, but not in men. In women, osteopenia is defined as a T score of between -1 and -2.5, and osteoporosis as a T-score of -2.5 or more. The T-score is the number of standard deviations by which the bone mineral density of an individual person differs from that of the young, normal mean of the same population. A strength of this diagnostic threshold has been the fashioning of a common approach to describe the disease. The WHO have supported the use of a single reference population (white women aged 20-29 years), a single reference site (the femoral neck) and a single technology (DXA)

Osteoporosis in men would be defined as a BMD value at the femoral neck that lay 2.5 SD or more below the average value of young healthy women The many studies that have examined fracture risk in men have come to disparate conclusions concerning the relationship between fracture risk and BMD. The relation between BMD and fracture risk changes with age, so that age-adjustment is required. The comparative studies show that the

Multiple myeloma ot lymphoma

Family history of osteoporosis

Chronic renal and liver diseases

Gastric or bowel resection, celiac disease

Infrequent causes

Cushing's disease Low body mass index

Table 1. Risk factors for osteoporosis in men.

**7. Diagnosis** 

(Kanis et al. 2008).

Hyperthyroidism and hypothyroidism

High risk causes

Hypogonadism

Hyperparathyroidism Medium risk causes Anticonvulsant grug use

consistent with hypogonadism become increasingly prevalent but also less specific (Zitzmann et al. 2006). The minimal testosterone levels needs in the elderly are not clearly established and may to vary between individuals. In this elderly population, effects of testosterone treatment on BMD have been inconsistent, with the most convincing effects being observed in men with very low serum testosterone (Katznelson et al. 1996; Snyder et al. 2000; Wang et al. 2001). In elderly men, testosterone treatment may have additional beneficial effects on muscle mass and strength, which may help decrease fracture risk through a reduced propensity to fall (Snyder et al.1999).

#### **6.4 Estradiol deficiency**

Declining levels of estrogens levels with age contribute to bone loss and fracture risk in men. Estrogen plays an important role in regulating bone density, bone resorption and bone loss in elderly men as well as in the acquisition of peak bone mass. It seems, that free estradiol and SHBG, but not free testosterone, are independently associated with fracture risk, clinical vertebral fractures, non-vertebral osteoporotic fractures and hip fractures. Specifically, the yearly incidence of fractures was inversely associated with serum estradiol levels at estradiol levels less than 16 pg/ml; above this level, there was no relationship between fracture incidence and estradiol levels (Amin et al., 2000, Barrett-Connor et al. 2000) These findings have clinical implications: measurement of serum sex steroid levels, particularly estradiol levels, in men with osteoporosis and use of selective estrogen receptor modulators (SERMs) in preventing bone loss in aging men may be usefull.

#### **6.5 Vitamin D deficiency**

Vitamin D deficiency is a widespread condition and still poses a major problem to bone health in wide population. Vitamin D and its hydroxylated derivatives can be produced either endogenously after exposure to sunlight or gained from dietary intake. Vitamin D deficiency results in secondary hyperparathyroidism (elevated parathyroid hormone serum levels) followed by demineralization of bone. Secondary hyperparathyroidism usually arises from hypocalcemia, as seen in chronic renal failure or vitamin D deficiency (Campion & Maricic 2003). Significant correlations between low spinal bone mineral density and low vitamin D levels can be observed. Vitamin D represents a key regulator of intestinal calcium absorption and its association with bone metabolism and osteoporosis - osteomalacia - is clear. Aging in males is connected with decreased renal hydroxylation of 25OH-vitamin D to calcitriol what is associated with low action of 1-alpha hydroxylase and may lead to low serum levels of active vitamin D derivatives . In patients with osteomalacia the material properties of properly mineralized bone are not reached and these patients are predisposed to fractures (Bilezikian 1999; Khosla et al. 2006).

#### **6.6 Others risk factors and causes of secondary osteoporosis in men**

Others risk factors which also could be associated with idiopathic osteoporosis in men (Harper & Weber 1998) and the more common others medical conditions which could lead to osteoporosis are listed in Table 1.

Secondary causes of osteoporosis should be excluded before the diagnosis of idiopathic osteoporosis can be made. Low bone mass and falls are both determinants of osteoporotic fracture in men. The following are a list of risk factors which could be associated with idiopathic osteoporosis in men.

consistent with hypogonadism become increasingly prevalent but also less specific (Zitzmann et al. 2006). The minimal testosterone levels needs in the elderly are not clearly established and may to vary between individuals. In this elderly population, effects of testosterone treatment on BMD have been inconsistent, with the most convincing effects being observed in men with very low serum testosterone (Katznelson et al. 1996; Snyder et al. 2000; Wang et al. 2001). In elderly men, testosterone treatment may have additional beneficial effects on muscle mass and strength, which may help decrease fracture risk

Declining levels of estrogens levels with age contribute to bone loss and fracture risk in men. Estrogen plays an important role in regulating bone density, bone resorption and bone loss in elderly men as well as in the acquisition of peak bone mass. It seems, that free estradiol and SHBG, but not free testosterone, are independently associated with fracture risk, clinical vertebral fractures, non-vertebral osteoporotic fractures and hip fractures. Specifically, the yearly incidence of fractures was inversely associated with serum estradiol levels at estradiol levels less than 16 pg/ml; above this level, there was no relationship between

These findings have clinical implications: measurement of serum sex steroid levels, particularly estradiol levels, in men with osteoporosis and use of selective estrogen receptor

Vitamin D deficiency is a widespread condition and still poses a major problem to bone health in wide population. Vitamin D and its hydroxylated derivatives can be produced either endogenously after exposure to sunlight or gained from dietary intake. Vitamin D deficiency results in secondary hyperparathyroidism (elevated parathyroid hormone serum levels) followed by demineralization of bone. Secondary hyperparathyroidism usually arises from hypocalcemia, as seen in chronic renal failure or vitamin D deficiency (Campion & Maricic 2003). Significant correlations between low spinal bone mineral density and low vitamin D levels can be observed. Vitamin D represents a key regulator of intestinal calcium absorption and its association with bone metabolism and osteoporosis - osteomalacia - is clear. Aging in males is connected with decreased renal hydroxylation of 25OH-vitamin D to calcitriol what is associated with low action of 1-alpha hydroxylase and may lead to low serum levels of active vitamin D derivatives . In patients with osteomalacia the material properties of properly mineralized bone are not reached and these patients are predisposed

Others risk factors which also could be associated with idiopathic osteoporosis in men (Harper & Weber 1998) and the more common others medical conditions which could lead

Secondary causes of osteoporosis should be excluded before the diagnosis of idiopathic osteoporosis can be made. Low bone mass and falls are both determinants of osteoporotic fracture in men. The following are a list of risk factors which could be associated with

fracture incidence and estradiol levels (Amin et al., 2000, Barrett-Connor et al. 2000)

modulators (SERMs) in preventing bone loss in aging men may be usefull.

**6.6 Others risk factors and causes of secondary osteoporosis in men** 

through a reduced propensity to fall (Snyder et al.1999).

**6.4 Estradiol deficiency** 

**6.5 Vitamin D deficiency** 

to fractures (Bilezikian 1999; Khosla et al. 2006).

to osteoporosis are listed in Table 1.

idiopathic osteoporosis in men.


Table 1. Risk factors for osteoporosis in men.

#### **7. Diagnosis**

Osteoporosis can be defined as ''a skeletal disease'' characterized by low bone mass and micro-architectural deterioration of bone tissue, leading to enhanced bone fragility and a consequent increase in fracture risk. The World Health Organization has definitions for osteopenia and osteoporosis in women, but not in men. In women, osteopenia is defined as a T score of between -1 and -2.5, and osteoporosis as a T-score of -2.5 or more. The T-score is the number of standard deviations by which the bone mineral density of an individual person differs from that of the young, normal mean of the same population. A strength of this diagnostic threshold has been the fashioning of a common approach to describe the disease. The WHO have supported the use of a single reference population (white women aged 20-29 years), a single reference site (the femoral neck) and a single technology (DXA) (Kanis et al. 2008).

Osteoporosis in men would be defined as a BMD value at the femoral neck that lay 2.5 SD or more below the average value of young healthy women The many studies that have examined fracture risk in men have come to disparate conclusions concerning the relationship between fracture risk and BMD. The relation between BMD and fracture risk changes with age, so that age-adjustment is required. The comparative studies show that the

Osteoporosis in Men - A Crucial Role of Sex Hormones 207

Few randomized controlled clinical trials on drug treatment for osteoporosis have been conducted in men. This is due to the fact that data from earlier trials on mixed female–male populations are not accepted as a source of guidelines for men and only a few of the requested randomized controlled studies on purely male cohorts have been performed. Older drugs were approved for osteoporosis in general without the need for separate trials in men (e.g. calcium, fluoride, calcitonin, alfacalcidol) and, therefore, these are still available for treating men. It was suggested that significant differences in bone biology might exist between the sexes. This is a severe therapeutic disadvantage for men with osteoporosis. Interestingly, so far, for all drugs that have also been studied in men, similar therapeutic results, in terms of BMD and fracture reducing potency, have been reported in men and women, disproving the argument of significant differences in bone biology of the female

Calcium and vitamin D intake probably provide beneficial effects on bone mass and fractures. Reducing modifiable individual risk factors of diet and lifestyle, including alcohol and nicotine intake, remain important throughout life. For men suffering from one or more diseases or medical conditions with a high risk for the development of secondary osteoporosis (see early detectionand counteractionmeasures are important, e.g. reduction of glucocorticoid dosage if possible, androgen therapy in cases of hypogonadism, thiazides for idiopathic hypercalcuria and early surgical treatment of primary hyperparathyroidism. In the elderly who are at risk for falls (e.g. those with reduced muscle strength, poor balance, previous falls) attempts to increase strength and balance or the use of a hip protector may be beneficial. Table 3 summarises general recommendations for the prevention of osteoporotic

Maintenance of adequate calcium and vitamin D intake (total intake 1000–1500 mg

Since about 50% of men are diagnosed with secondary osteoporosis, an aetiology-adapted treatment is more important in male than in female. The treatment in such cases is often complicated. eg: glucocorticoids cause bone lose in dose-dependent manner, in corticotherapy of rheumatic disease however, too great a reduction in corticoids may increase the risk of osteoporosis, since an insufficient immunosuppressive effect will allow further degradation of bone tissue by proinflammatory cytokines. Furthermore, insufficient

**8. Treatment of osteoporosis in men** 

**8.1 General measures for prevention of osteoporosis in men.** 

Long-term regular physical activity and exercise

Routine calcium/vitamin D supplementation after age 70 years

Identify other risk factors and consider specific prophylactic measures

Table 3. General measures for the prevention of osteoporotic fractures in men

calcium and 600–800 IU vitamin D per day)

Recognize and treat testosterone deficiency

**8.2 Aetiological therapy in secondary osteoporosis** 

Limit alcohol intake and smoking

and male skeleton.

fractures in men.

risk of hip fracture is similar in men and women for any given absolute value for BMD measured mainly at the hip. In the meta-analysis described above the relationship between hip fracture incidence and BMD at the proximal femur was identical in men and women at any given age (Johnell 2005). These studies indicate that a similar cut-off value for femoral neck BMD that is used in women should be used in the diagnosis of osteoporosis in men. The implementation of probability based fracture risk assessment (e.g. FRAX) will decrease the clinical utility of the T-score but diagnostic criteria remain of value in diagnostic of osteoporosis in men (Orwoll 2000).

Osteoporosis in men commonly presents with vertebral body fracture or hip fracture, whereas in women it is often diagnosed by routine bone density screening, but osteoporosis can be identified in men before fractures occur. Men with history of nontraumatic fracture, particularly of the hip, vertebral body, or distal wrist; radiographic evidence of osteopenia (because 30 to 50 percent of bone mass must be lost before evidence of loss is seen on a plain radiograph); long-term glucocorticoid use; hypogonadism; hyperparathyroidism; and other risk factors for osteoporosis, including disease states, medications affecting bone metabolism, or gait disorder, should be considered for formal osteoporosis testing (Khosla et al.2006).

Physicians might consider routinely screening men aged 70 or older, because this is the age when fracture rates increase most rapidly. Men with asymptomatic vertebral body fractures, who are at substantially increased risk of future fractures, can be identified using serial measurements of height. A man with a loss of height or whose distal ribs touch the pelvic brim should be considered for thoracic and lumbar spine radiographs to look for vertebral fractures. Screening measures include diagnostic radiologic studies using dual-energy x-ray absorptiometry (DXA) of the hip and spine, heel ultrasonography, or quantitative computed tomography. In men, declining BMD and T scores (the comparison with peak BMD adjusted for sex and race) correlate with an increased risk of hip and other fractures similar to that occurring in women.

Once osteoporosis is diagnosed, the cause should be determined, if possible, to identify the various risk factors and medical conditions causing secondary osteoporosis. The search for risk factors should include review of the history and physical examination findings plus a laboratory evaluation. Routine laboratory tests include complete blood cell count, liver and kidney panels, and measurement of calcium, phosphorus, alkaline phosphatase, thyroidstimulating hormone level, and total testosterone levels - table 2.


Table 2. Laboratory evaluation for osteoporosis in men

#### **8. Treatment of osteoporosis in men**

206 Sex Hormones

risk of hip fracture is similar in men and women for any given absolute value for BMD measured mainly at the hip. In the meta-analysis described above the relationship between hip fracture incidence and BMD at the proximal femur was identical in men and women at any given age (Johnell 2005). These studies indicate that a similar cut-off value for femoral neck BMD that is used in women should be used in the diagnosis of osteoporosis in men. The implementation of probability based fracture risk assessment (e.g. FRAX) will decrease the clinical utility of the T-score but diagnostic criteria remain of value in diagnostic of

Osteoporosis in men commonly presents with vertebral body fracture or hip fracture, whereas in women it is often diagnosed by routine bone density screening, but osteoporosis can be identified in men before fractures occur. Men with history of nontraumatic fracture, particularly of the hip, vertebral body, or distal wrist; radiographic evidence of osteopenia (because 30 to 50 percent of bone mass must be lost before evidence of loss is seen on a plain radiograph); long-term glucocorticoid use; hypogonadism; hyperparathyroidism; and other risk factors for osteoporosis, including disease states, medications affecting bone metabolism, or gait disorder, should be considered for formal osteoporosis testing (Khosla

Physicians might consider routinely screening men aged 70 or older, because this is the age when fracture rates increase most rapidly. Men with asymptomatic vertebral body fractures, who are at substantially increased risk of future fractures, can be identified using serial measurements of height. A man with a loss of height or whose distal ribs touch the pelvic brim should be considered for thoracic and lumbar spine radiographs to look for vertebral fractures. Screening measures include diagnostic radiologic studies using dual-energy x-ray absorptiometry (DXA) of the hip and spine, heel ultrasonography, or quantitative computed tomography. In men, declining BMD and T scores (the comparison with peak BMD adjusted for sex and race) correlate with an increased risk of hip and other fractures similar to that

Once osteoporosis is diagnosed, the cause should be determined, if possible, to identify the various risk factors and medical conditions causing secondary osteoporosis. The search for risk factors should include review of the history and physical examination findings plus a laboratory evaluation. Routine laboratory tests include complete blood cell count, liver and kidney panels, and measurement of calcium, phosphorus, alkaline phosphatase, thyroid-

stimulating hormone level, and total testosterone levels - table 2.

Kidney and liver function tests

Table 2. Laboratory evaluation for osteoporosis in men

25-hydroxyvitamin D

Total testosterone

TSH

Initial screening Additional tests Complete blood cell cunt Serum protein

Phosphorus Estradiol level

Calcium 24+hour urine calciuria

Alkaline phosphatase Parathyroid hormone level

osteoporosis in men (Orwoll 2000).

et al.2006).

occurring in women.

Few randomized controlled clinical trials on drug treatment for osteoporosis have been conducted in men. This is due to the fact that data from earlier trials on mixed female–male populations are not accepted as a source of guidelines for men and only a few of the requested randomized controlled studies on purely male cohorts have been performed. Older drugs were approved for osteoporosis in general without the need for separate trials in men (e.g. calcium, fluoride, calcitonin, alfacalcidol) and, therefore, these are still available for treating men. It was suggested that significant differences in bone biology might exist between the sexes. This is a severe therapeutic disadvantage for men with osteoporosis. Interestingly, so far, for all drugs that have also been studied in men, similar therapeutic results, in terms of BMD and fracture reducing potency, have been reported in men and women, disproving the argument of significant differences in bone biology of the female and male skeleton.

#### **8.1 General measures for prevention of osteoporosis in men.**

Calcium and vitamin D intake probably provide beneficial effects on bone mass and fractures. Reducing modifiable individual risk factors of diet and lifestyle, including alcohol and nicotine intake, remain important throughout life. For men suffering from one or more diseases or medical conditions with a high risk for the development of secondary osteoporosis (see early detectionand counteractionmeasures are important, e.g. reduction of glucocorticoid dosage if possible, androgen therapy in cases of hypogonadism, thiazides for idiopathic hypercalcuria and early surgical treatment of primary hyperparathyroidism. In the elderly who are at risk for falls (e.g. those with reduced muscle strength, poor balance, previous falls) attempts to increase strength and balance or the use of a hip protector may be beneficial. Table 3 summarises general recommendations for the prevention of osteoporotic fractures in men.


Table 3. General measures for the prevention of osteoporotic fractures in men

#### **8.2 Aetiological therapy in secondary osteoporosis**

Since about 50% of men are diagnosed with secondary osteoporosis, an aetiology-adapted treatment is more important in male than in female. The treatment in such cases is often complicated. eg: glucocorticoids cause bone lose in dose-dependent manner, in corticotherapy of rheumatic disease however, too great a reduction in corticoids may increase the risk of osteoporosis, since an insufficient immunosuppressive effect will allow further degradation of bone tissue by proinflammatory cytokines. Furthermore, insufficient

2010).

osteoporosis in men.

required on these atypical fractures

**8.6 Calcium and vitamin D** 

to sunlight.

**8.7 Strontium ranelate** 

**8.8 Androgen replacement therapy** 

on non-vertebral and hip fractures in men (Orwoll et al. 2004).

remains controversial (Heikinheimo et al. 1992; Orwoll et al.1992).

antifraxture effect of SR in men must be proved in further studies.

Osteoporosis in Men - A Crucial Role of Sex Hormones 209

men and women who had recently suffered a hip fracture. Yearly administration of 5 mg of zoledronic acid for a median of 1.9 years within 3 months of the fracture reduced the occurrence of overall new clinical fractures and mortality, but not hip fractures (Orwoll et al.

Bisphosphonates increased BMD and reduced fracture risk in men with glucocorticoid or leuprolide-induced bone loss. They are currently the treatment of choice for idiopathic

Bisphosphonates are generally well tolerated. However, both intravenous and oral bisphosphonates have been linked in rare cases to osteonecrosis of the jaw, although current limited data suggest no clear increase in the risk of this complication in patients with osteoporosis. Rare case reports of atypical femoral diaphyseal fractures in patients on bisphosphonate therapy have also recently emerged in the literature and raise concerns about the long-term safety of this treatment in some individuals. However, more data are

Although most trials of oral bisphosphonates in men have been either underpowered or not primarily designed to assess their effect on fracture incidence, some trials showed a significant 60%–88% reduction in the occurrence of new radiologic vertebral fractures. Although the increase in BMD with bisphosphonate therapy is similar in men and women and could therefore theoretically translate into a reduction of fracture risk similar to the one observed in women, more data are required to ascertain the benefits of oral bisphosphonates

In two recent studies, injections of vitamin D2 (ergocalciferol) in men living in nursing homes resulted in reduced appendicular fractures. On the other hand, the antifracture efficacy of D3 (calciferol) 1,25 (OH)2D3 (calcitriol) in men without serious D3 deficiency

Calcium intake should be 1,000 to 1,500 mg per day, and vitamin D intake should be 400 to 800 IU per day. Only 50 to 60 percent of older adults meet recommendations for calcium intake (Amin & Felson 2001). Older adults also have decreased vitamin D levels because skin synthesis, oral intake, and gastrointestinal absorption are diminished. Skin synthesis of vitamin D decreases because older patients tend to remain indoors and incur less exposure

Strontium ranelate exerts dual – antiresorptive and anabolic action in bone. In recent study in men with primary osteoporosis it produced even significantly greater mean increases in BMD over 12 months compared with alendronate (Meunier et al.2004). However, the

Symptomatic hypogonadism is considered an indication for testosterone substitution at all ages (Wang 2009). In clinical trials in patients with Klinefelter syndorme and hypogonadism hypogonadotropic with low bone mineral density testosterone replacement therapy was associated with increase of BMD and decreasing of resorptions markers (Finkelstein et al. 1989). These effects may probably reduce the risk of fracture in part of patients. Also in

disease control is associated with less mobility. For a number of mono-aetiolgical, and for the majority of polyaetiological, secondary osteoporoses no aetiological therapies are available, i.e. the therapeutic strategy is no different from that used in idiopathic osteoporosis.

#### **8.3 Treatment of idiopathic osteoporosis**

In men with secondary osteoporosis, with no options for aetiology-related treatment, and in all cases of primary or idiopathic osteoporosis, an individually adapted therapeutic strategy has to be planned. Since osteoporosis is a chronic disease that has to be treated over several years, it is important to inform the patient carefully about modifiable risk factors, future fracture risk, chances of improving pain, therapeutic mechanism of the selected medications and their possible side effects. With the exception of estrogen and raloxifen, the same specific drugs as those used with women can be adopted in men. However, not all are approved for this application in men. Calcitonin, alfacalcidol and fluoride do not exclude male osteoporosis and they are listed as second-line treatments. Bisphosphonates, strontium ranelate and teriparatide are firstline treatments, but only alendronate and risedronate have been approved for men. Teriparatide is not approved for men in the European Union countries.

#### **8.4 Calcitonin and fluoride**

There are only small studies on these two treatments in men with osteoporosis. There is one double blind, placebo-controlled study with the physiological osteoclast inhibitor calcitonin. In this study 28 men with osteoporosis received either 200 IU of salmon calcitonin nasal spray plus 500 mg calcium per day or a placebo nasal spray plus calcium. Lumbar spine BMD increase of 7.1% in the calcitonin group vs. 2.4% in the controls, in parallel with a higher decrease in bone resorption markers (Trovas et al.2002). In prospective controlled, 3 year trial of 60 men with primary osteoporosis a significantly lower vertebral fracture rate with low-dose-intermittent fluoride therapy compared to controls receiving only calcium plus vitamin D was found (Ringe et al. 1998).

#### **8.5 Bisphosphonates**

Bisphosphonate therapy has been shown to be effective in increasing BMD in men with primary osteoporosis, as well as in men with secondary osteoporosis, including hypogonadism and glucocorticoid-induced osteoporosis. The results of a randomized, controlled clinical trial showed that alendronate, given with calcium and vitamin D, was effective in preventing bone loss in men. The BMD increased by 7.3% at the lumbar spine and 2.5% at the femoral neck in the treatment group. Moreover, vertebral fracture risk was significantly reduced (0.8% in the treatment group compared with 7.1% in the placebo group). The efficacy of alendronate in treating osteoporosis in men has been confirmed (Orwoll et al.2000; Miller et al. 2004; Ringe et al. 2004) .

Risedronate is a potent bisphosphonate that has been demonstrated to reduce vertebral fractures in patients with glucocorticoid-induced osteoporosis within 1 year. Risedronate 35 mg once weekly has been approved recently as a second bisphosphonate for the treatment of men with a high fracture risk (Boonen et all.2006, Harrington et al.2004; McClung et al. 2001; Ringe et al. 2006).

Zoledronic acid is a potent intravenously administered bisphosphonate whose effects on fracture risk been assessed in a recent randomized placebo-controlled trial involving elderly

disease control is associated with less mobility. For a number of mono-aetiolgical, and for the majority of polyaetiological, secondary osteoporoses no aetiological therapies are available, i.e. the therapeutic strategy is no different from that used in idiopathic

In men with secondary osteoporosis, with no options for aetiology-related treatment, and in all cases of primary or idiopathic osteoporosis, an individually adapted therapeutic strategy has to be planned. Since osteoporosis is a chronic disease that has to be treated over several years, it is important to inform the patient carefully about modifiable risk factors, future fracture risk, chances of improving pain, therapeutic mechanism of the selected medications and their possible side effects. With the exception of estrogen and raloxifen, the same specific drugs as those used with women can be adopted in men. However, not all are approved for this application in men. Calcitonin, alfacalcidol and fluoride do not exclude male osteoporosis and they are listed as second-line treatments. Bisphosphonates, strontium ranelate and teriparatide are firstline treatments, but only alendronate and risedronate have been approved for men.

There are only small studies on these two treatments in men with osteoporosis. There is one double blind, placebo-controlled study with the physiological osteoclast inhibitor calcitonin. In this study 28 men with osteoporosis received either 200 IU of salmon calcitonin nasal spray plus 500 mg calcium per day or a placebo nasal spray plus calcium. Lumbar spine BMD increase of 7.1% in the calcitonin group vs. 2.4% in the controls, in parallel with a higher decrease in bone resorption markers (Trovas et al.2002). In prospective controlled, 3 year trial of 60 men with primary osteoporosis a significantly lower vertebral fracture rate with low-dose-intermittent fluoride therapy compared to controls receiving only calcium

Bisphosphonate therapy has been shown to be effective in increasing BMD in men with primary osteoporosis, as well as in men with secondary osteoporosis, including hypogonadism and glucocorticoid-induced osteoporosis. The results of a randomized, controlled clinical trial showed that alendronate, given with calcium and vitamin D, was effective in preventing bone loss in men. The BMD increased by 7.3% at the lumbar spine and 2.5% at the femoral neck in the treatment group. Moreover, vertebral fracture risk was significantly reduced (0.8% in the treatment group compared with 7.1% in the placebo group). The efficacy of alendronate in treating osteoporosis in men has been confirmed

Risedronate is a potent bisphosphonate that has been demonstrated to reduce vertebral fractures in patients with glucocorticoid-induced osteoporosis within 1 year. Risedronate 35 mg once weekly has been approved recently as a second bisphosphonate for the treatment of men with a high fracture risk (Boonen et all.2006, Harrington et al.2004; McClung et al.

Zoledronic acid is a potent intravenously administered bisphosphonate whose effects on fracture risk been assessed in a recent randomized placebo-controlled trial involving elderly

Teriparatide is not approved for men in the European Union countries.

osteoporosis.

**8.3 Treatment of idiopathic osteoporosis** 

plus vitamin D was found (Ringe et al. 1998).

(Orwoll et al.2000; Miller et al. 2004; Ringe et al. 2004) .

**8.4 Calcitonin and fluoride** 

**8.5 Bisphosphonates** 

2001; Ringe et al. 2006).

men and women who had recently suffered a hip fracture. Yearly administration of 5 mg of zoledronic acid for a median of 1.9 years within 3 months of the fracture reduced the occurrence of overall new clinical fractures and mortality, but not hip fractures (Orwoll et al. 2010).

Bisphosphonates increased BMD and reduced fracture risk in men with glucocorticoid or leuprolide-induced bone loss. They are currently the treatment of choice for idiopathic osteoporosis in men.

Bisphosphonates are generally well tolerated. However, both intravenous and oral bisphosphonates have been linked in rare cases to osteonecrosis of the jaw, although current limited data suggest no clear increase in the risk of this complication in patients with osteoporosis. Rare case reports of atypical femoral diaphyseal fractures in patients on bisphosphonate therapy have also recently emerged in the literature and raise concerns about the long-term safety of this treatment in some individuals. However, more data are required on these atypical fractures

Although most trials of oral bisphosphonates in men have been either underpowered or not primarily designed to assess their effect on fracture incidence, some trials showed a significant 60%–88% reduction in the occurrence of new radiologic vertebral fractures. Although the increase in BMD with bisphosphonate therapy is similar in men and women and could therefore theoretically translate into a reduction of fracture risk similar to the one observed in women, more data are required to ascertain the benefits of oral bisphosphonates on non-vertebral and hip fractures in men (Orwoll et al. 2004).

#### **8.6 Calcium and vitamin D**

In two recent studies, injections of vitamin D2 (ergocalciferol) in men living in nursing homes resulted in reduced appendicular fractures. On the other hand, the antifracture efficacy of D3 (calciferol) 1,25 (OH)2D3 (calcitriol) in men without serious D3 deficiency remains controversial (Heikinheimo et al. 1992; Orwoll et al.1992).

Calcium intake should be 1,000 to 1,500 mg per day, and vitamin D intake should be 400 to 800 IU per day. Only 50 to 60 percent of older adults meet recommendations for calcium intake (Amin & Felson 2001). Older adults also have decreased vitamin D levels because skin synthesis, oral intake, and gastrointestinal absorption are diminished. Skin synthesis of vitamin D decreases because older patients tend to remain indoors and incur less exposure to sunlight.

#### **8.7 Strontium ranelate**

Strontium ranelate exerts dual – antiresorptive and anabolic action in bone. In recent study in men with primary osteoporosis it produced even significantly greater mean increases in BMD over 12 months compared with alendronate (Meunier et al.2004). However, the antifraxture effect of SR in men must be proved in further studies.

#### **8.8 Androgen replacement therapy**

Symptomatic hypogonadism is considered an indication for testosterone substitution at all ages (Wang 2009). In clinical trials in patients with Klinefelter syndorme and hypogonadism hypogonadotropic with low bone mineral density testosterone replacement therapy was associated with increase of BMD and decreasing of resorptions markers (Finkelstein et al. 1989). These effects may probably reduce the risk of fracture in part of patients. Also in

Osteoporosis in Men - A Crucial Role of Sex Hormones 211

In conclusion, osteoporosis is a prevalent health problem in men. An evidence-based approach should be adopted in the clinical investigation and drug treatment for this condition. Further research into the validity of diagnostic criteria, risk factors and emerging

Adler SA. Management of osteoporosis in men on androgen deprivation therapy. Maturitas.

Amin S, Zhang Y, Sawin CT et al. Association of hypogonadism and estradiol levels with

Anderson FH, Cooper C. Hip and vertebral fractures. In: Orwoll ES, ed. Osteoporosis in

Araujo AB, O'Donnell AB, Brambilla DJ et al. Prevalence and incidence of androgen

Bass E, French DD, Bradham DD et al. Risk adjusted mortality rates of elderly veterans with

Barrett-Connor E. Mueller JE, von Muhlen DG et al. Low levels of estradiol are associated

Bateman TA, Zimmerman RJ, Ayers RA et al. Histomorphometric, physical, and mechanical

Behre HM, Kliesch S, Leifke E et al. Long-term effect of testosterone therapy on bone

Benito M, Gomberg B, Wehrli RH et al. Deterioration of trabecular architecture in

Boonen S, Delmas PD, Wenderoth D et all. Risedronate shown to be safe and effective in

Center JR, Nguyen TV, Schneider D et al. Mortality after all major types of osteoporotic

Cooper C, Campion G, Melton LJ 3d. Hip fractures in the elderly: a worldwide projection.

Cooper C, Melton LJ 3d. Epidemiology of osteoporosis. Trends Endocrinol Metab

Journal of Clinical Endocrinology and Metabolism 2000 85 219–223

Bilezikian JP. Osteoporosis in men. J Clin Endocrinol Metab 1999;84:3431-3434

multicenter study. Osteoporos Int 2006;17(Suppl 2):S230–1

Campion JM, Maricic MJ. Osteoporosis in men. Am Fam Physician 2003;67(7):1521–6. Carani C, Qin K, Simoni M et al. Effect of testosterone and estradiol in a man with aromatase

Amin S, Felson DT. Osteoporosis in men. Rheum Dis Clin North Am 2001;27:19-47.

Aging Study. J Clin Endocrinol Metab 2004;89:5925-5926

men. San Diego, Calif.: Academic, 1999:29-49

hip fractures. Ann Epidemiol 2007;17:514–519

bone mineral density in elderly men from the Framingham study. Ann Intern Med

deficiency in middle-aged and older man: estimates from the Massachusetts Male

with vertebral fractures in older men, but not women: the Rancho Bernardo Study.

effects of spaceflight and insulin-like growth factor-I on rat long bones. Bone

mineral density in hypogonadal men. Journal of Clinical Endocrinology and

hypogonadal men. Journal of Clinical Endocrinology and Metabolism

men with osteoporosis in a 2-year, double-blind, randomized, placebo-controlled,

fracture in men and women: an observational study. Lancet 1999;353(9156):878–882

therapeutic agents for osteoporosis in men is however urgently required.

**10. Conclusion** 

**11. References** 

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patiens witj primary and secondary hypogonadism due to testis diseases or hypothalamopituitary disriders, testosterone replacement therapy may reduce bone turnover and prevent further bone loss, and even increase BMD, at least in some patients (Behre et al.1997; Snyder et al. 2000). Effects on fracture risk have not been assessed.

In the case of low serum testosterone due to long-term treatment with systemic glucocorticoid administration, beneficial effects of testosterone treatment on BMD were observed (Crawford et al. 2003). In view of the lack of documentation of antifracture efficacy of testosterone, treatment of osteoporosis should include bisfosfonates or other antiosteoporotic agents, whether or not on testosterone substitution (Khosla et al.2006). In men with profound hypogonadism due to androgen deprivation therapy for prostate cancer, treatment with testosterone is contraindicated and the other therapeutic options should be considered such as SERMs, bisphosphonates and denosumab, can effectively reduce bone turnover, prevent bone loss and reduce fracture risk (Adlet et al. 2011; Khosla rt al. 2006).

As noted above, aging is accompanied by progressive moderate decrease in the population mean serum concentration of testosterone and increasing prevalence of men with serum (free, bioavailable) testosterone levels that lie below the range for young men (Araujo et al.2004). In this elderly population, effects of testosterone treatment on BMD have been inconsistent, with the most convincing effects being observed in men with very low serum testosterone (Katznelson et al.1996; Snyder et al. 2000; Wang et al 2001). In elderly men, testosterone treatment may have additional beneficial effects on muscle mass and strength, which may help decrease fracture risk through a reduced propensity to fall (Snyder et al. 1999). Nevertheless, the effect of testosterone treatment on fracture risk is unknown. By contrast, treatment with bisphosphonates and teriparatide has been shown to be effective in men with low baseline serum testosterone in subgroup analysis of clinical trials of treatment.

In this context, hypogonadism in older men requires a conservative approach and testosterone treatment should be considered only for men with frankly low serum testosterone, in the presence of unequivocal signs and symptoms of hypogonadism, In view of the lack of documentation of anti-fracture efficacy of testosterone, treatment of osteoporosis should include established osteoporosis treatments whether or not on testosterone substitution. Nevertheless, the effect of testosterone treatment on fracture risk is unknown and the long-term risk-benefit ratio of prolonged treatment in elderly men is not yet established. The potential adverse effects on haematocrit, prostate and cardiovascular risk requires alertness.

#### **9. Teriparatade**

A favourable effect of treatment with parathyroid hormone (PTH) on BMD in men with advanced osteoporosis has also been demonstrated in a small pilot study with daily injections of 400 IU PTH (1–34) [Teriparatide]. After 18 months, the average lumbar spine BMD had increased by 13.5% in the PTH-group and was unchanged in placebo group. In a larger trial of 437 men with osteoporosis (daily dose of 20 mg or 40 mg rhPTH or placebo, subcutaneously) over 11 months plus 18 months follow-up similar effects on BMD and a significantly lower rate of vertebral fractures for the pooled PTH groups were found. The similarity of those effects on BMD with the effects observed in clinical studies of women where the influence of the treatment on fracture incidence was assessed, clearly indicates the therapeutic usefulness of teriparatide in both sexes (Kaudman 2001; Kurland 2000)

#### **10. Conclusion**

210 Sex Hormones

patiens witj primary and secondary hypogonadism due to testis diseases or hypothalamopituitary disriders, testosterone replacement therapy may reduce bone turnover and prevent further bone loss, and even increase BMD, at least in some patients (Behre et al.1997; Snyder

In the case of low serum testosterone due to long-term treatment with systemic glucocorticoid administration, beneficial effects of testosterone treatment on BMD were observed (Crawford et al. 2003). In view of the lack of documentation of antifracture efficacy of testosterone, treatment of osteoporosis should include bisfosfonates or other antiosteoporotic agents, whether or not on testosterone substitution (Khosla et al.2006). In men with profound hypogonadism due to androgen deprivation therapy for prostate cancer, treatment with testosterone is contraindicated and the other therapeutic options should be considered such as SERMs, bisphosphonates and denosumab, can effectively reduce bone turnover, prevent bone

As noted above, aging is accompanied by progressive moderate decrease in the population mean serum concentration of testosterone and increasing prevalence of men with serum (free, bioavailable) testosterone levels that lie below the range for young men (Araujo et al.2004). In this elderly population, effects of testosterone treatment on BMD have been inconsistent, with the most convincing effects being observed in men with very low serum testosterone (Katznelson et al.1996; Snyder et al. 2000; Wang et al 2001). In elderly men, testosterone treatment may have additional beneficial effects on muscle mass and strength, which may help decrease fracture risk through a reduced propensity to fall (Snyder et al. 1999). Nevertheless, the effect of testosterone treatment on fracture risk is unknown. By contrast, treatment with bisphosphonates and teriparatide has been shown to be effective in men with low baseline

In this context, hypogonadism in older men requires a conservative approach and testosterone treatment should be considered only for men with frankly low serum testosterone, in the presence of unequivocal signs and symptoms of hypogonadism, In view of the lack of documentation of anti-fracture efficacy of testosterone, treatment of osteoporosis should include established osteoporosis treatments whether or not on testosterone substitution. Nevertheless, the effect of testosterone treatment on fracture risk is unknown and the long-term risk-benefit ratio of prolonged treatment in elderly men is not yet established. The potential adverse effects on haematocrit, prostate and cardiovascular

A favourable effect of treatment with parathyroid hormone (PTH) on BMD in men with advanced osteoporosis has also been demonstrated in a small pilot study with daily injections of 400 IU PTH (1–34) [Teriparatide]. After 18 months, the average lumbar spine BMD had increased by 13.5% in the PTH-group and was unchanged in placebo group. In a larger trial of 437 men with osteoporosis (daily dose of 20 mg or 40 mg rhPTH or placebo, subcutaneously) over 11 months plus 18 months follow-up similar effects on BMD and a significantly lower rate of vertebral fractures for the pooled PTH groups were found. The similarity of those effects on BMD with the effects observed in clinical studies of women where the influence of the treatment on fracture incidence was assessed, clearly indicates the

therapeutic usefulness of teriparatide in both sexes (Kaudman 2001; Kurland 2000)

et al. 2000). Effects on fracture risk have not been assessed.

loss and reduce fracture risk (Adlet et al. 2011; Khosla rt al. 2006).

serum testosterone in subgroup analysis of clinical trials of treatment.

risk requires alertness.

**9. Teriparatade** 

In conclusion, osteoporosis is a prevalent health problem in men. An evidence-based approach should be adopted in the clinical investigation and drug treatment for this condition. Further research into the validity of diagnostic criteria, risk factors and emerging therapeutic agents for osteoporosis in men is however urgently required.

#### **11. References**


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**9** 

Sae Chul Kim

*Korea* 

**Testosterone Deficiency Linked** 

**to Lower Urinary Tract Symptoms** 

*Department of Urology College of Medicine Chung-Ang University Seoul,* 

Over the past decade, it has become clear many of the age-related health problems of men, that have hitherto been treated using different medical disciplines, are actually inter-related and require a more integrative approach in the aging male. Lower urinary tract symptoms (LUTS) may serve as an example. LUTS consists of storage, voiding, and post micturition symptoms affecting the lower urinary tract. Storage symptoms (daytime frequency, nocturia, urgency, incontinence) are experienced during the storage phase of the bladder. Voiding symptoms (weak stream, splitting or spraying, abdominal straining, hesitancy, intermittency, terminal dribble) are experienced during the voiding phase. Post-micturition symptoms (feeling of incomplete emptying, post micturition dribble) are experienced immediately after micturition. Individuals with LUTS often experience urinary incontinence (UI) or overactive bladder (OAB) symptoms. OAB is a subset of storage LUTS defined as urgency, with or without urgency UI, usually with frequency and nocturia. Men may report one or any combination of the symptoms and LUTS, including UI and OAB, have

The prevalence of LUTS increases from 8% in the fourth decade of life to more than 70% in the seventh decade. In a large population-based cross-sectional survey the prevalence of storage LUTS (men, 51.3%; women, 59.2%) was greater than that for voiding (men, 25.7%; women, 19.5%) and post micturition (men, 16.9%; women, 14.2%) symptoms combined

Once symptoms arise, their progress is variable and unpredictable with about one third of patients improving, one third remaining stable and one third deteriorating. LUTS may point to serious pathology of the urogenital tract but are often nonspecific and large studies of patients have failed to show any correlation between LUTS and a specific diagnosis. An allencompassing view of LUTS that focuses on the lower urinary tract as an integrated functional unit, but simultaneously reflects pathophysiology in the body as a whole, is more likely to improve a clinician's ability to manage the symptoms and therefore improve patient outcomes. Benign prostatic hyperplasia (BPH), which occurs more frequently with aging, is the most common cause of LUTS in middle-aged and elderly men, although many other diseases such as detrusor muscle weakness and/or instability, urinary tract infection, chronic prostatitis, urinary stone, prostate cancer, bladder cancer, neurological disease, e.g. multiple sclerosis, spinal cord injury, cauda equina syndrome, and cardiac and renal diseases may

**1. Introduction** 

(Irwin et al., 2008).

accompany LUTS.

detrimental effects on health-related quality of life.

estradiol on bone mineral density and hormonal parameters. J Clin Endocrinol Metab 2000;85:1841–1845


### **Testosterone Deficiency Linked to Lower Urinary Tract Symptoms**

Sae Chul Kim

*Department of Urology College of Medicine Chung-Ang University Seoul, Korea* 

#### **1. Introduction**

214 Sex Hormones

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Seeman E Pathogenesis of bone fragility in women and men. Lancet 2002;359:1841–1850 Snyder PJ, Peachey TW, Hannoush P et al. Effects of testosterone treatment on body

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turnover markers and bone mineral density in hypogonadal men. Clin Endocrinol

recommendations: investigation, treatment and monitoring of late-onset

reconsidering the roles of androgens and estrogens in periosteal expansion. J Clin

Over the past decade, it has become clear many of the age-related health problems of men, that have hitherto been treated using different medical disciplines, are actually inter-related and require a more integrative approach in the aging male. Lower urinary tract symptoms (LUTS) may serve as an example. LUTS consists of storage, voiding, and post micturition symptoms affecting the lower urinary tract. Storage symptoms (daytime frequency, nocturia, urgency, incontinence) are experienced during the storage phase of the bladder. Voiding symptoms (weak stream, splitting or spraying, abdominal straining, hesitancy, intermittency, terminal dribble) are experienced during the voiding phase. Post-micturition symptoms (feeling of incomplete emptying, post micturition dribble) are experienced immediately after micturition. Individuals with LUTS often experience urinary incontinence (UI) or overactive bladder (OAB) symptoms. OAB is a subset of storage LUTS defined as urgency, with or without urgency UI, usually with frequency and nocturia. Men may report one or any combination of the symptoms and LUTS, including UI and OAB, have detrimental effects on health-related quality of life.

The prevalence of LUTS increases from 8% in the fourth decade of life to more than 70% in the seventh decade. In a large population-based cross-sectional survey the prevalence of storage LUTS (men, 51.3%; women, 59.2%) was greater than that for voiding (men, 25.7%; women, 19.5%) and post micturition (men, 16.9%; women, 14.2%) symptoms combined (Irwin et al., 2008).

Once symptoms arise, their progress is variable and unpredictable with about one third of patients improving, one third remaining stable and one third deteriorating. LUTS may point to serious pathology of the urogenital tract but are often nonspecific and large studies of patients have failed to show any correlation between LUTS and a specific diagnosis. An allencompassing view of LUTS that focuses on the lower urinary tract as an integrated functional unit, but simultaneously reflects pathophysiology in the body as a whole, is more likely to improve a clinician's ability to manage the symptoms and therefore improve patient outcomes. Benign prostatic hyperplasia (BPH), which occurs more frequently with aging, is the most common cause of LUTS in middle-aged and elderly men, although many other diseases such as detrusor muscle weakness and/or instability, urinary tract infection, chronic prostatitis, urinary stone, prostate cancer, bladder cancer, neurological disease, e.g. multiple sclerosis, spinal cord injury, cauda equina syndrome, and cardiac and renal diseases may accompany LUTS.

Testosterone Deficiency Linked to Lower Urinary Tract Symptoms 217

prostate volume in elderly men is less pronounced. Joseph et al. (2002) reported that a large prostate volume was marginally associated with increased total testosterone level in African-American men, but Meikle et al. (1997) found an inverse correlation between prostate volume and total testosterone level in 214 male twins based on white populations. Others have not found a significant association between total testosterone level and prostate volume. Partin et al. (1991) correlated 23 hormonal factors, including total testosterone/free testosterone to BPH volume assessed histologically. After correcting for age, the BPH volume correlated significantly with free testosterone, but not total testosterone. If not corrected for age, neither testosterone nor free testosterone correlated with BPH volume. It has been demonstrated that 5 alpha-reductase activity (Bruchovsky et al., 1998), and androgen receptor levels (Barrack et al., 1983) increase with aging, which means that prostatic cells may gradually become more sensitive to DHT during aging, and that this stimulates cell replication in the prostate. Estradiol has been hypothesized to potentiate the effects of androgens in inducing BPH by inducing the androgen receptor, which thereby sensitizes the prostate to free testosterone. In dogs, estrogens have been shown to induce the androgen receptor, alter steroid metabolism resulting in higher levels of intraprostatic DHT, inhibit cell death when given in the presence of androgens, and stimulate stromal collagen production. Schatzl et al. (2000) found hypogonadism in approximately one fifth of elderly men with LUTS but it had no impact on LUTS status, PSA level, prostate volume, uroflowmetry, or endocrine parameters. In contrast to testosterone, they observed an agerelated increase in estradiol (+0.86 pg/mL per decade), the only hormone to correlate with prostate volume, thus suggesting its significance for BPH and BPE. Gann et al. (1995) assessed the relation of steroid hormone levels with subsequent surgical treatment for BPH among participants in the Physicians Health Study and found a strong correlation for increasing risk and serum estrogen levels. The relevance of estrogens is underscored by the fact that, within the prostate, the highest concentrations have been detected in the stroma, the predominant tissue found in BPH. The rate-limiting step in estrogen biosynthesis is the conversion of androgens to estrogens, which is catalyzed by the enzyme aromatase. This enzyme is expressed in the human prostate and is regulated by follicle-stimulating hormone (FSH). The recent demonstration of a coexpression of gonadotropin hormones and their corresponding receptors in the human prostate suggests that FSH receptor activation acts in an endocrine fashion by way of age-dependent, elevated FSH (Schatzl et al., 2000). Alternatively, FSH might act in a para-autocrine fashion by way of locally produced FSH,

From the above mentioned facts, the age-related growth of the prostate cannot be explained by a mere increase or decrease in serum androgens. Schultheiss et al. (2004) reviewed previous studies and concluded that the link between androgens and age-related growth of the prostate might be explained by a shift of the hormonal ratio (e.g. the androgen/estrogen ratio), the changing intraprostatic hormonal level, or a modified action of hormones and their respective receptors, as well as of intraprostatic enzymes (e.g. 5α reductase).

Historically, bladder outlet obstruction (BOO), LUTS, and BPH has been considered to be almost synonymous, however, an increasing number of studies now demonstrate that the correlations between these parameters are weak and symptoms may arise from many

Additional large studies are needed to evaluate the above mechanisms.

potentially stimulating aromatase activity.

**3. Testosterone deficiency linked to LUTS** 

The role of testosterone in voiding function remains obscure, although it plays a definite role in the etiopathogenesis of BPH. The indirect relation could obscure an interrelation between circulating levels of testosterone and symptoms of LUTS at a statistically significant level which nevertheless is biologically plausible. This chapter will discuss possible relationships between androgens and LUTS

#### **2. Sex hormone involvement in the etiopathognesis of BPH**

The two factors that are generally accepted to play a role in the etiopathogenesis of BPH are aging and androgen. While serum testosterone level steadily decreases after 40 years of age, the prevalence of histologic BPH in autopsy studies rises from approximately 20% in men aged 41-50 to 50% in men aged 51-60, and to over 90% in men older than 80 (Berry et al., 1984). An enlarged prostate (BPE) is detectable in approximately on half of the patients with histologic evidence of BPH. The gradual reduction of plasma testosterone in middle-aged and older men from mid-life onwards coincides paradoxically with the time when there is progressive growth of the prostate, a highly androgen-dependent organ. In the prostate, testosterone is converted into the more potent androgen, dihydrotestosterone (DHT) by 5α reductase. It is thought that DHT has a central role in BPH development and maintenance because the inhibition of 5α reductase activity is associated with decreased serum DHT concentration and decreased prostate size (Figure 1).

Fig. 1. Androgen regulation of cell growth in the prostate cell

Observations and clinical studies in men have clearly demonstrated that BPH is under endocrine control. Men castrated before puberty (eunuchs) develop neither BPH nor BPE, and individuals with an inherited deficiency of 5α reductase have only a vestigial prostate gland. Castration results in the regression of established BPH and improvement in urinary symptoms. Administration of a gonadotropin releasing hormone (GnRH) analog in men reversibly shrinks established BPH, resulting in objective improvement in urinary flow rate and subjective improvement in symptoms. Finally, clinical experience with finasteride, a 5α reductase type II inhibitor has documented the relevance of DHT on prostate size (Marberger, 1998). Despite these convicting data, the correlation between androgens and

The role of testosterone in voiding function remains obscure, although it plays a definite role in the etiopathogenesis of BPH. The indirect relation could obscure an interrelation between circulating levels of testosterone and symptoms of LUTS at a statistically significant level which nevertheless is biologically plausible. This chapter will discuss possible relationships

The two factors that are generally accepted to play a role in the etiopathogenesis of BPH are aging and androgen. While serum testosterone level steadily decreases after 40 years of age, the prevalence of histologic BPH in autopsy studies rises from approximately 20% in men aged 41-50 to 50% in men aged 51-60, and to over 90% in men older than 80 (Berry et al., 1984). An enlarged prostate (BPE) is detectable in approximately on half of the patients with histologic evidence of BPH. The gradual reduction of plasma testosterone in middle-aged and older men from mid-life onwards coincides paradoxically with the time when there is progressive growth of the prostate, a highly androgen-dependent organ. In the prostate, testosterone is converted into the more potent androgen, dihydrotestosterone (DHT) by 5α reductase. It is thought that DHT has a central role in BPH development and maintenance because the inhibition of 5α reductase activity is associated with decreased serum DHT

**2. Sex hormone involvement in the etiopathognesis of BPH** 

concentration and decreased prostate size (Figure 1).

Fig. 1. Androgen regulation of cell growth in the prostate cell

Observations and clinical studies in men have clearly demonstrated that BPH is under endocrine control. Men castrated before puberty (eunuchs) develop neither BPH nor BPE, and individuals with an inherited deficiency of 5α reductase have only a vestigial prostate gland. Castration results in the regression of established BPH and improvement in urinary symptoms. Administration of a gonadotropin releasing hormone (GnRH) analog in men reversibly shrinks established BPH, resulting in objective improvement in urinary flow rate and subjective improvement in symptoms. Finally, clinical experience with finasteride, a 5α reductase type II inhibitor has documented the relevance of DHT on prostate size (Marberger, 1998). Despite these convicting data, the correlation between androgens and

between androgens and LUTS

prostate volume in elderly men is less pronounced. Joseph et al. (2002) reported that a large prostate volume was marginally associated with increased total testosterone level in African-American men, but Meikle et al. (1997) found an inverse correlation between prostate volume and total testosterone level in 214 male twins based on white populations. Others have not found a significant association between total testosterone level and prostate volume. Partin et al. (1991) correlated 23 hormonal factors, including total testosterone/free testosterone to BPH volume assessed histologically. After correcting for age, the BPH volume correlated significantly with free testosterone, but not total testosterone. If not corrected for age, neither testosterone nor free testosterone correlated with BPH volume. It has been demonstrated that 5 alpha-reductase activity (Bruchovsky et al., 1998), and androgen receptor levels (Barrack et al., 1983) increase with aging, which means that prostatic cells may gradually become more sensitive to DHT during aging, and that this stimulates cell replication in the prostate. Estradiol has been hypothesized to potentiate the effects of androgens in inducing BPH by inducing the androgen receptor, which thereby sensitizes the prostate to free testosterone. In dogs, estrogens have been shown to induce the androgen receptor, alter steroid metabolism resulting in higher levels of intraprostatic DHT, inhibit cell death when given in the presence of androgens, and stimulate stromal collagen production. Schatzl et al. (2000) found hypogonadism in approximately one fifth of elderly men with LUTS but it had no impact on LUTS status, PSA level, prostate volume, uroflowmetry, or endocrine parameters. In contrast to testosterone, they observed an agerelated increase in estradiol (+0.86 pg/mL per decade), the only hormone to correlate with prostate volume, thus suggesting its significance for BPH and BPE. Gann et al. (1995) assessed the relation of steroid hormone levels with subsequent surgical treatment for BPH among participants in the Physicians Health Study and found a strong correlation for increasing risk and serum estrogen levels. The relevance of estrogens is underscored by the fact that, within the prostate, the highest concentrations have been detected in the stroma, the predominant tissue found in BPH. The rate-limiting step in estrogen biosynthesis is the conversion of androgens to estrogens, which is catalyzed by the enzyme aromatase. This enzyme is expressed in the human prostate and is regulated by follicle-stimulating hormone (FSH). The recent demonstration of a coexpression of gonadotropin hormones and their corresponding receptors in the human prostate suggests that FSH receptor activation acts in an endocrine fashion by way of age-dependent, elevated FSH (Schatzl et al., 2000). Alternatively, FSH might act in a para-autocrine fashion by way of locally produced FSH, potentially stimulating aromatase activity.

From the above mentioned facts, the age-related growth of the prostate cannot be explained by a mere increase or decrease in serum androgens. Schultheiss et al. (2004) reviewed previous studies and concluded that the link between androgens and age-related growth of the prostate might be explained by a shift of the hormonal ratio (e.g. the androgen/estrogen ratio), the changing intraprostatic hormonal level, or a modified action of hormones and their respective receptors, as well as of intraprostatic enzymes (e.g. 5α reductase). Additional large studies are needed to evaluate the above mechanisms.

#### **3. Testosterone deficiency linked to LUTS**

Historically, bladder outlet obstruction (BOO), LUTS, and BPH has been considered to be almost synonymous, however, an increasing number of studies now demonstrate that the correlations between these parameters are weak and symptoms may arise from many

Testosterone Deficiency Linked to Lower Urinary Tract Symptoms 219

≥20) even after adjusting for confounding factors (Figure 3). However, the odds ratio of bioavailable testosterone was lower than that of free testosterone on multivariable analysis

Fig. 3. Blood levels of free testosterone (pg/ml) depending on degree of LUTS severity

mild moderate severe

Chang IH, Kim SC. J Urol, 2009

Fig. 4. Odds ratios (95% Cl) of total (T), free (FT) and bioavailable (BT) testosterone, and sex hormone binding globulin (SHBG) on log scales for prediction of severe LUTS (Adopted

A few studies have investigated serum DHT and LUTS. Of the studies examining this association, DHT has not been related with LUTS or BPH in prospective (Meigs et al., 2001; Gann et al., 1995) or cross sectional studies (Platz et al., 1999; Litman et al., 2007). Trifiro et al. (2010) found there was a weak (and unadjusted) association only, suggesting an increase in mean serum DHT concentration among men with LUTS compared with those without. However, serum DHT may not be an ideal measurement of intraprostatic androgen concentrations, as serum DHT does not necessarily correlate to prostate tissue concentrations of DHT. Because of this observation, the serum DHT metabolites 17 betadiol-glucuronide and androrstanediol glucuronide (AAG) are often used to estimate DHT activity. A cross sectional and a prospective study showed higher levels of the DHT

(Figure 4).

(Adopted from Chang, et al., 2009)

6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8

free testosterone

from Chang, et al., 2009)

different etiologies. Although LUTS is commonly attributed to BOO caused by BPE, LUTS is not associated with BOO in a third to a half of men (Figure 2) and furthermore, the severity of LUTS secondary to BPH is not necessarily correlated with prostate volume (Chang et al., 2009). Some men with greatly enlarged glands may have little obstruction and few symptoms while others with prostate glands less enlarged have more outlet obstruction and severe symptoms. Asian men have a smaller prostate than Caucasians, but may have similar or higher symptom scores and a more impaired quality of life (Homma et al., 1997).

Fig. 2. Correlation between lower urinary tract symptoms (LUTS) and histologic benign prostatic hyperplasia (BPH), benign prostatic enlargement (BPE) and bladder outlet obstruction (BOO)

Many studies have tried to establish a relationship between sex steroids and BPH, but a few studies have analyzed the relationship between circulating testosterone and LUTS. At the epidemiological level, an association between central obesity in adulthood, the metabolic syndrome, erectile dysfunction (ED) and LUTS has been established (Rohrmann et al., 2007). A common denominator of the ailments is lower-than-normal testosterone levels occurring in a significant proportion of elderly men. However, there is no consensus on possible effects of testosterone on LUTS. Schatzl et al. (2003) found that hypogonadism was seen approximately one-fifth of elderly men with LUTS, but it had no impact on symptom status. Litman et al. (2007) found an inverse correlation between symptoms of LUTS and plasma total and bioavailable testosterone but this relationship disappeared after statistical adjustment for age. Roberts et al. (2004) reported a negative association between total testosterone and American Urological Association Symptom Index (AUA-SI) but not with bioavailable testosterone. Miwa et al. (2008) noted an inverse association of free testosterone, but not total testosterone, to International Prostate Symptom Score (IPSS). Recently, author (2009) found free and bioavailable testosterone had significant negative relationships with IPSS total scores and subscores for voiding symptoms even after adjusting for age, prostate total volume and transitional zone volume, high sensitivity C-reactive protein (CRP) and homeostasis model assessment of insulin resistance (HOMA-IR). In addition, free and bioavailable testosterone were significantly related to the presence of severe LUTS (IPSS

different etiologies. Although LUTS is commonly attributed to BOO caused by BPE, LUTS is not associated with BOO in a third to a half of men (Figure 2) and furthermore, the severity of LUTS secondary to BPH is not necessarily correlated with prostate volume (Chang et al., 2009). Some men with greatly enlarged glands may have little obstruction and few symptoms while others with prostate glands less enlarged have more outlet obstruction and severe symptoms. Asian men have a smaller prostate than Caucasians, but may have similar

**BPE**

**All men ≥ 40 years of age**

**BOO**

or higher symptom scores and a more impaired quality of life (Homma et al., 1997).

Fig. 2. Correlation between lower urinary tract symptoms (LUTS) and histologic benign prostatic hyperplasia (BPH), benign prostatic enlargement (BPE) and bladder outlet

Many studies have tried to establish a relationship between sex steroids and BPH, but a few studies have analyzed the relationship between circulating testosterone and LUTS. At the epidemiological level, an association between central obesity in adulthood, the metabolic syndrome, erectile dysfunction (ED) and LUTS has been established (Rohrmann et al., 2007). A common denominator of the ailments is lower-than-normal testosterone levels occurring in a significant proportion of elderly men. However, there is no consensus on possible effects of testosterone on LUTS. Schatzl et al. (2003) found that hypogonadism was seen approximately one-fifth of elderly men with LUTS, but it had no impact on symptom status. Litman et al. (2007) found an inverse correlation between symptoms of LUTS and plasma total and bioavailable testosterone but this relationship disappeared after statistical adjustment for age. Roberts et al. (2004) reported a negative association between total testosterone and American Urological Association Symptom Index (AUA-SI) but not with bioavailable testosterone. Miwa et al. (2008) noted an inverse association of free testosterone, but not total testosterone, to International Prostate Symptom Score (IPSS). Recently, author (2009) found free and bioavailable testosterone had significant negative relationships with IPSS total scores and subscores for voiding symptoms even after adjusting for age, prostate total volume and transitional zone volume, high sensitivity C-reactive protein (CRP) and homeostasis model assessment of insulin resistance (HOMA-IR). In addition, free and bioavailable testosterone were significantly related to the presence of severe LUTS (IPSS

obstruction (BOO)

**Histologic BPH**

**LUTS**

≥20) even after adjusting for confounding factors (Figure 3). However, the odds ratio of bioavailable testosterone was lower than that of free testosterone on multivariable analysis (Figure 4).

Fig. 3. Blood levels of free testosterone (pg/ml) depending on degree of LUTS severity (Adopted from Chang, et al., 2009)

Fig. 4. Odds ratios (95% Cl) of total (T), free (FT) and bioavailable (BT) testosterone, and sex hormone binding globulin (SHBG) on log scales for prediction of severe LUTS (Adopted from Chang, et al., 2009)

A few studies have investigated serum DHT and LUTS. Of the studies examining this association, DHT has not been related with LUTS or BPH in prospective (Meigs et al., 2001; Gann et al., 1995) or cross sectional studies (Platz et al., 1999; Litman et al., 2007). Trifiro et al. (2010) found there was a weak (and unadjusted) association only, suggesting an increase in mean serum DHT concentration among men with LUTS compared with those without. However, serum DHT may not be an ideal measurement of intraprostatic androgen concentrations, as serum DHT does not necessarily correlate to prostate tissue concentrations of DHT. Because of this observation, the serum DHT metabolites 17 betadiol-glucuronide and androrstanediol glucuronide (AAG) are often used to estimate DHT activity. A cross sectional and a prospective study showed higher levels of the DHT

Testosterone Deficiency Linked to Lower Urinary Tract Symptoms 221

pharmacological treatment modalities of LUTS as in an adjunctive therapeutic effect of testosterone supplement to phosphodiesterase type 5 (PDE5) inhibitors for ED treatment

There is ample evidence from many epidemiological studies that LUTS and ED are strongly linked, independently of age. ED assessed by a questionnaire, International Index of Erectile Function (IIEF) score was strongly related to LUTS severity. When controlling for age, LUTS severity was by far the strongest predictor of erectile function, with an odd ratio for severe vs mild LUTS of 8.99 followed by diabetes, 3.01; cardiac disease, 2.17; hypertension, 1.83 and hyperlipidemia, 1.57 (McVary., 2006). The fact that LUTS severity is the strongest predictor of ED suggests that LUTS and ED may share their underlying causes and the underlying causes may explain the reasons why severity of LUTS does not correlates with prostate size. Although a direct causal relationship is not established yet, four pathophysiological mechanisms can explain the relationship. These include 1) insulin resistance and autonomic hyperactivity, 2) alteration in nitric oxide bioavailability, 3) Rho-kinase activation/endothelin pathway, 4) pelvic atherosclerosis, all of which are known to be

It was proposed that LUTS is a part of the metabolic syndrome which includes hyperglycemia, obesity, dyslipidemia and hypertension. Recently, testosterone deficiency has captured attention to be a possible risk factor of metabolic syndrome. The basis of this concept came from increased insulin resistance found in both hypergonadotropic and hypogonadotropic hypogonadism. Patients of un-treated Klinefelter's syndrome, compared with control subjects, showed a significantly higher prevalence of metabolic risk factors (Bojesen et al., 2006). GnRH agonist-treated men with prostate cancer also showed significantly decreased insulin sensitivity (Smith et al., 2006). Long-term survivors of testicular cancer have an increased risk for cardiovascular events 10 or more years after chemotherapy (Huddart et al., 2003). Nuver et al. (2005) found lower testosterone associated with a higher body mass index (BMI) pretreatment and a larger BMI increase during followup in testicular cancer survivors following cisplatin–based chemotherapy than controls, suggesting testosterone may play a role in the development of the metabolic syndrome. Plasma testosterone levels decline with aging in healthy men and features of the metabolic syndrome also show age-related deteriorations, suggesting that testosterone is an important regulator of insulin sensitivity in men. There are lots of evidences that late onset hypogonadism is associated with metabolic syndrome. Blouin et al. (2006) investigated whether this decline or the aging process per se accounts for the risk of metabolic syndrome. They observed that patients with a high testosterone level were more likely to have fewer than three components of metabolic syndrome than those with a low testosterone level. Author (2009) also found that HOMA-IR correlated negatively with serum total, free and bioavailable testosterone. Studies showed that blood levels of insulin and metabolic risk factors increased with lower testosterone levels in middle aged men (Laaksonen, et al., 2003). And patients with metabolic syndrome had significantly lower serum testosterone level than those without metabolic syndrome. Men who developed both metabolic syndrome and diabetes mellitus at 11-year follow-up were especially likely to have low testosterone levels already at baseline (Laaksonen, et al., 2004). Pitteloud et al (2005a)

accompanied by the testosterone deficiency (Buvat et al., 2011; Blute et al., 2009).

**4. Insulin resistance and autonomic hyperactivity** 

androgen-dependent.

metabolites and testosterone to DHT metabolite ratios to be directly associated with LUTS or BPH (Kristal et al., 2008; Platz et al., 1999). Kristal et al (2008) showed stronger associations as a ratio suggesting that the testosterone : DHT metabolite or testosterone : DHT ratio may therefore be a more sensitive marker of LUTS/BPH than either hormone individually. Platz et al. (1999) estimated the risk of BPH and severe LUTS in relation to testosterone, DHT, estradiol and AAG in 300 men with severe LUTS. There was a positive relation with AAG and an inverse one with estradiol for the risk of BPH surgery/LUTS, the correlations persisting even after adjusting for steroid hormones and sex hormone binding globulin (SHBG).

A study showed at the 105th Annual Scientific Meeting of the AUA that as testosterone levels decreased, LUTS severity increased and maximum flow rate decreased, while prostate volume remained the same as hormone levels decreased (Sauver, et al., 2010). In another study to determine the relationship between androgens, LUTS and urodynamic variables of BOO in patients with LUTS/BPH, free testosterone was negatively correlated with detrusor pressure at the end of urinary flow (closure detrusor pressure) and pressure at the maximum urinary flow rate (Qmax) (Koritsiadis et al., 2008). Mean closure detrusor pressure and detrusor pressure at Qmax differed significantly between patients with low and normal free testosterone levels. Detrusor overactivity (DO) was noted in patients who had significantly lower free testosterone levels than those with no DO. Although several cross-sectional and prospective studies showed no consensus of association of testosterone with LUTS or BPH, it is notable that no studies have as yet reported an increased risk of LUTS with higher testosterone.

In the rabbit bladder outlet obstruction study, bladder dysfunction is mainly mediated by three cellular processes; 1) progressive denervation, 2) cellular mitochondria malfunction, 3) dysregulation of intracellular calcium storage and release from the sarcoplasmic reticulum (SR). Biomarkers for these three functions are calcium adenosine triphosphatase (ATPase) for calcium release, citrate synthase for mitochondrial function, and choline acetyl-transferse for cholinergic innervations (Juan et al., 2007). Bladder contraction can be divided into phasic and tonic period. The phasic response depends on the adenosine triphosphate (ATP) concentration in the bladder wall, whereas the tonic phase requires mitochondrial oxidative activity to generate energy. Castration of adult male rabbits resulted in a significant decrease in the activities of the mitochondria specific enzyme, citrate synthase of the bladder body and base, muscle and mucosa, urethra and corpora, while choline acetyl-transferse activity and calcium ATPase activity showed different responses depending on the sites (Juan et al., 2007).

Preliminary evidence indicates that men with LUTS benefit from testosterone treatment, and pilot studies have also shown that testosterone therapy has a positive effect on LUTS in lateonset hypogonadism. A study found that the higher plasma levels of testosterone generated with oral testosterone undecanoate than with testosterone gel (50mg/day) were more effective in reducing the score on the IPSS, probably indicating that there is a relationship between plasma levels of testosterone and their effects on LUTS (Yassin et al., 2008). Although this inverse association is contrary to the commonly held clinical opinion that higher serum androgen levels may worsen clinical LUTS and BPH, the risk would not applicable to normalization of the serum testosterone levels in late onset hypogonadism. Clinical trials are needed to confirm that testosterone replacement therapy in older men does not increase the risks of LUTS or clinical BPH. It would also be significant to explore whether normalization of plasma testosterone has an adjunctive therapeutic effect to the more established

metabolites and testosterone to DHT metabolite ratios to be directly associated with LUTS or BPH (Kristal et al., 2008; Platz et al., 1999). Kristal et al (2008) showed stronger associations as a ratio suggesting that the testosterone : DHT metabolite or testosterone : DHT ratio may therefore be a more sensitive marker of LUTS/BPH than either hormone individually. Platz et al. (1999) estimated the risk of BPH and severe LUTS in relation to testosterone, DHT, estradiol and AAG in 300 men with severe LUTS. There was a positive relation with AAG and an inverse one with estradiol for the risk of BPH surgery/LUTS, the correlations persisting even after adjusting for steroid hormones and sex hormone binding

A study showed at the 105th Annual Scientific Meeting of the AUA that as testosterone levels decreased, LUTS severity increased and maximum flow rate decreased, while prostate volume remained the same as hormone levels decreased (Sauver, et al., 2010). In another study to determine the relationship between androgens, LUTS and urodynamic variables of BOO in patients with LUTS/BPH, free testosterone was negatively correlated with detrusor pressure at the end of urinary flow (closure detrusor pressure) and pressure at the maximum urinary flow rate (Qmax) (Koritsiadis et al., 2008). Mean closure detrusor pressure and detrusor pressure at Qmax differed significantly between patients with low and normal free testosterone levels. Detrusor overactivity (DO) was noted in patients who had significantly lower free testosterone levels than those with no DO. Although several cross-sectional and prospective studies showed no consensus of association of testosterone with LUTS or BPH, it is notable that no studies have as yet reported an increased risk of

In the rabbit bladder outlet obstruction study, bladder dysfunction is mainly mediated by three cellular processes; 1) progressive denervation, 2) cellular mitochondria malfunction, 3) dysregulation of intracellular calcium storage and release from the sarcoplasmic reticulum (SR). Biomarkers for these three functions are calcium adenosine triphosphatase (ATPase) for calcium release, citrate synthase for mitochondrial function, and choline acetyl-transferse for cholinergic innervations (Juan et al., 2007). Bladder contraction can be divided into phasic and tonic period. The phasic response depends on the adenosine triphosphate (ATP) concentration in the bladder wall, whereas the tonic phase requires mitochondrial oxidative activity to generate energy. Castration of adult male rabbits resulted in a significant decrease in the activities of the mitochondria specific enzyme, citrate synthase of the bladder body and base, muscle and mucosa, urethra and corpora, while choline acetyl-transferse activity and calcium

ATPase activity showed different responses depending on the sites (Juan et al., 2007).

Preliminary evidence indicates that men with LUTS benefit from testosterone treatment, and pilot studies have also shown that testosterone therapy has a positive effect on LUTS in lateonset hypogonadism. A study found that the higher plasma levels of testosterone generated with oral testosterone undecanoate than with testosterone gel (50mg/day) were more effective in reducing the score on the IPSS, probably indicating that there is a relationship between plasma levels of testosterone and their effects on LUTS (Yassin et al., 2008). Although this inverse association is contrary to the commonly held clinical opinion that higher serum androgen levels may worsen clinical LUTS and BPH, the risk would not applicable to normalization of the serum testosterone levels in late onset hypogonadism. Clinical trials are needed to confirm that testosterone replacement therapy in older men does not increase the risks of LUTS or clinical BPH. It would also be significant to explore whether normalization of plasma testosterone has an adjunctive therapeutic effect to the more established

globulin (SHBG).

LUTS with higher testosterone.

pharmacological treatment modalities of LUTS as in an adjunctive therapeutic effect of testosterone supplement to phosphodiesterase type 5 (PDE5) inhibitors for ED treatment accompanied by the testosterone deficiency (Buvat et al., 2011; Blute et al., 2009).

There is ample evidence from many epidemiological studies that LUTS and ED are strongly linked, independently of age. ED assessed by a questionnaire, International Index of Erectile Function (IIEF) score was strongly related to LUTS severity. When controlling for age, LUTS severity was by far the strongest predictor of erectile function, with an odd ratio for severe vs mild LUTS of 8.99 followed by diabetes, 3.01; cardiac disease, 2.17; hypertension, 1.83 and hyperlipidemia, 1.57 (McVary., 2006). The fact that LUTS severity is the strongest predictor of ED suggests that LUTS and ED may share their underlying causes and the underlying causes may explain the reasons why severity of LUTS does not correlates with prostate size. Although a direct causal relationship is not established yet, four pathophysiological mechanisms can explain the relationship. These include 1) insulin resistance and autonomic hyperactivity, 2) alteration in nitric oxide bioavailability, 3) Rho-kinase activation/endothelin pathway, 4) pelvic atherosclerosis, all of which are known to be androgen-dependent.

#### **4. Insulin resistance and autonomic hyperactivity**

It was proposed that LUTS is a part of the metabolic syndrome which includes hyperglycemia, obesity, dyslipidemia and hypertension. Recently, testosterone deficiency has captured attention to be a possible risk factor of metabolic syndrome. The basis of this concept came from increased insulin resistance found in both hypergonadotropic and hypogonadotropic hypogonadism. Patients of un-treated Klinefelter's syndrome, compared with control subjects, showed a significantly higher prevalence of metabolic risk factors (Bojesen et al., 2006). GnRH agonist-treated men with prostate cancer also showed significantly decreased insulin sensitivity (Smith et al., 2006). Long-term survivors of testicular cancer have an increased risk for cardiovascular events 10 or more years after chemotherapy (Huddart et al., 2003). Nuver et al. (2005) found lower testosterone associated with a higher body mass index (BMI) pretreatment and a larger BMI increase during followup in testicular cancer survivors following cisplatin–based chemotherapy than controls, suggesting testosterone may play a role in the development of the metabolic syndrome.

Plasma testosterone levels decline with aging in healthy men and features of the metabolic syndrome also show age-related deteriorations, suggesting that testosterone is an important regulator of insulin sensitivity in men. There are lots of evidences that late onset hypogonadism is associated with metabolic syndrome. Blouin et al. (2006) investigated whether this decline or the aging process per se accounts for the risk of metabolic syndrome. They observed that patients with a high testosterone level were more likely to have fewer than three components of metabolic syndrome than those with a low testosterone level. Author (2009) also found that HOMA-IR correlated negatively with serum total, free and bioavailable testosterone. Studies showed that blood levels of insulin and metabolic risk factors increased with lower testosterone levels in middle aged men (Laaksonen, et al., 2003). And patients with metabolic syndrome had significantly lower serum testosterone level than those without metabolic syndrome. Men who developed both metabolic syndrome and diabetes mellitus at 11-year follow-up were especially likely to have low testosterone levels already at baseline (Laaksonen, et al., 2004). Pitteloud et al (2005a)

Testosterone Deficiency Linked to Lower Urinary Tract Symptoms 223

used. One explanation for this discrepancy is the SHBG is mediating the link between testosterone and insulin sensitivity. Proponents of the use of free testosterone argue that it is the best index of androgenicity in insulin-resistant men given the low SHBG levels that pertain in this setting (Plymate et al., 1988). Another explanation is that the assays used to measure free testosterone have serious methodological limitations (Matsumoto & Bremner, 2004). In a recent study of the epidemiological relationship between metabolic syndrome and LUTS, it was hypothesized that metabolic syndrome is associated with overactivity of the autonomic nervous system, and that insulin resistance, a key element of metabolic syndrome, might be responsible. Hyperinsulinemia promotes endothelin-1 (ET-1) secretion through mitogen-activated protein (MAP)-kinase pathway leading to increase in smooth muscle contraction. In addition, it activates sympathetic nerve system contributing to further increase in the contraction. Overactivity of the autonomic nervous system is supposedly not responsible for the development of LUTS, but rather is believed to play a key role in increasing LUTS severity above an intrinsic basal intensity (McVary et al., 2005;

The studies of Meusburger & Keast (2001) and Keast et al. (2002) have provided elegant demonstrations on the potential role of androgens in maintaining the structure and function of many pelvic ganglion neurons. Giuliano et al. (1993) suggested that testosterone acting peripherally to the spinal cord enhances the erectile response of the cavernous nerve. Traish et al. (2007) showed that testosterone treatment of castrated animals restored the cavernosal nerve fibers and myelin sheath structure, similar to that observed in the sham group. Considering these reports and the hypothesis that noradrenaline and α1-AR that mediate adrenergic contraction of smooth muscles in the prostate, bladder neck, urethra and corpus cavernosum, contribute to the common link, testosterone deficiency could be a causative

Another possible mechanism includes a direct relaxation effect of testosterone on smooth muscle cells, a change in the number of receptors for sympathetic affecter molecules or a change in sensitivity of smooth muscle cell α-adrenergic receptors (ARs). Comparative binding and functional studies of lower genitourinary tissues have demonstrated that α1- ARs are abundant in the bladder base and prostate but spare in the bladder body (Lepor & Shapiro, 1984). In certain human arteries, α1-AR expression increases and the relative proportion of α1-AR subtypes is modulated by aging (Rudner et al., 1999). These changes might be happening as well in the human prostatic, bladder and erectile tissue. The contractile response of the cavernosal strips of older men with ED was greater than for those isolated from younger men with ED, suggesting that aging has a role in adrenergic sensitivity in patients with ED (Christ et al., 1991). It has also been suggested that α1-ARs are up-regulated in patients with LUTS associated with BPH, resulting in increased smooth

Theoretically, selective α1-AR antagonists are ideally suited for the treatment of the dynamic component of bladder outlet obstruction, because the resistance along the bladder outlet can be selectively reduced without impairing detrusor contraction. Virtually, α1-AR antagonists have been widely used in practice for treating symptomatic BPH/LUTS. Despite the clinical availability of α1-AR antagonists for treating LUTS, as yet it is not possible to provide a comprehensive picture of the impact of testosterone on α1- ARs. While androgen effect on central and peripheral nervous system are well known, the local effect of androgen on bladder and urethra has not been extensively studied. Even reports on the density of α1- ARs after castration are not consistent. Takyu (1993) found the density of α1-ARs in rabbit

muscle tone in the prostatic capsule and bladder neck (Medina et al., 1999).

Kasturi et al., 2006).

factor of LUTS via autonomic hyperactivity.

demonstrated men with hypogonadal testosterone levels were twice as insulin resistant as their eugonadal counterparts and 90% fulfill criteria for the metabolic syndrome.

Recent studies provide insight into the role of mitochondrial function in the pathogenesis of insulin resistance. Mitochondria have a critical role in Ca2+ buffering in bladder smooth muscle. Pitteloud et al. (2005a) demonstrated testosterone levels were positively correlated not only with insulin sensitivity but also with genetic (oxidative phosphorylation gene expression) and functional (maximal aerobic capacity) markers of mitochondrial function, suggesting a novel molecular mechanism whereby testosterone might modulate insulin sensitivity in men. Pitteloud et al. (2005b) found in another study that insulin resistance was associated with a decrease in Leydig cell testosterone secretion by evaluating the hypothalamic-pituitary-gonadal axis in men with a spectrum of insulin sensitivity.

The most common cause of ED is penile vascular insufficiency. This is usually part of a generalized endothelial dysfunction and is related to several conditions, including type 2 diabetes mellitus, hypertension, hyperlipidemia, and obesity, of which conditions underlie the pathophysiology of metabolic syndrome. There is evidence from multiple epidemiological studies that LUTS and ED are correlated, independent of age or comorbidities as diabetes or hypertension. The prevalence of LUTS was 72% in men with ED versus 38% in those without ED. And the presence of LUTS was a risk factor of ED. Therefore, men seeking care for one condition should always be screened for complaints of the other condition.

Metabolic risk factors induce testosterone deficiency. Obesity-related conditions such as obstructive sleep apnea, insulin resistance and type 2 diabetes mellitus are independently associated with decreased plasma testosterone. Possible mechanisms include decreased LH pulse amplitude, inhibitory effects of estrogen at the hypothalamus and pituitary and the effects of leptin and other peptides centrally and on Leydig cells. Diabetes induces testicular oxidative stress and damage,

Summing up, metabolic syndrome is a risk factor of LUTS and ED and may reduce the blood level of testosterone. Contrariwise, testosterone deficiency may be a causative factor of metabolic risk factors and may accompany LUTS and ED. LUTS is a risk factor of ED and vice versa. This interrelationship suggests that these ailments may be a symptom complex and may share the underlying causes (Figure 5).

Fig. 5. Interrelationship between metabolic syndrme (MS) testosterone deficiency (TD) lower urinary tract symptom (LUTS) and erectile dysfunction (ED)

However, previous studies on the relationship between androgens and insulin sensitivity in men gave conflicting results depending on whether total or free testosterone levels were

demonstrated men with hypogonadal testosterone levels were twice as insulin resistant as

Recent studies provide insight into the role of mitochondrial function in the pathogenesis of insulin resistance. Mitochondria have a critical role in Ca2+ buffering in bladder smooth muscle. Pitteloud et al. (2005a) demonstrated testosterone levels were positively correlated not only with insulin sensitivity but also with genetic (oxidative phosphorylation gene expression) and functional (maximal aerobic capacity) markers of mitochondrial function, suggesting a novel molecular mechanism whereby testosterone might modulate insulin sensitivity in men. Pitteloud et al. (2005b) found in another study that insulin resistance was associated with a decrease in Leydig cell testosterone secretion by evaluating the

The most common cause of ED is penile vascular insufficiency. This is usually part of a generalized endothelial dysfunction and is related to several conditions, including type 2 diabetes mellitus, hypertension, hyperlipidemia, and obesity, of which conditions underlie the pathophysiology of metabolic syndrome. There is evidence from multiple epidemiological studies that LUTS and ED are correlated, independent of age or comorbidities as diabetes or hypertension. The prevalence of LUTS was 72% in men with ED versus 38% in those without ED. And the presence of LUTS was a risk factor of ED. Therefore, men seeking care for one condition should always be screened for complaints of

Metabolic risk factors induce testosterone deficiency. Obesity-related conditions such as obstructive sleep apnea, insulin resistance and type 2 diabetes mellitus are independently associated with decreased plasma testosterone. Possible mechanisms include decreased LH pulse amplitude, inhibitory effects of estrogen at the hypothalamus and pituitary and the effects of leptin and other peptides centrally and on Leydig cells. Diabetes induces testicular

Summing up, metabolic syndrome is a risk factor of LUTS and ED and may reduce the blood level of testosterone. Contrariwise, testosterone deficiency may be a causative factor of metabolic risk factors and may accompany LUTS and ED. LUTS is a risk factor of ED and vice versa. This interrelationship suggests that these ailments may be a symptom complex

**MS TD**

**LUTS**

**ED**

Fig. 5. Interrelationship between metabolic syndrme (MS) testosterone deficiency (TD) lower

However, previous studies on the relationship between androgens and insulin sensitivity in men gave conflicting results depending on whether total or free testosterone levels were

their eugonadal counterparts and 90% fulfill criteria for the metabolic syndrome.

hypothalamic-pituitary-gonadal axis in men with a spectrum of insulin sensitivity.

the other condition.

oxidative stress and damage,

and may share the underlying causes (Figure 5).

urinary tract symptom (LUTS) and erectile dysfunction (ED)

used. One explanation for this discrepancy is the SHBG is mediating the link between testosterone and insulin sensitivity. Proponents of the use of free testosterone argue that it is the best index of androgenicity in insulin-resistant men given the low SHBG levels that pertain in this setting (Plymate et al., 1988). Another explanation is that the assays used to measure free testosterone have serious methodological limitations (Matsumoto & Bremner, 2004).

In a recent study of the epidemiological relationship between metabolic syndrome and LUTS, it was hypothesized that metabolic syndrome is associated with overactivity of the autonomic nervous system, and that insulin resistance, a key element of metabolic syndrome, might be responsible. Hyperinsulinemia promotes endothelin-1 (ET-1) secretion through mitogen-activated protein (MAP)-kinase pathway leading to increase in smooth muscle contraction. In addition, it activates sympathetic nerve system contributing to further increase in the contraction. Overactivity of the autonomic nervous system is supposedly not responsible for the development of LUTS, but rather is believed to play a key role in increasing LUTS severity above an intrinsic basal intensity (McVary et al., 2005; Kasturi et al., 2006).

The studies of Meusburger & Keast (2001) and Keast et al. (2002) have provided elegant demonstrations on the potential role of androgens in maintaining the structure and function of many pelvic ganglion neurons. Giuliano et al. (1993) suggested that testosterone acting peripherally to the spinal cord enhances the erectile response of the cavernous nerve. Traish et al. (2007) showed that testosterone treatment of castrated animals restored the cavernosal nerve fibers and myelin sheath structure, similar to that observed in the sham group. Considering these reports and the hypothesis that noradrenaline and α1-AR that mediate adrenergic contraction of smooth muscles in the prostate, bladder neck, urethra and corpus cavernosum, contribute to the common link, testosterone deficiency could be a causative factor of LUTS via autonomic hyperactivity.

Another possible mechanism includes a direct relaxation effect of testosterone on smooth muscle cells, a change in the number of receptors for sympathetic affecter molecules or a change in sensitivity of smooth muscle cell α-adrenergic receptors (ARs). Comparative binding and functional studies of lower genitourinary tissues have demonstrated that α1- ARs are abundant in the bladder base and prostate but spare in the bladder body (Lepor & Shapiro, 1984). In certain human arteries, α1-AR expression increases and the relative proportion of α1-AR subtypes is modulated by aging (Rudner et al., 1999). These changes might be happening as well in the human prostatic, bladder and erectile tissue. The contractile response of the cavernosal strips of older men with ED was greater than for those isolated from younger men with ED, suggesting that aging has a role in adrenergic sensitivity in patients with ED (Christ et al., 1991). It has also been suggested that α1-ARs are up-regulated in patients with LUTS associated with BPH, resulting in increased smooth muscle tone in the prostatic capsule and bladder neck (Medina et al., 1999).

Theoretically, selective α1-AR antagonists are ideally suited for the treatment of the dynamic component of bladder outlet obstruction, because the resistance along the bladder outlet can be selectively reduced without impairing detrusor contraction. Virtually, α1-AR antagonists have been widely used in practice for treating symptomatic BPH/LUTS. Despite the clinical availability of α1-AR antagonists for treating LUTS, as yet it is not possible to provide a comprehensive picture of the impact of testosterone on α1- ARs. While androgen effect on central and peripheral nervous system are well known, the local effect of androgen on bladder and urethra has not been extensively studied. Even reports on the density of α1- ARs after castration are not consistent. Takyu (1993) found the density of α1-ARs in rabbit

al., 2003).

Testosterone Deficiency Linked to Lower Urinary Tract Symptoms 225

than in normal prostate, and this may also contribute to affect voiding function (Podlasek et

Fig. 6. Potential regulation of nitric oxide synthase (NOS) and hosphodiesterase type 5

tract, given that the same enzymes and androgen receptors are also present.

Fig. 7. NOS/NO theory for LUTS/ED (Adopted from McVary, et al., 2006)

It is known that the lower urinary and genital tracts are embryologically and anatomically closely related, and that both are sensitive to sex steroids. A preponderance of evidence reports a role for androgens in regulating the expression and activity of NOS isoforms in the corpus cavernosum in animal models. In castrated animals, testosterone and DHT administration restored the erectile response and NOS expression in penis. It can be speculated that if testosterone participates in erectile mechanisms by modulating NOS and phosphodiesterase type 5 (PDE5), a similar interaction might be found in the lower urinary

(PDE5) by androgens (Adopted fromTraish, et al., 2007)

proximal urethra decreased after castration and testosterone supplementation restored the densities of α1-ARs to control levels. Morita et al. (1992) reported that α1-adrenergic and muscarinic cholinergic receptor densities decreased significantly after castration. Yono et al (2006) found the density of α1-ARs in the rat prostate decreased with aging. Because the percent of muscle density shows no significant change throughout life in the rat prostate (Moriyama et al., 1995), the age related decrease in the density of α1-ARs appears to result from direct down-regulation of α1-AR protein. Auger-Pourmarin et al. (1998) presented that testosterone administration produced a 23% decrease of α1-AR density, likely by an increase of prostatic glandular epithelium and a decrease in the relative proportion of smooth muscle, thus of α1-AR density. Lacey et al. (1996) found there was an apparent increase in α1-AR density in dog prostate after castration which returned to baseline with testosterone replacement. However, the increase in α1-ARs density resulted from relative increases in the ratio of smooth muscle to epithelium rather from direct up-regulation of α1-AR protein.

Lastly, recent studies provided an evidence that the inflammatory infiltrates, which are frequently found in and around nodules of BPH (Rohrmann et al, 2005), elevate serum CRP concentration, a non-specific marker of inflammation. Furthermore, the presence of metabolic syndrome might mediate intraprostatic inflammation because of its association with an elevated serum CRP concentration, which would link metabolic syndrome to symptomatic BPH (Rohrmann et al., 2005).

#### **5. Alteration in nitric oxide bioavailability**

It is widely accepted that NO, which is synthesized from its precursor L-arginine via NO synthase (NOS), is important in the relaxation of corpus cavernosum smooth muscle and vasculature. By activating gualnylate cyclase, with resultant elevation of cyclic guanylate monophosphate (cGMP), NO results in a lowering of intracellular calcium and smooth muscle relaxation. cGMP is an important secondary messenger of NO involved in modulating the contractility of various smooth muscles. It stimulates protein kinase G, which in turn initiates phosphorylation of membrane-bound proteins at the potassium channels. This leads to potassium ion outflow into the extracellular space resulting in hyperpolarization. Hyperpolarization leads to closure of the L-type calcium channels subsequently resulting in a decrease in the intracellular Ca++ ion concentration and consequent smooth muscle cell relaxation. PDE plays important roles in this process by modulating the levels of cGMP and their duration of action (Traish et al., 2007) (Figure 6).

NO is also present in the human prostate and bladder and putatively modulates smooth muscle tone. NOS activity has been reported to be highest in the prostatic urethra, intermediate in the bladder neck, and less pronounced in detrusor muscle. It has been reported that NOS expression, and thus NO production, of the prostate is reduced in the transition zone of the prostate in BPH compared with normal prostate (Bloch et al., 1997). The proposed reduction in expression of NOS isoforms results in increased smooth muscle cell contractile forces at the bladder neck and prostatic urethra leading to the subsequent development of LUTS or LUTS without BPH. On the other hand, reduced NOS/NO results in smooth muscle cell proliferation and may result in structural changes in the prostate and simultaneously increased contraction and affects outlet resistance and bladder compliance (Figure 7). Histochemical staining and immunohistochemistry confirmed dense nitrinergic innervations of glandular epithelium, fibromuscular stroma and blood vessels in the normal human prostate. The nitrinergic innervations is also less in hyperplastic human prostate

proximal urethra decreased after castration and testosterone supplementation restored the densities of α1-ARs to control levels. Morita et al. (1992) reported that α1-adrenergic and muscarinic cholinergic receptor densities decreased significantly after castration. Yono et al (2006) found the density of α1-ARs in the rat prostate decreased with aging. Because the percent of muscle density shows no significant change throughout life in the rat prostate (Moriyama et al., 1995), the age related decrease in the density of α1-ARs appears to result from direct down-regulation of α1-AR protein. Auger-Pourmarin et al. (1998) presented that testosterone administration produced a 23% decrease of α1-AR density, likely by an increase of prostatic glandular epithelium and a decrease in the relative proportion of smooth muscle, thus of α1-AR density. Lacey et al. (1996) found there was an apparent increase in α1-AR density in dog prostate after castration which returned to baseline with testosterone replacement. However, the increase in α1-ARs density resulted from relative increases in the ratio of smooth muscle to epithelium rather from direct up-regulation of α1-AR protein. Lastly, recent studies provided an evidence that the inflammatory infiltrates, which are frequently found in and around nodules of BPH (Rohrmann et al, 2005), elevate serum CRP concentration, a non-specific marker of inflammation. Furthermore, the presence of metabolic syndrome might mediate intraprostatic inflammation because of its association with an elevated serum CRP concentration, which would link metabolic syndrome to

It is widely accepted that NO, which is synthesized from its precursor L-arginine via NO synthase (NOS), is important in the relaxation of corpus cavernosum smooth muscle and vasculature. By activating gualnylate cyclase, with resultant elevation of cyclic guanylate monophosphate (cGMP), NO results in a lowering of intracellular calcium and smooth muscle relaxation. cGMP is an important secondary messenger of NO involved in modulating the contractility of various smooth muscles. It stimulates protein kinase G, which in turn initiates phosphorylation of membrane-bound proteins at the potassium channels. This leads to potassium ion outflow into the extracellular space resulting in hyperpolarization. Hyperpolarization leads to closure of the L-type calcium channels subsequently resulting in a decrease in the intracellular Ca++ ion concentration and consequent smooth muscle cell relaxation. PDE plays important roles in this process by modulating the levels of cGMP and their duration of action (Traish et al., 2007) (Figure 6). NO is also present in the human prostate and bladder and putatively modulates smooth muscle tone. NOS activity has been reported to be highest in the prostatic urethra, intermediate in the bladder neck, and less pronounced in detrusor muscle. It has been reported that NOS expression, and thus NO production, of the prostate is reduced in the transition zone of the prostate in BPH compared with normal prostate (Bloch et al., 1997). The proposed reduction in expression of NOS isoforms results in increased smooth muscle cell contractile forces at the bladder neck and prostatic urethra leading to the subsequent development of LUTS or LUTS without BPH. On the other hand, reduced NOS/NO results in smooth muscle cell proliferation and may result in structural changes in the prostate and simultaneously increased contraction and affects outlet resistance and bladder compliance (Figure 7). Histochemical staining and immunohistochemistry confirmed dense nitrinergic innervations of glandular epithelium, fibromuscular stroma and blood vessels in the normal human prostate. The nitrinergic innervations is also less in hyperplastic human prostate

symptomatic BPH (Rohrmann et al., 2005).

**5. Alteration in nitric oxide bioavailability** 

than in normal prostate, and this may also contribute to affect voiding function (Podlasek et al., 2003).

Fig. 6. Potential regulation of nitric oxide synthase (NOS) and hosphodiesterase type 5 (PDE5) by androgens (Adopted fromTraish, et al., 2007)

It is known that the lower urinary and genital tracts are embryologically and anatomically closely related, and that both are sensitive to sex steroids. A preponderance of evidence reports a role for androgens in regulating the expression and activity of NOS isoforms in the corpus cavernosum in animal models. In castrated animals, testosterone and DHT administration restored the erectile response and NOS expression in penis. It can be speculated that if testosterone participates in erectile mechanisms by modulating NOS and phosphodiesterase type 5 (PDE5), a similar interaction might be found in the lower urinary tract, given that the same enzymes and androgen receptors are also present.

Fig. 7. NOS/NO theory for LUTS/ED (Adopted from McVary, et al., 2006)

Testosterone Deficiency Linked to Lower Urinary Tract Symptoms 227

prostatic smooth muscle cells, and inhibited noradrenergic contractions elicited by electrical

In many pathological cases, hyperactivity of Rho/Rho-kinase signaling has been observed. Increased Rho-kinase activity, and consequently increased calcium sensitivity of the contractile machinery, can be found in the detrusor of rabbits with partial BOO (Bing et al., 2003) and in the corpus cavernosum smooth muscle of rabbits with partial BOO (Chang et al., 2005). Increased smooth muscle myosin basal phosphorylation necessary for smooth muscle contraction in the corpus cavernosum smooth muscle of partial BOO, mediated via an increase in Rho-kinase expression/activity, would be expected to make the corpus cavernosum smooth muscle more difficult to relax, which suggests that the RhoA/Rho-

In various animal species estrogen receptors are shown to be expressed in central nervous structures involved in micturition (VanderHorst et al., 2001), as well as in the bladder and urethra (Williams & Papka, 1996; Makela et al., 2000). Chavalmane et al. (2010) found that estrogen supplementation significantly increased the relaxing response of carbacolprecontracted rat bladder strips to Y-27632, an inhibitor of Rho-kinase. On the contrary, testosterone administration in the same animal model did not increase responsiveness to Y-27632, but even reduced it. This was in apparent contrast with the observation in isolated human bladder cells, where testosterone mimicked estrogen effects. However, in human bladder cells co-incubation with letrozole, an inhibitor of aromatase, reverted all the testosterone-induced effects. In addition, DHT, a non-aromatizable androgen, did not substantially stimulate smooth muscle gene expression and cell motility. These findings suggest that active aromatization is operating in human bladder cells and that estrogen and not androgen receptors are involved in stimulating cell migration and expression of genes related to the smooth muscle phenotype. In contrast to human bladder, rat bladder does not express aromatase/CYP19A1 mRNA and testosterone presumably acts only through the androgen receptors. These data indicate that estrogen, more than androgen, receptors upregulate Rho/Rho-kinase signaling and might have a role in calcium sensitization in human male bladder and androgens have the opposite effect. Clinical studies showing that androgen deficiency is associated with male bladder instability are in keeping with this view. Since an altered estrogen/androgen ratio characterizes several physiological and pathological conditions, often associated to bladder hyperactivity and LUTS, it is possible that a relative hyperesterogenism might induce bladder overactivity, through an activation

The actions of several factors beside noradrenaline (e.g. endothelin-1, angiotensin II), possibly involved in the increased smooth muscle activity found in both LUTS/BPH and ED, are dependent on Rho-kinase activity that acts downstream from these receptors (Anderson, 2003). Although the exact mechanisms by which angiotensin II elicits its cellular effects are not known, the mechanism of action of angiotensin II in the cells is related to the Rho/Rho-kinase pathway. While estradiol did not change the number of human aortic endothelial cells secreting endothelin-1 but decreased the number of secreting cells stimulated with angiotensin-II, testosterone induced an increase in the number of cells secreting endothelin-1 and up-regulated endothelin-1 mRNA, indicating that testosterone, estradiol and angiotensin-II have parallel effects on the production of endothelin-1 (Pearson

field stimulation and exogenous phenylephrine in rat prostatic tissue.

kinase pathway as being involved in the mechanism for LUTS-associated ED.

of the Rho/Rho-kinase pathway.

et al., 2008).

Jones & Schoenberg (1985) suggested that aging itself might be an underlying cause of DO, besides BOO. In experimental studies, the inhibition of NO production causes bladder hyperactivity in rats in vivo (Persson et al., 1992), indicating a possible relation with changes in NOS activity in the lower urinary tract. Koritsiadis et al. (2008) found men with no DO had higher free testosterone levels, and all hypogonadal patients had DO, providing evidence that a decline in androgen levels might be a causative factor in DO, by triggering overactivity in an otherwise pathological bladder.

PDE5 hydrolyzes cGMP in vascular and cavernous smooth muscle into GMP. Activation of PDE5 terminates NO-induced, cGMP-mediated smooth muscle relaxation. In penile tissue, the balance between the intracellular levels of cGMP and GMP is primarily regulated by the activities of NOS and PDE5. Thus, it is likely that any disruption in the expression or activity of these enzymes will lead to pathophysiology. Castration has been shown to induce the expression and activity of PDE5 in rabbits and rats, and androgen supplementation has been shown to up-regulate the expression and activity of PDE5 (Zhang et al., 2005; Morelli et al., 2004). Further, administration of PDE inhibitor to castrated animals has little effect on the intracavernosal pressure in response to pelvic nerve stimulation (Zhang et al., 2005), suggesting that androgens are critical not only for regulating NOS activity, but also in modulating PDE5 activity. The presence of PDE in the urinary bladder was identified in studies of the rat (Qiu, et al., 2001) and the human (Werkstrom, et al., 2006).

A recent study, investigating PDE5 expression and activity in the human bladder, elegantly demonstrated that PDE5 regulates smooth muscles tone of the bladder. Vardenafil appeared to block PDE5 activity, and therefore, may be a possible therapeutic option for bladder dysfunction by ameliorating irritative LUTS. The study also found that castration decreased, and testosterone supplementation restored, PDE5 gene expression in rat bladder (Morelli, et al., 2009). Meanwhile, a large number of clinical studies have convincingly shown that PDE inhibitors have a beneficial effect on LUTS. Sairam et al (2002) found the overall trend in the IPSS was towards improvement after treatment with sildenafil. In a recent 12-week global dose-finding study conducted in 1058 men with BPH-LUTS, tadalafil was associated with statistically significant and clinically meaningful improvements in multiple measures of LUTS, including quality of life and ED improvement, compared to placebo (Roehrborn et al., 2008). Kim et al (2011) reported men with BPH-LUTS treated with tadalafil 5 mg once daily experienced a reduction in BPH-LUTS which was comparable to tamsulosin.

#### **6. Rho-kinase activation/endothelin activity**

Smooth muscle contraction has been attributed to an increase in the intracellular calcium concentration. However, some regulatory mechanisms can modify the sensitivity of contractile and regulatory proteins to calcium, leading to a smooth muscle contraction without changing intracellular calcium concentration (Somlyo A.P. & Somlyo A.V., 2000). One of these mechanisms is the Rho-kinase pathway, which is thought to be a major calcium-sensitizing mechanism in smooth muscle. The Rho-kinase is activated by a Gprotein, RhoA, thought to be coupled to excitatory α1-adrenoceptors. The major contractile process in bladder is under acetylcholine control, through the activation of M3 muscarinic receptors. RhoA/Rho-kinase calcium sensitization pathway plays a major role in maintaining the contractile actions in bladder smooth muscle tone. Rees et al. (2003) found that a specific inhibitor of Rho-kinase, Y-27632, decreased the proliferation of human and rat

Jones & Schoenberg (1985) suggested that aging itself might be an underlying cause of DO, besides BOO. In experimental studies, the inhibition of NO production causes bladder hyperactivity in rats in vivo (Persson et al., 1992), indicating a possible relation with changes in NOS activity in the lower urinary tract. Koritsiadis et al. (2008) found men with no DO had higher free testosterone levels, and all hypogonadal patients had DO, providing evidence that a decline in androgen levels might be a causative factor in DO, by triggering

PDE5 hydrolyzes cGMP in vascular and cavernous smooth muscle into GMP. Activation of PDE5 terminates NO-induced, cGMP-mediated smooth muscle relaxation. In penile tissue, the balance between the intracellular levels of cGMP and GMP is primarily regulated by the activities of NOS and PDE5. Thus, it is likely that any disruption in the expression or activity of these enzymes will lead to pathophysiology. Castration has been shown to induce the expression and activity of PDE5 in rabbits and rats, and androgen supplementation has been shown to up-regulate the expression and activity of PDE5 (Zhang et al., 2005; Morelli et al., 2004). Further, administration of PDE inhibitor to castrated animals has little effect on the intracavernosal pressure in response to pelvic nerve stimulation (Zhang et al., 2005), suggesting that androgens are critical not only for regulating NOS activity, but also in modulating PDE5 activity. The presence of PDE in the urinary bladder was identified in

A recent study, investigating PDE5 expression and activity in the human bladder, elegantly demonstrated that PDE5 regulates smooth muscles tone of the bladder. Vardenafil appeared to block PDE5 activity, and therefore, may be a possible therapeutic option for bladder dysfunction by ameliorating irritative LUTS. The study also found that castration decreased, and testosterone supplementation restored, PDE5 gene expression in rat bladder (Morelli, et al., 2009). Meanwhile, a large number of clinical studies have convincingly shown that PDE inhibitors have a beneficial effect on LUTS. Sairam et al (2002) found the overall trend in the IPSS was towards improvement after treatment with sildenafil. In a recent 12-week global dose-finding study conducted in 1058 men with BPH-LUTS, tadalafil was associated with statistically significant and clinically meaningful improvements in multiple measures of LUTS, including quality of life and ED improvement, compared to placebo (Roehrborn et al., 2008). Kim et al (2011) reported men with BPH-LUTS treated with tadalafil 5 mg once

studies of the rat (Qiu, et al., 2001) and the human (Werkstrom, et al., 2006).

daily experienced a reduction in BPH-LUTS which was comparable to tamsulosin.

Smooth muscle contraction has been attributed to an increase in the intracellular calcium concentration. However, some regulatory mechanisms can modify the sensitivity of contractile and regulatory proteins to calcium, leading to a smooth muscle contraction without changing intracellular calcium concentration (Somlyo A.P. & Somlyo A.V., 2000). One of these mechanisms is the Rho-kinase pathway, which is thought to be a major calcium-sensitizing mechanism in smooth muscle. The Rho-kinase is activated by a Gprotein, RhoA, thought to be coupled to excitatory α1-adrenoceptors. The major contractile process in bladder is under acetylcholine control, through the activation of M3 muscarinic receptors. RhoA/Rho-kinase calcium sensitization pathway plays a major role in maintaining the contractile actions in bladder smooth muscle tone. Rees et al. (2003) found that a specific inhibitor of Rho-kinase, Y-27632, decreased the proliferation of human and rat

**6. Rho-kinase activation/endothelin activity** 

overactivity in an otherwise pathological bladder.

prostatic smooth muscle cells, and inhibited noradrenergic contractions elicited by electrical field stimulation and exogenous phenylephrine in rat prostatic tissue.

In many pathological cases, hyperactivity of Rho/Rho-kinase signaling has been observed. Increased Rho-kinase activity, and consequently increased calcium sensitivity of the contractile machinery, can be found in the detrusor of rabbits with partial BOO (Bing et al., 2003) and in the corpus cavernosum smooth muscle of rabbits with partial BOO (Chang et al., 2005). Increased smooth muscle myosin basal phosphorylation necessary for smooth muscle contraction in the corpus cavernosum smooth muscle of partial BOO, mediated via an increase in Rho-kinase expression/activity, would be expected to make the corpus cavernosum smooth muscle more difficult to relax, which suggests that the RhoA/Rhokinase pathway as being involved in the mechanism for LUTS-associated ED.

In various animal species estrogen receptors are shown to be expressed in central nervous structures involved in micturition (VanderHorst et al., 2001), as well as in the bladder and urethra (Williams & Papka, 1996; Makela et al., 2000). Chavalmane et al. (2010) found that estrogen supplementation significantly increased the relaxing response of carbacolprecontracted rat bladder strips to Y-27632, an inhibitor of Rho-kinase. On the contrary, testosterone administration in the same animal model did not increase responsiveness to Y-27632, but even reduced it. This was in apparent contrast with the observation in isolated human bladder cells, where testosterone mimicked estrogen effects. However, in human bladder cells co-incubation with letrozole, an inhibitor of aromatase, reverted all the testosterone-induced effects. In addition, DHT, a non-aromatizable androgen, did not substantially stimulate smooth muscle gene expression and cell motility. These findings suggest that active aromatization is operating in human bladder cells and that estrogen and not androgen receptors are involved in stimulating cell migration and expression of genes related to the smooth muscle phenotype. In contrast to human bladder, rat bladder does not express aromatase/CYP19A1 mRNA and testosterone presumably acts only through the androgen receptors. These data indicate that estrogen, more than androgen, receptors upregulate Rho/Rho-kinase signaling and might have a role in calcium sensitization in human male bladder and androgens have the opposite effect. Clinical studies showing that androgen deficiency is associated with male bladder instability are in keeping with this view. Since an altered estrogen/androgen ratio characterizes several physiological and pathological conditions, often associated to bladder hyperactivity and LUTS, it is possible that a relative hyperesterogenism might induce bladder overactivity, through an activation of the Rho/Rho-kinase pathway.

The actions of several factors beside noradrenaline (e.g. endothelin-1, angiotensin II), possibly involved in the increased smooth muscle activity found in both LUTS/BPH and ED, are dependent on Rho-kinase activity that acts downstream from these receptors (Anderson, 2003). Although the exact mechanisms by which angiotensin II elicits its cellular effects are not known, the mechanism of action of angiotensin II in the cells is related to the Rho/Rho-kinase pathway. While estradiol did not change the number of human aortic endothelial cells secreting endothelin-1 but decreased the number of secreting cells stimulated with angiotensin-II, testosterone induced an increase in the number of cells secreting endothelin-1 and up-regulated endothelin-1 mRNA, indicating that testosterone, estradiol and angiotensin-II have parallel effects on the production of endothelin-1 (Pearson et al., 2008).

Testosterone Deficiency Linked to Lower Urinary Tract Symptoms 229

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Berry, SJ.; Coffey, DS.; Walsh, PC. & Ewing, LL. (1984). The development of human

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Bloch, W.; Klotz, T.; Loch, C.; Schmidt, G.; Engelmann, U. & Addicks, K. (1997). Distribution

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#### **7. Pelvic atherosclerosis**

Various studies show that bladder dysfunction can be caused by ischemia and suggest that atherosclerosis and hypercholesterolemia might be associated with LUTS. Ischemia in rabbit urinary bladder caused a marked reduction in the compliance and capacity of the bladder (Gill et al., 1988). Azadzoi et al. (1999) found that atherosclerosis-induced chronic bladder ischemia increased transforming growth factor-β1 expression in the bladder leading to fibrosis, smooth muscle atrophy and non-compliance. Hypercholesterolemia also interfered with bladder structure and compliance but to a significantly lesser extent compared with chronic bladder ischemia. It is known atherosclerosis and hypercholesterolemia are associated with low serum androgen levels. Bladder outlet obstruction results in bladder hypertrophy which induces ischemia. Levin et al. (1997) hypothesized that this leads to a release of intracellular calcium, leading to activation of specific enzymes and generation of free radicals. These then attack the membranes of nerves, sarcoplasmic reticulum and mitochondria.

In two cross-sectional studies of elderly men, intima-media thickness (IMT), an indicator of general atherosclerosis, was associated with lower testosterone levels (Hak et al., 2002; van den Beld et al., 2003). In a prospective population-based study, free but not total, testosterone levels were inversely related to IMT (Muller et al., 2004). In a logistic regression model adjusted for the confounding effect of cardiovascular risk factors, men with testosterone levels in the lowest quintile (<9.0 nmol L-1) had an independent OR =1.51 (P=0.015) of being in the highest IMT quintile (Svartberg et al., 2006). Besides the possible modulating effect of testosterone on cardiovascular disease risk factors, a few other possible explanations for the association between testosterone and atherosclerosis have been suggested. A direct beneficial effect of testosterone on plaque development, probably mediated by the vascular androgen receptor has been reported in an animal study (Hanke et al., 2001). Testosterone has also been shown to enhance endothelium-independent and endothelium-dependent vasodilation (Kang et al., 2002 )

#### **8. Conclusion**

Many studies have tried to establish a relationship between sex steroids and BPH, but a few studies have analyzed the relationship between circulating testosterone and LUTS. Although there is no consensus on possible effects of testosterone on LUTS, endogenous testosterone may have a beneficial effect on the lower urinary tract function, and testosterone deficiency may provide a pathophysiologic basis for the link between LUTS. Preliminary evidence indicates that men with LUTS benefit from testosterone treatment. Four pathophysiological mechanisms can explain the relationship; insulin resistance and autonomic hyperactivity, alteration in NO/NOS/PDE activity, Rho-kinase activation/endothelin pathway, pelvic atherosclerosis, all of which are known to be androgen-dependent.

#### **9. References**

Andersson, KE. (2003). Erectile physiological and pathophysiological pathways involved in erectile dysfunction. *Journal of Urology*, 170:S6-S14.

Various studies show that bladder dysfunction can be caused by ischemia and suggest that atherosclerosis and hypercholesterolemia might be associated with LUTS. Ischemia in rabbit urinary bladder caused a marked reduction in the compliance and capacity of the bladder (Gill et al., 1988). Azadzoi et al. (1999) found that atherosclerosis-induced chronic bladder ischemia increased transforming growth factor-β1 expression in the bladder leading to fibrosis, smooth muscle atrophy and non-compliance. Hypercholesterolemia also interfered with bladder structure and compliance but to a significantly lesser extent compared with chronic bladder ischemia. It is known atherosclerosis and hypercholesterolemia are associated with low serum androgen levels. Bladder outlet obstruction results in bladder hypertrophy which induces ischemia. Levin et al. (1997) hypothesized that this leads to a release of intracellular calcium, leading to activation of specific enzymes and generation of free radicals. These then attack the membranes of nerves, sarcoplasmic reticulum and

In two cross-sectional studies of elderly men, intima-media thickness (IMT), an indicator of general atherosclerosis, was associated with lower testosterone levels (Hak et al., 2002; van den Beld et al., 2003). In a prospective population-based study, free but not total, testosterone levels were inversely related to IMT (Muller et al., 2004). In a logistic regression model adjusted for the confounding effect of cardiovascular risk factors, men with testosterone levels in the lowest quintile (<9.0 nmol L-1) had an independent OR =1.51 (P=0.015) of being in the highest IMT quintile (Svartberg et al., 2006). Besides the possible modulating effect of testosterone on cardiovascular disease risk factors, a few other possible explanations for the association between testosterone and atherosclerosis have been suggested. A direct beneficial effect of testosterone on plaque development, probably mediated by the vascular androgen receptor has been reported in an animal study (Hanke et al., 2001). Testosterone has also been shown to enhance endothelium-independent and

Many studies have tried to establish a relationship between sex steroids and BPH, but a few studies have analyzed the relationship between circulating testosterone and LUTS. Although there is no consensus on possible effects of testosterone on LUTS, endogenous testosterone may have a beneficial effect on the lower urinary tract function, and testosterone deficiency may provide a pathophysiologic basis for the link between LUTS. Preliminary evidence indicates that men with LUTS benefit from testosterone treatment. Four pathophysiological mechanisms can explain the relationship; insulin resistance and autonomic hyperactivity, alteration in NO/NOS/PDE activity, Rho-kinase activation/endothelin pathway, pelvic atherosclerosis, all of which are known to be

Andersson, KE. (2003). Erectile physiological and pathophysiological pathways involved in

erectile dysfunction. *Journal of Urology*, 170:S6-S14.

endothelium-dependent vasodilation (Kang et al., 2002 )

**7. Pelvic atherosclerosis** 

mitochondria.

**8. Conclusion** 

androgen-dependent.

**9. References** 


Testosterone Deficiency Linked to Lower Urinary Tract Symptoms 231

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**10** 

*France* 

**Sex Hormones and Bacterial Infections** 

*1Unité de Recherche sur les Maladies Infectieuses Tropicales et Emergentes, Centre National de la Recherche Scientifique Unité Mixte de Recherche 6236,* 

Epidemiological and experimental data suggest the association of gender and sex with susceptibility and severity of infectious diseases (Moss, 2005). Gender and sex likely affect viral and parasitic infectious diseases (Morales-Montor *et al.*, 2004; Fish, 2008; Snider *et al.*, 2009). Here we will review the effect of gender and sex on bacterial infectious diseases (sepsis, mycobacterial diseases and Q fever). We will differentiate gender and sex by considering that gender refers to differences determined by cultural and societal factors and sex refers to the biological differences between males and females (Fish, 2008). Indeed, variables such as poverty, occupational status and marital status affect differently men and women in different countries (Theobald *et al.*, 2006), leading to different risks of exposition to infectious pathogens and accesses to efficient treatment. This is illustrated by the decreased prevalence of tuberculosis in industrialized countries associated with socioeconomic changes including reduced malnutrition and overcrowding, improved sanitary conditions in the workplaces before the use of chemotherapy (Davies *et al.*, 1999). Sex-based differences in the susceptibility to pathogens include what is due to chromosome effect and sex hormones. Thus, it is critical to delineate the respective roles of gender and sex on bacterial infections. The present review focuses on four features of the association between sex and bacterial infections with a special attention for bacterial sepsis, mycobacterial

**2. Epidemiological approach of susceptibility to bacterial infections** 

Epidemiological data show that the susceptibility to bacterial infectious diseases is unequally distributed in men and women. In sepsis, an infectious process associated with systemic inflammatory response syndrome, large-scale studies reported higher incidence in men than in women (Angus *et al.*, 2001; Martin *et al.*, 2003; Pietropaoli *et al.*, 2010). Men also develop more frequently sepsis episodes among patients with trauma (Osborn *et al.*, 2004; Wafaisade *et al.*, 2011) or acute kidney injury (Lopes *et al.*, 2010). In addition, men are over-

**1. Introduction** 

infections and *Coxiella burnetii* infection.

Marc Leone1,2, Julien Textoris1,2,

*Faculté de Médecine, Marseille,* 

*Aix-Marseille Université, Marseille,* 

Christian Capo1 and Jean-Louis Mege1

*Assistance Publique-Hôpitaux de Marseille,* 

*2Service d'Anesthésie et de Réanimation, Hôpital Nord,* 

Zhang, XH.; Morelli, A.; Luconi, M.; Vignozzi, L.; Filippi, S.; Marini, M.; Vannelli, GB.; Mancina, R.; Forti, G.& Maggi, M. (2005). Testosterone regulates PDE5 expression and in vivo responsiveness to tadalafil in rat corpus cavernosum. *European Urology.* Vol. 47, No. 3, pp. 409-416.

### **Sex Hormones and Bacterial Infections**

Marc Leone1,2, Julien Textoris1,2,

Christian Capo1 and Jean-Louis Mege1 *1Unité de Recherche sur les Maladies Infectieuses Tropicales et Emergentes, Centre National de la Recherche Scientifique Unité Mixte de Recherche 6236, Faculté de Médecine, Marseille, 2Service d'Anesthésie et de Réanimation, Hôpital Nord, Assistance Publique-Hôpitaux de Marseille, Aix-Marseille Université, Marseille, France* 

#### **1. Introduction**

236 Sex Hormones

Zhang, XH.; Morelli, A.; Luconi, M.; Vignozzi, L.; Filippi, S.; Marini, M.; Vannelli, GB.;

Vol. 47, No. 3, pp. 409-416.

Mancina, R.; Forti, G.& Maggi, M. (2005). Testosterone regulates PDE5 expression and in vivo responsiveness to tadalafil in rat corpus cavernosum. *European Urology.*

> Epidemiological and experimental data suggest the association of gender and sex with susceptibility and severity of infectious diseases (Moss, 2005). Gender and sex likely affect viral and parasitic infectious diseases (Morales-Montor *et al.*, 2004; Fish, 2008; Snider *et al.*, 2009). Here we will review the effect of gender and sex on bacterial infectious diseases (sepsis, mycobacterial diseases and Q fever). We will differentiate gender and sex by considering that gender refers to differences determined by cultural and societal factors and sex refers to the biological differences between males and females (Fish, 2008). Indeed, variables such as poverty, occupational status and marital status affect differently men and women in different countries (Theobald *et al.*, 2006), leading to different risks of exposition to infectious pathogens and accesses to efficient treatment. This is illustrated by the decreased prevalence of tuberculosis in industrialized countries associated with socioeconomic changes including reduced malnutrition and overcrowding, improved sanitary conditions in the workplaces before the use of chemotherapy (Davies *et al.*, 1999). Sex-based differences in the susceptibility to pathogens include what is due to chromosome effect and sex hormones. Thus, it is critical to delineate the respective roles of gender and sex on bacterial infections. The present review focuses on four features of the association between sex and bacterial infections with a special attention for bacterial sepsis, mycobacterial infections and *Coxiella burnetii* infection.

#### **2. Epidemiological approach of susceptibility to bacterial infections**

Epidemiological data show that the susceptibility to bacterial infectious diseases is unequally distributed in men and women. In sepsis, an infectious process associated with systemic inflammatory response syndrome, large-scale studies reported higher incidence in men than in women (Angus *et al.*, 2001; Martin *et al.*, 2003; Pietropaoli *et al.*, 2010). Men also develop more frequently sepsis episodes among patients with trauma (Osborn *et al.*, 2004; Wafaisade *et al.*, 2011) or acute kidney injury (Lopes *et al.*, 2010). In addition, men are over-

Sex Hormones and Bacterial Infections 239

With respect to Q fever, the male-to-female ratio of patients admitted to hospital is 2.45 in adults (Tissot Dupont *et al.*, 1992). The rate of Q fever-related complications is higher in males than in females (Raoult *et al.*, 2000). Men represent 75% patients diagnosed as having *C. burnetii* endocarditis (Houpikian & Raoult, 2005), the most severe manifestation of chronic Q fever. Note that females have fewer symptoms during pregnancy (Tissot-Dupont

In contrast to susceptibility to bacterial infections, studying the role of sex in the mortality provided contrasting evidence. Some epidemiological studies did not report any gender differences in sepsis-related death (Crabtree *et al.*, 1999; Martin *et al.*, 2003; Laupland *et al.*, 2004; Esper *et al.*, 2006) whereas other found either increased mortality in men (Osborn *et al.*, 2004; Melamed & Sorvillo, 2009; Wafaisade *et al.*, 2011) or women (Combes *et al.*, 2009; Pietropaoli *et al.*, 2010; Nachtigall *et al.*, 2011). As men are also at increased risk of death due to trauma, cancer and cardiovascular diseases as compared with women, the analysis of epidemiological data should integrate these potential biases (Micheli *et al.*, 2009; Pinkhasov

The literature provides evidence that sex hormones may account for the differences of susceptibility to bacterial infections and their prognosis between men and women. The production of sex hormones evolves with aging, suggesting that susceptibility to infection will change along the life. In adults, the widest difference in sepsis incidence occurs between 25-30 years of age when sex hormones play a key role in the sexual dimorphism (Angus *et al.*, 2001). In elderly patients, sepsis tends to occur later in women than in men (Martin *et al.*, 2003). Most studies show that the infection distribution is similar in children independently of sex (Tissot Dupont *et al.*, 1992; Rose *et al.*, 2001; Odetola *et al.*, 2007). No difference is found in young boys and girls with Q fever (Maltezou & Raoult, 2002) or with tuberculosis (Che &

Whether sex hormones govern the susceptibility to and the severity of bacterial infections, infection modulates the amounts of sex hormones. In patients with sepsis (Christeff *et al.*, 1988; Fourrier *et al.*, 1994; Majetschak *et al.*, 2000) or tuberculosis (Bottasso *et al.*, 2007; Rey *et al.*, 2007) circulating levels of estrogens are increased whereas those of testosterone dramatically fall. It has been also found that increased levels of estrogens are especially marked in males with sepsis (Fourrier *et al.*, 1994; Majetschak *et al.*, 2000). In addition, high circulating levels of estrogens seem efficient predictors of death in sepsis (Christeff *et al.*, 1988; Dossett *et al.*, 2008; May *et al.*, 2008). The mortality of elderly patients with severe infections is related to increased estradiol levels in both men and women, whereas increased

testosterone levels are found in females who do not survive (Angstwurm *et al.*, 2005).

Pregnancy represents a remarkable model for investigating the effect of sex hormones on the development of infectious diseases. Indeed, the developing placenta produces human chorionic gonadotrophin that stimulates the ovaries to supply higher levels of estrogen and progesterone. It is well known that pregnancy is associated with listeriosis. Pregnant women with listeriosis account for 27% of all listeriosis cases and 60% of listeriosis cases among 10- 40 years old persons (Lorber, 1997). Less spectacular is the case of Q fever. The primoinfection to *C. burnetii* during pregnancy increases the risk to develop a chronic evolution of the disease with potential fatal evolution (Carcopino *et al.*, 2009). The true incidence of sepsis or severe sepsis in parturient women is difficult to assess in part because the standard criteria used for sepsis identification are not effective to predict sepsis during pregnancy

*et al.*, 2007).

Antoine, 2011).

(Lappen *et al.*, 2010).

*et al.*, 2010; Coronado *et al.*, 2011).


represented among patients with respiratory infections (Esper *et al.*, 2006) or bloodstream infections (Laupland *et al.*, 2004) (Table 1).

Table 1. Gender effect in epidemiological studies (Result are expressed as either absolute number, percentage, or relative risk and 95% confident interval as required (and available in the original study))

In the context of infectious diseases due to intracellular bacteria, such as tuberculosis (*Mycobacterium tuberculosis*) (Che & Antoine, 2011), Q fever (*C. burnetii*) (Tissot Dupont *et al.*, 1992; Anderson *et al.*, 2009) and Legionnaires' disease (*Legionella pneumophila)* (Campese *et al.*, 2011), men represent the majority of patients (Table 2).


Table 2. Epidemiological data show that males and females are differently affected by bacterial infections. The reasons that explain this difference in susceptibility to and/or severity of the disease may be multiple. They include different risks of pathogen exposure, social behavior associated with gender such as smoking or drinking and biological parameters such as sex hormones.

represented among patients with respiratory infections (Esper *et al.*, 2006) or bloodstream

**Sepsis incidence** 

All 1.3% Men

15.7/100,000 /year

hospitalizations

6,621,559 All 3/1000 Men *vs.* Women

30,303 Trauma 2% Women

All 1.3% of all

Unknown All Unknown Men *vs.* Women

Table 1. Gender effect in epidemiological studies (Result are expressed as either absolute number, percentage, or relative risk and 95% confident interval as required (and available in

In the context of infectious diseases due to intracellular bacteria, such as tuberculosis (*Mycobacterium tuberculosis*) (Che & Antoine, 2011), Q fever (*C. burnetii*) (Tissot Dupont *et al.*, 1992; Anderson *et al.*, 2009) and Legionnaires' disease (*Legionella pneumophila)* (Campese *et* 

**Sepsis: Sex effect** 

2.83 vs. 2.87 /1000

1.28 [1.24 to 1.32]

0.65 [0.49 to 0.86]

Men *vs.* Women 17.7 vs. 13.5 / 100,000/year

1.27 [1.24 to 1.30]

54 vs. 46%

72.5 vs. 27.5%

1.81 [1.61 to 2.03]

Men

Unknown Men *vs.* Women

**Mortality: Sex effect** 

Men *vs.* Women 29.3 vs. 27.9%

Men *vs.* Women 22.0 vs. 21.8%

0.76 [0.66 to 0.88]

Men *vs.* Women 20.1 vs. 21.0%

Men *vs.* Women 33 vs. 35% (p = 0.01)

Men 1.1 [0.5-2.3]

Unknown

Women

No listed as independent risk factor

infections (Laupland *et al.*, 2004) (Table 1).

**Patient selection** 

blood culture

kidney injury

*al.*, 2011), men represent the majority of patients (Table 2).

29,829 Trauma 10.2% Men

**Infection due to intracellular bacteria Sex ratio Reference** 

Tuberculosis 1.4 Che & Antoine, 2011 Q fever 2.4 Tissot-Dupont, 1992 Legionnaires' disease 2.9 Campese, 2011

Table 2. Epidemiological data show that males and females are differently affected by bacterial infections. The reasons that explain this difference in susceptibility to and/or severity of the disease may be multiple. They include different risks of pathogen exposure,

social behavior associated with gender such as smoking or drinking and biological

**Reference Number of** 

Angus *et al*. 2001

Martin *et al*. 2003

Osborn *et al*. 2004

Laupland *et al*. 2004

Pietropaoli *et al*. 2010

Wafaisade *et al*. 2011

the original study))

parameters such as sex hormones.

Esper *et al*. 2006

Lopes *et al*. 2010 **patients** 

750 million hospital admissions

930 million hospitalizations

9,266 Positive

Unknown Acute

With respect to Q fever, the male-to-female ratio of patients admitted to hospital is 2.45 in adults (Tissot Dupont *et al.*, 1992). The rate of Q fever-related complications is higher in males than in females (Raoult *et al.*, 2000). Men represent 75% patients diagnosed as having *C. burnetii* endocarditis (Houpikian & Raoult, 2005), the most severe manifestation of chronic Q fever. Note that females have fewer symptoms during pregnancy (Tissot-Dupont *et al.*, 2007).

In contrast to susceptibility to bacterial infections, studying the role of sex in the mortality provided contrasting evidence. Some epidemiological studies did not report any gender differences in sepsis-related death (Crabtree *et al.*, 1999; Martin *et al.*, 2003; Laupland *et al.*, 2004; Esper *et al.*, 2006) whereas other found either increased mortality in men (Osborn *et al.*, 2004; Melamed & Sorvillo, 2009; Wafaisade *et al.*, 2011) or women (Combes *et al.*, 2009; Pietropaoli *et al.*, 2010; Nachtigall *et al.*, 2011). As men are also at increased risk of death due to trauma, cancer and cardiovascular diseases as compared with women, the analysis of epidemiological data should integrate these potential biases (Micheli *et al.*, 2009; Pinkhasov *et al.*, 2010; Coronado *et al.*, 2011).

The literature provides evidence that sex hormones may account for the differences of susceptibility to bacterial infections and their prognosis between men and women. The production of sex hormones evolves with aging, suggesting that susceptibility to infection will change along the life. In adults, the widest difference in sepsis incidence occurs between 25-30 years of age when sex hormones play a key role in the sexual dimorphism (Angus *et al.*, 2001). In elderly patients, sepsis tends to occur later in women than in men (Martin *et al.*, 2003). Most studies show that the infection distribution is similar in children independently of sex (Tissot Dupont *et al.*, 1992; Rose *et al.*, 2001; Odetola *et al.*, 2007). No difference is found in young boys and girls with Q fever (Maltezou & Raoult, 2002) or with tuberculosis (Che & Antoine, 2011).

Whether sex hormones govern the susceptibility to and the severity of bacterial infections, infection modulates the amounts of sex hormones. In patients with sepsis (Christeff *et al.*, 1988; Fourrier *et al.*, 1994; Majetschak *et al.*, 2000) or tuberculosis (Bottasso *et al.*, 2007; Rey *et al.*, 2007) circulating levels of estrogens are increased whereas those of testosterone dramatically fall. It has been also found that increased levels of estrogens are especially marked in males with sepsis (Fourrier *et al.*, 1994; Majetschak *et al.*, 2000). In addition, high circulating levels of estrogens seem efficient predictors of death in sepsis (Christeff *et al.*, 1988; Dossett *et al.*, 2008; May *et al.*, 2008). The mortality of elderly patients with severe infections is related to increased estradiol levels in both men and women, whereas increased testosterone levels are found in females who do not survive (Angstwurm *et al.*, 2005).

Pregnancy represents a remarkable model for investigating the effect of sex hormones on the development of infectious diseases. Indeed, the developing placenta produces human chorionic gonadotrophin that stimulates the ovaries to supply higher levels of estrogen and progesterone. It is well known that pregnancy is associated with listeriosis. Pregnant women with listeriosis account for 27% of all listeriosis cases and 60% of listeriosis cases among 10- 40 years old persons (Lorber, 1997). Less spectacular is the case of Q fever. The primoinfection to *C. burnetii* during pregnancy increases the risk to develop a chronic evolution of the disease with potential fatal evolution (Carcopino *et al.*, 2009). The true incidence of sepsis or severe sepsis in parturient women is difficult to assess in part because the standard criteria used for sepsis identification are not effective to predict sepsis during pregnancy (Lappen *et al.*, 2010).

Sex Hormones and Bacterial Infections 241

*al.*, 2009). The role of testosterone appears variable according to the experimental conditions. Orchiectomy does not alter the mortality of wild-type male mice but, after orchiectomy, increased mortality is observed in knockout (KO) male mice that are phenotypically normal but lack the ability to produce increased nitric oxide during endotoxemia. This increased mortality in orchiectomized KO males is prevented by the administration of exogenous testosterone (Laubach *et al.*, 1998), demonstrating that exogenous testosterone is potentially protective for the host when nitric oxide production is deficient. This experiment also suggests that testosterone may play a different role in healthy individuals and patients in

In humans, 30 young volunteers including 15 males and 15 females received 2 ng/kg LPS. The females were studied in the follicular phase and nine of them used oral contraceptives. During endotoxemia, the decrease in blood pressure is more pronounced in females than in males. Norepinephrine sensitivity remains unchanged in females but decreases in males, suggesting that the clinical picture is more evident and the response to treatment is more effective in females than in males. The administration of LPS results in increased circulating levels of TNF, IL-6, interferon (IFN)-gamma and IL-10 in males and females, but TNF and IFN-gamma levels are significantly higher in females than in males (van Eijk *et al.*, 2007). This study suggests that the hypotension that likely occurs earlier in females than in males may be related to a more marked immune response to LPS

Taken together, murine models of LPS-mediated inflammation and endotoxemia in humans highlight the role of estrogens, and testosterone to a lesser extent, in host responses to LPS

The models of sepsis using cecal ligation and puncture (CLP) seem to be more accurate than those using LPS injection (Dyson & Singer, 2009). The most frequent scenario used to evaluate the effect of sex is a two-hit model consisting of hemorrhages followed by sepsis. Male and proestrus female C3H/HeN mice are subjected to hemorrhage or sham operation and to polymicrobial sepsis by CLP twenty-four hours after. Animals subjected to hemorrhage followed by CLP show depressed splenocyte and macrophage functions as compared with sham animals (Angele *et al.*, 1997). After CLP, females have lower mortality than males, irrespective of prior hemorrhage or sham operation. Unlike males, hemorrhages

Female sex hormones likely play a major role in protection against CLP sepsis. Indeed, after CLP, the mortality of ovariectomized CBA/J mice is significantly higher than in intact mice. Ovariectomy results in a decreased production of IL-1 and IL-6 by splenic and peritoneal macrophages, but the production of IL-1 and IL-6 by macrophages from intact female mice is maintained after trauma-hemorrhage (Knöferl *et al.*, 2002). Thus, in septic conditions, female sex hormones protect female mice by producing increased levels of pro-

The modulation of estrogen receptors is a convenient way to study the role of estrogens in sepsis models. In a CLP model, multiple oral doses of a nonsteroidal selective estrogen receptor-beta agonist increase survival of mice, decrease systemic bacteremia, reduce peritoneal IL-6 and TNF levels. Interestingly, the estrogen receptor-beta agonist provides a comparable level of protection in both males and females (Cristofaro *et al.*, 2006). On another

do not increase the mortality rate in females (Diodato *et al.*, 2001).

severe conditions.

administration.

and TLR-4 activation.

**3.2 Mouse model of sepsis** 

inflammatory cytokines.

The analysis of epidemiological data indicates that male gender predisposes to develop sepsis and chronic bacterial infectious diseases whereas women are relatively protected. Although sex hormones seem to be critical in the sex dimorphism, we have to integrate other variables related to the host such as chromosomal factors and exposure to bacterial pathogens.

#### **3. Sex hormones and experimental models of bacterial infection**

The use of experimental models of infection was the easiest approach to study the role of sex hormones in bacterial infections; the availability of castrated animals with or without hormonal substitution has been largely contributive.

#### **3.1 Experimental endotoxemia**

Endotoxemia may be experimentally reproduced by the administration of lipopolysaccharide (LPS) to animals or human volunteers. LPS are present in the outer membrane of Gram-negative bacteria and act as endotoxins inducing strong inflammatory response in animals. Females produce a more vigorous pro-inflammatory response than males as demonstrated by higher circulating levels of Tumor Necrosis Factor (TNF) (Trentzsch *et al.*, 2003). The effect of sexual dimorphism in the outcome of endotoxemia depends on LPS dose. The intraperitoneal administration of low doses of LPS (5 mg/kg) to C57BL/6 mice leads to a lower survival of males compared to females (Laubach *et al.*, 1998) whereas the administration of higher doses (12.5 mg/kg) leads to similar mortality in males and females.

The study of the role of sex hormones in this dimorphism has benefited from the modulation of hormonal context. First, the circulating levels of TNF are higher in castrated C57BL/6 male mice than in intact males treated with LPS (Trentzsch *et al.*, 2003), suggesting that testosterone may reduce TNF production. Reinforcing this hypothesis, the LPS-induced response observed in castrated mice is reduced after testosterone treatment, regardless of sex (Spinedi *et al.*, 1992; Torres *et al.*, 2005). Second, the intraperitoneal injection of LPS in ovariectomized females is associated with reduced levels of TNF, interleukin (IL)-6, and IL-10 as compared with intact females. This is accompanied by the reduced macrophage expression of Toll-like receptor (TLR)-4, a pattern recognition receptor interacting with LPS (Rettew *et al.*, 2009). The role of estrogens in LPS response has been shown by complementation of castrated mice with sex hormones. Indeed, ovariectomized animals receiving exogenous 17β-estradiol showed higher TNF levels after endotoxin challenge than untreated gonadectomized or intact animals. In addition, peritoneal macrophages isolated from ovariectomized mice receiving 17β-estradiol replacement bind LPS more efficiently than untreated animals because of upregulated expression of CD14 and TLR-4 (Rettew *et al.*, 2009). Finally, the administration of 17β-estradiol to castrated C57BL/6J male mice increases the circulating levels of LPS-induced TNF production, as compared with control male or female mice (Trentzsch *et al.*, 2003). These findings should be analyzed according to the genetic background of the animals. Castrated males react differently according to the mouse strain (A/J, DBA/2J, AKR/J, BALB/cJ) (Trentzsch *et al.*, 2003; Torres *et al.*, 2005) after LPS challenge.

In contrast to estrogen treatment, the exogenous treatment of mice with progesteronecontaining implants fails to increase circulating levels of LPS-binding protein, cell surface levels of CD14 on peritoneal macrophages or total TLR-4 content in macrophages (Rettew *et* 

The analysis of epidemiological data indicates that male gender predisposes to develop sepsis and chronic bacterial infectious diseases whereas women are relatively protected. Although sex hormones seem to be critical in the sex dimorphism, we have to integrate other variables

The use of experimental models of infection was the easiest approach to study the role of sex hormones in bacterial infections; the availability of castrated animals with or without

Endotoxemia may be experimentally reproduced by the administration of lipopolysaccharide (LPS) to animals or human volunteers. LPS are present in the outer membrane of Gram-negative bacteria and act as endotoxins inducing strong inflammatory response in animals. Females produce a more vigorous pro-inflammatory response than males as demonstrated by higher circulating levels of Tumor Necrosis Factor (TNF) (Trentzsch *et al.*, 2003). The effect of sexual dimorphism in the outcome of endotoxemia depends on LPS dose. The intraperitoneal administration of low doses of LPS (5 mg/kg) to C57BL/6 mice leads to a lower survival of males compared to females (Laubach *et al.*, 1998) whereas the administration of higher doses (12.5 mg/kg) leads to similar mortality in males

The study of the role of sex hormones in this dimorphism has benefited from the modulation of hormonal context. First, the circulating levels of TNF are higher in castrated C57BL/6 male mice than in intact males treated with LPS (Trentzsch *et al.*, 2003), suggesting that testosterone may reduce TNF production. Reinforcing this hypothesis, the LPS-induced response observed in castrated mice is reduced after testosterone treatment, regardless of sex (Spinedi *et al.*, 1992; Torres *et al.*, 2005). Second, the intraperitoneal injection of LPS in ovariectomized females is associated with reduced levels of TNF, interleukin (IL)-6, and IL-10 as compared with intact females. This is accompanied by the reduced macrophage expression of Toll-like receptor (TLR)-4, a pattern recognition receptor interacting with LPS (Rettew *et al.*, 2009). The role of estrogens in LPS response has been shown by complementation of castrated mice with sex hormones. Indeed, ovariectomized animals receiving exogenous 17β-estradiol showed higher TNF levels after endotoxin challenge than untreated gonadectomized or intact animals. In addition, peritoneal macrophages isolated from ovariectomized mice receiving 17β-estradiol replacement bind LPS more efficiently than untreated animals because of upregulated expression of CD14 and TLR-4 (Rettew *et al.*, 2009). Finally, the administration of 17β-estradiol to castrated C57BL/6J male mice increases the circulating levels of LPS-induced TNF production, as compared with control male or female mice (Trentzsch *et al.*, 2003). These findings should be analyzed according to the genetic background of the animals. Castrated males react differently according to the mouse strain (A/J, DBA/2J, AKR/J, BALB/cJ) (Trentzsch *et al.*, 2003; Torres *et al.*, 2005) after LPS

In contrast to estrogen treatment, the exogenous treatment of mice with progesteronecontaining implants fails to increase circulating levels of LPS-binding protein, cell surface levels of CD14 on peritoneal macrophages or total TLR-4 content in macrophages (Rettew *et* 

related to the host such as chromosomal factors and exposure to bacterial pathogens.

**3. Sex hormones and experimental models of bacterial infection** 

hormonal substitution has been largely contributive.

**3.1 Experimental endotoxemia** 

and females.

challenge.

*al.*, 2009). The role of testosterone appears variable according to the experimental conditions. Orchiectomy does not alter the mortality of wild-type male mice but, after orchiectomy, increased mortality is observed in knockout (KO) male mice that are phenotypically normal but lack the ability to produce increased nitric oxide during endotoxemia. This increased mortality in orchiectomized KO males is prevented by the administration of exogenous testosterone (Laubach *et al.*, 1998), demonstrating that exogenous testosterone is potentially protective for the host when nitric oxide production is deficient. This experiment also suggests that testosterone may play a different role in healthy individuals and patients in severe conditions.

In humans, 30 young volunteers including 15 males and 15 females received 2 ng/kg LPS. The females were studied in the follicular phase and nine of them used oral contraceptives. During endotoxemia, the decrease in blood pressure is more pronounced in females than in males. Norepinephrine sensitivity remains unchanged in females but decreases in males, suggesting that the clinical picture is more evident and the response to treatment is more effective in females than in males. The administration of LPS results in increased circulating levels of TNF, IL-6, interferon (IFN)-gamma and IL-10 in males and females, but TNF and IFN-gamma levels are significantly higher in females than in males (van Eijk *et al.*, 2007). This study suggests that the hypotension that likely occurs earlier in females than in males may be related to a more marked immune response to LPS administration.

Taken together, murine models of LPS-mediated inflammation and endotoxemia in humans highlight the role of estrogens, and testosterone to a lesser extent, in host responses to LPS and TLR-4 activation.

#### **3.2 Mouse model of sepsis**

The models of sepsis using cecal ligation and puncture (CLP) seem to be more accurate than those using LPS injection (Dyson & Singer, 2009). The most frequent scenario used to evaluate the effect of sex is a two-hit model consisting of hemorrhages followed by sepsis. Male and proestrus female C3H/HeN mice are subjected to hemorrhage or sham operation and to polymicrobial sepsis by CLP twenty-four hours after. Animals subjected to hemorrhage followed by CLP show depressed splenocyte and macrophage functions as compared with sham animals (Angele *et al.*, 1997). After CLP, females have lower mortality than males, irrespective of prior hemorrhage or sham operation. Unlike males, hemorrhages do not increase the mortality rate in females (Diodato *et al.*, 2001).

Female sex hormones likely play a major role in protection against CLP sepsis. Indeed, after CLP, the mortality of ovariectomized CBA/J mice is significantly higher than in intact mice. Ovariectomy results in a decreased production of IL-1 and IL-6 by splenic and peritoneal macrophages, but the production of IL-1 and IL-6 by macrophages from intact female mice is maintained after trauma-hemorrhage (Knöferl *et al.*, 2002). Thus, in septic conditions, female sex hormones protect female mice by producing increased levels of proinflammatory cytokines.

The modulation of estrogen receptors is a convenient way to study the role of estrogens in sepsis models. In a CLP model, multiple oral doses of a nonsteroidal selective estrogen receptor-beta agonist increase survival of mice, decrease systemic bacteremia, reduce peritoneal IL-6 and TNF levels. Interestingly, the estrogen receptor-beta agonist provides a comparable level of protection in both males and females (Cristofaro *et al.*, 2006). On another

Sex Hormones and Bacterial Infections 243

Testosterone decreases the intensity of the pro-inflammatory response, resulting in an inappropriate response to septic insult, progression of the infectious process and death. In some cases, the limitation of an excessive pro-inflammatory response may appear beneficial by limiting the occurrence of multiple organ failure. This can lead to a latent infection with infectious complications, resulting in either death or recovery. Note that sepsis induces a negative feedback on testosterone production. (IL: interleukin,

*Pseudomonas aeruginosa* is one of the predominant Gram negative bacteria responsible for pulmonary infection in intensive care units (Leone *et al.*, 2007). The role of gender is not specifically reported in epidemiological studies although men are more prone to develop lung infection than women (Leone *et al.*, 2007). The importance of sex hormones has been assessed in a model of C57BL/6 mice challenged to pulmonary infection with *P. aeruginosa*. At variance with other models, the weight loss, bacterial load and inflammatory mediators in the lungs are higher in females than in males. The number of bacteria found in the lungs of IL-10-deficient males is higher than that observed in wild type males. These findings clearly show that female mice are more susceptible to *P. aeruginosa* lung infection than males and that IL-10 modulates host response to infection as described above in sepsis models

On the other hand, *P. aeruginosa* is the predominant bacterium found in the course of cystic fibrosis. Importantly, the outcomes are worse for cystic fibrosis women infected with *P. aeruginosa* as compared with men (Demko *et al.*, 1995). A mouse model has been dedicated to investigate the effect of gender on cystic fibrosis. The administration of exogenous estrogen to adult cystic fibrosis males with *P. aeruginosa* pneumonia leads to more severe manifestations of inflammation in both lung tissue and bronchial alveolar lavage fluid.

TNF: tumor necrosis factor).

(Guilbault *et al.*, 2002).

**3.3 Infection by extracellular bacteria** 

Fig. 2. Interaction between testosterone and host response

hand, the administration of an estrogen receptor alpha agonist entirely prevents the rise in plasma IL-6 and IL-10 levels induced by a sequence of trauma-hemorrhage whereas the administration of an estrogen receptor beta agonist is only in part effective. Similar conclusions can be drawn from experiments at the cell level. The effects of an estrogen receptor beta agonist on Kupffer cells, splenic macrophages, alveolar macrophages and peripheral blood mononuclear cells following trauma-hemorrhage are less pronounced than that of an estrogen receptor alpha agonist (Dienstknecht *et al.*, 2004). In all cases, the beneficial effects of 17β-estradiol are limited to tissue-fixed macrophages, suggesting the compartmentalization of host response (Suzuki *et al.*, 2007). In figure 1, we hypothesized possible mechanisms for explaining the dual role of estrogens in sepsis.

Estrogens reduce the production of caspase-12 and then increase that of pro-inflammatory mediators. This is associated either with a rapid limitation of the infectious process leading to fast recovery or with the occurrence of multiple organ failure related to an excessive pro-inflammatory response leading to death. Note that sepsis induces a positive feedback on estrogen production. (IL: interleukin, TNF: tumor necrosis factor).

Fig. 1. Interaction between estrogens and host response

Testosterone has detrimental effect on survival of male mice by attenuating immune response. Indeed, after CLP, the survival rate of male mice treated with flutamide, an androgen receptor blocker, is higher than that of vehicle-treated mice. Flutamide treatment also restores splenocyte proliferation and IL-2 release as well as the release of IL-1 by splenic macrophages (Angele *et al.*, 1997). The effect of testosterone on experimental sepsis may be controlled by IL-10. Indeed, after CLP early IL-10 treatment is associated with increased survival of males, but not of females (Kahlke *et al.*, 2000). In figure 2, we hypothesized possible mechanisms for explaining the role of testosterone in sepsis.

hand, the administration of an estrogen receptor alpha agonist entirely prevents the rise in plasma IL-6 and IL-10 levels induced by a sequence of trauma-hemorrhage whereas the administration of an estrogen receptor beta agonist is only in part effective. Similar conclusions can be drawn from experiments at the cell level. The effects of an estrogen receptor beta agonist on Kupffer cells, splenic macrophages, alveolar macrophages and peripheral blood mononuclear cells following trauma-hemorrhage are less pronounced than that of an estrogen receptor alpha agonist (Dienstknecht *et al.*, 2004). In all cases, the beneficial effects of 17β-estradiol are limited to tissue-fixed macrophages, suggesting the compartmentalization of host response (Suzuki *et al.*, 2007). In figure 1, we hypothesized

Estrogens reduce the production of caspase-12 and then increase that of pro-inflammatory mediators. This is associated either with a rapid limitation of the infectious process leading to fast recovery or with the occurrence of multiple organ failure related to an excessive pro-inflammatory response leading to death. Note that sepsis induces a positive feedback on estrogen production. (IL: interleukin, TNF: tumor

Testosterone has detrimental effect on survival of male mice by attenuating immune response. Indeed, after CLP, the survival rate of male mice treated with flutamide, an androgen receptor blocker, is higher than that of vehicle-treated mice. Flutamide treatment also restores splenocyte proliferation and IL-2 release as well as the release of IL-1 by splenic macrophages (Angele *et al.*, 1997). The effect of testosterone on experimental sepsis may be controlled by IL-10. Indeed, after CLP early IL-10 treatment is associated with increased survival of males, but not of females (Kahlke *et al.*, 2000). In figure 2, we hypothesized

necrosis factor).

Fig. 1. Interaction between estrogens and host response

possible mechanisms for explaining the role of testosterone in sepsis.

possible mechanisms for explaining the dual role of estrogens in sepsis.

Testosterone decreases the intensity of the pro-inflammatory response, resulting in an inappropriate response to septic insult, progression of the infectious process and death. In some cases, the limitation of an excessive pro-inflammatory response may appear beneficial by limiting the occurrence of multiple organ failure. This can lead to a latent infection with infectious complications, resulting in either death or recovery. Note that sepsis induces a negative feedback on testosterone production. (IL: interleukin, TNF: tumor necrosis factor).

Fig. 2. Interaction between testosterone and host response

#### **3.3 Infection by extracellular bacteria**

*Pseudomonas aeruginosa* is one of the predominant Gram negative bacteria responsible for pulmonary infection in intensive care units (Leone *et al.*, 2007). The role of gender is not specifically reported in epidemiological studies although men are more prone to develop lung infection than women (Leone *et al.*, 2007). The importance of sex hormones has been assessed in a model of C57BL/6 mice challenged to pulmonary infection with *P. aeruginosa*. At variance with other models, the weight loss, bacterial load and inflammatory mediators in the lungs are higher in females than in males. The number of bacteria found in the lungs of IL-10-deficient males is higher than that observed in wild type males. These findings clearly show that female mice are more susceptible to *P. aeruginosa* lung infection than males and that IL-10 modulates host response to infection as described above in sepsis models (Guilbault *et al.*, 2002).

On the other hand, *P. aeruginosa* is the predominant bacterium found in the course of cystic fibrosis. Importantly, the outcomes are worse for cystic fibrosis women infected with *P. aeruginosa* as compared with men (Demko *et al.*, 1995). A mouse model has been dedicated to investigate the effect of gender on cystic fibrosis. The administration of exogenous estrogen to adult cystic fibrosis males with *P. aeruginosa* pneumonia leads to more severe manifestations of inflammation in both lung tissue and bronchial alveolar lavage fluid.

Sex Hormones and Bacterial Infections 245

The pregnancy is characterized by dramatic changes in sex hormones with decreased estrogen and increased progesterone levels. Estrogens and progesterone are known to affect the susceptibility to pathogens likely through the modulation of the immune responses. Indeed, pregnancy is a transient period of tolerance in which the prevention of fetus rejection increases the susceptibility to intracellular bacteria (Munoz-Suano *et al.*, 2011).

In humans, Q fever is frequently asymptomatic in pregnant women but it may result in increased risk to develop a chronic form of the disease (Carcopino *et al.*, 2009). Female BALB/c mice have been infected with *C. burnetii* through the intraperitoneal route before repeated pregnancies over a 2-year period. Persistent infection associated with abortion and perinatal death is observed in these mice. The occurrence of endocarditis on native valves, which characterize chronic Q fever following *C. burnetii* infection during pregnancy, has

Pregnancy is a risk factor for typhoid infection (Olubuyide, 1992). After an infection challenge with *Salmonella enterica* serovar Typhimurium, the splenic bacterial load is markedly increased in pregnant mice compared with non-pregnant mice (Pejcic-Karapetrovic *et al.*, 2007). The increased bacterial load is related to sex hormones since a three-day treatment of virgin mice with 1 mg/day of estrogen increases their susceptibility to an intraperitoneal bacterial challenge as compared with control mice. In contrast, a threeday treatment of virgin mice with 1 mg/day of progesterone is associated with increased survival time (Kita *et al.*, 1989). It is likely that progesterone improves the resistance of mice by increasing the influx of peritoneal cells after infection whereas estrogen affects the acute

Listeriosis is an infection that occurs in about 30% of cases in pregnant women (Lorber, 1997). The impact of sex hormones on the course of the disease has been especially well described. Exposure to diethylstilbestrol, a synthetic nonsteroidal estrogen, precipitates a dramatic increase in *Listeria* susceptibility. To assess the interplay between diethylstilbestrol, sex hormones and immune response, *L. monocytogenes* has been intravenously administered to C3H/HeSlc female and male mice treated with diethylstilbestrol. The delayed-type hypersensitivity response is suppressed in females treated with diethylstilbestrol but not in males. After castration, a diethylstilbestrol-induced suppression is also observed in males. Testosterone inhibits this diethylstilbestrol-induced suppression (Kato *et al.*, 1988). Another report shows that the administration of estradiol or diethylstilbestrol is associated with an increased mortality of female B6C3F1 mice infected with *L. monocytogenes*. This effect of estradiol or diethylstilbestrol is due to their estrogenic activity since compounds such as 5αdihydrotestosterone or progesterone with little or no estrogenic activity do not affect the mortality of infected mice (Pung *et al.*, 1984). Female C3H/He mice treated with exogenous doses of estrogen have been inoculated with *L. monocytogenes* by the intraperitoneal route. On days 3, 5 and 7 after infection, the bacterial load in spleen and liver is higher in estrogentreated mice than in control mice (Salem *et al.*, 1999). Note that the sensitivity of mice to infection is highly dependent on the genetic background since the C57BL/6 mice are 100 times more resistant to intravenously injected *L. monocytogenes* than BALB/c mice, due to the action of a single gene, Lr (Mandel & Cheers, 1980). Taken together, these experimental studies demonstrate the role of sex hormones in the context of materno-fetal tolerance and

**4. Infectious diseases associated with pregnancy** 

Several examples of infectious diseases will illustrate this statement.

been observed in some infected pregnant mice (Stein *et al.*, 2000).

inflammatory responses (Kita *et al.*, 1989).

susceptibility to intracellular bacteria.

Inflammatory infiltrates are increased as determined by histological studies. The inflammatory response is accompanied by an increased lung tissue expression of both IL-23 and IL-17 (Wang *et al.*, 2010). Thus, sex hormones modulate *P. aeruginosa* infection with a marked inflammatory effect of estrogens.

#### **3.4 Infections by intracellular bacteria 3.4.1 Mycobacterial infections**

As tuberculosis occurs more frequently in males than in females, the effect of sex should be a common feature of mycobacterial infections. Male mice infected by *Mycobacterium marinum* are more susceptible than female mice in terms of mortality, incidence of gross skin lesions and bacterial load in lungs and spleen. The castration of males improves their resistance to infection and this effect is substantially reversed by continuous testosterone treatment. The testosterone treatment also increases the susceptibility of females to *M. marinum* infection, demonstrating that testosterone is partly responsible for the increased susceptibility of mice to *M. marinum* infection (Yamamoto *et al.*, 1991). In DBA/2 female mice infected with *Mycobacterium avium*, the number of bacilli in the lungs of infected mice increased after ovariectomy, suggesting a protective effect of female sex hormones. The treatment of ovariectomized mice with exogenous 17β-estradiol reduces the burden of bacilli to the level found in sham-operated mice. Estrogens enhance the bacteriostatic activity of IFN-gamma against *M. avium* via increased nitrite production by macrophages (Tsuyuguchi *et al.*, 2001). These findings show that estrogens enhance the host protection against mycobacterial infections but testosterone is detrimental.

#### **3.4.2** *C. burnetii* **infection**

From the epidemiological data in which mature adult men are more at risk to develop Q fever than women (Tissot-Dupont *et al.*, 2007), we showed that sex hormones play a role in the occurrence and the severity of *C. burnetii* infection. In C57BL/6 mice infected with *C. burnetii*, bacterial load and granuloma numbers are lower in females than in males and are increased in ovariectomized females to levels similar to those found in males. The treatment of ovariectomized mice with 17β-estradiol reduces both bacterial loads and granuloma numbers (Leone *et al.*, 2004), demonstrating that estrogens control *C. burnetii* infection. To analyze the differences between males and females, intact and castrated mice have been infected with *C. burnetii* for 24 hours, and gene expression has been measured in liver cells using whole-genome microarrays. The expression of a total of 2,777 probes is specifically modulated by *C. burnetti* infection. Surprisingly, 86% of them are differentially expressed in males and females. Castration of males and females shows that sex hormones are responsible for more than 60% of the observed gene modulation, and this effect of sex hormones is most pronounced in males. Using functional annotation of modulated genes, four clusters have been identified as enriched in males. These clusters are related to cell-cell adhesion, signal transduction, defensins and cytokine/Jak-Stat pathways (Textoris *et al.*, 2010). A major cluster of modulated genes has been identified in females consisting of the circadian rhythm pathway with positive (Clock, Arntl) and negative (Per) limbs of a feedback loop. Clock and Arntl are down-modulated whereas Per is up-regulated. These changes may be associated with efficient bacterial elimination in females but not in males, in which the immune response would be inefficient.

Inflammatory infiltrates are increased as determined by histological studies. The inflammatory response is accompanied by an increased lung tissue expression of both IL-23 and IL-17 (Wang *et al.*, 2010). Thus, sex hormones modulate *P. aeruginosa* infection with a

As tuberculosis occurs more frequently in males than in females, the effect of sex should be a common feature of mycobacterial infections. Male mice infected by *Mycobacterium marinum* are more susceptible than female mice in terms of mortality, incidence of gross skin lesions and bacterial load in lungs and spleen. The castration of males improves their resistance to infection and this effect is substantially reversed by continuous testosterone treatment. The testosterone treatment also increases the susceptibility of females to *M. marinum* infection, demonstrating that testosterone is partly responsible for the increased susceptibility of mice to *M. marinum* infection (Yamamoto *et al.*, 1991). In DBA/2 female mice infected with *Mycobacterium avium*, the number of bacilli in the lungs of infected mice increased after ovariectomy, suggesting a protective effect of female sex hormones. The treatment of ovariectomized mice with exogenous 17β-estradiol reduces the burden of bacilli to the level found in sham-operated mice. Estrogens enhance the bacteriostatic activity of IFN-gamma against *M. avium* via increased nitrite production by macrophages (Tsuyuguchi *et al.*, 2001). These findings show that estrogens enhance the host protection

From the epidemiological data in which mature adult men are more at risk to develop Q fever than women (Tissot-Dupont *et al.*, 2007), we showed that sex hormones play a role in the occurrence and the severity of *C. burnetii* infection. In C57BL/6 mice infected with *C. burnetii*, bacterial load and granuloma numbers are lower in females than in males and are increased in ovariectomized females to levels similar to those found in males. The treatment of ovariectomized mice with 17β-estradiol reduces both bacterial loads and granuloma numbers (Leone *et al.*, 2004), demonstrating that estrogens control *C. burnetii* infection. To analyze the differences between males and females, intact and castrated mice have been infected with *C. burnetii* for 24 hours, and gene expression has been measured in liver cells using whole-genome microarrays. The expression of a total of 2,777 probes is specifically modulated by *C. burnetti* infection. Surprisingly, 86% of them are differentially expressed in males and females. Castration of males and females shows that sex hormones are responsible for more than 60% of the observed gene modulation, and this effect of sex hormones is most pronounced in males. Using functional annotation of modulated genes, four clusters have been identified as enriched in males. These clusters are related to cell-cell adhesion, signal transduction, defensins and cytokine/Jak-Stat pathways (Textoris *et al.*, 2010). A major cluster of modulated genes has been identified in females consisting of the circadian rhythm pathway with positive (Clock, Arntl) and negative (Per) limbs of a feedback loop. Clock and Arntl are down-modulated whereas Per is up-regulated. These changes may be associated with efficient bacterial elimination in females but not in males, in

marked inflammatory effect of estrogens.

**3.4 Infections by intracellular bacteria** 

against mycobacterial infections but testosterone is detrimental.

which the immune response would be inefficient.

**3.4.1 Mycobacterial infections** 

**3.4.2** *C. burnetii* **infection** 

#### **4. Infectious diseases associated with pregnancy**

The pregnancy is characterized by dramatic changes in sex hormones with decreased estrogen and increased progesterone levels. Estrogens and progesterone are known to affect the susceptibility to pathogens likely through the modulation of the immune responses. Indeed, pregnancy is a transient period of tolerance in which the prevention of fetus rejection increases the susceptibility to intracellular bacteria (Munoz-Suano *et al.*, 2011). Several examples of infectious diseases will illustrate this statement.

In humans, Q fever is frequently asymptomatic in pregnant women but it may result in increased risk to develop a chronic form of the disease (Carcopino *et al.*, 2009). Female BALB/c mice have been infected with *C. burnetii* through the intraperitoneal route before repeated pregnancies over a 2-year period. Persistent infection associated with abortion and perinatal death is observed in these mice. The occurrence of endocarditis on native valves, which characterize chronic Q fever following *C. burnetii* infection during pregnancy, has been observed in some infected pregnant mice (Stein *et al.*, 2000).

Pregnancy is a risk factor for typhoid infection (Olubuyide, 1992). After an infection challenge with *Salmonella enterica* serovar Typhimurium, the splenic bacterial load is markedly increased in pregnant mice compared with non-pregnant mice (Pejcic-Karapetrovic *et al.*, 2007). The increased bacterial load is related to sex hormones since a three-day treatment of virgin mice with 1 mg/day of estrogen increases their susceptibility to an intraperitoneal bacterial challenge as compared with control mice. In contrast, a threeday treatment of virgin mice with 1 mg/day of progesterone is associated with increased survival time (Kita *et al.*, 1989). It is likely that progesterone improves the resistance of mice by increasing the influx of peritoneal cells after infection whereas estrogen affects the acute inflammatory responses (Kita *et al.*, 1989).

Listeriosis is an infection that occurs in about 30% of cases in pregnant women (Lorber, 1997). The impact of sex hormones on the course of the disease has been especially well described. Exposure to diethylstilbestrol, a synthetic nonsteroidal estrogen, precipitates a dramatic increase in *Listeria* susceptibility. To assess the interplay between diethylstilbestrol, sex hormones and immune response, *L. monocytogenes* has been intravenously administered to C3H/HeSlc female and male mice treated with diethylstilbestrol. The delayed-type hypersensitivity response is suppressed in females treated with diethylstilbestrol but not in males. After castration, a diethylstilbestrol-induced suppression is also observed in males. Testosterone inhibits this diethylstilbestrol-induced suppression (Kato *et al.*, 1988). Another report shows that the administration of estradiol or diethylstilbestrol is associated with an increased mortality of female B6C3F1 mice infected with *L. monocytogenes*. This effect of estradiol or diethylstilbestrol is due to their estrogenic activity since compounds such as 5αdihydrotestosterone or progesterone with little or no estrogenic activity do not affect the mortality of infected mice (Pung *et al.*, 1984). Female C3H/He mice treated with exogenous doses of estrogen have been inoculated with *L. monocytogenes* by the intraperitoneal route. On days 3, 5 and 7 after infection, the bacterial load in spleen and liver is higher in estrogentreated mice than in control mice (Salem *et al.*, 1999). Note that the sensitivity of mice to infection is highly dependent on the genetic background since the C57BL/6 mice are 100 times more resistant to intravenously injected *L. monocytogenes* than BALB/c mice, due to the action of a single gene, Lr (Mandel & Cheers, 1980). Taken together, these experimental studies demonstrate the role of sex hormones in the context of materno-fetal tolerance and susceptibility to intracellular bacteria.

Sex Hormones and Bacterial Infections 247

doses of estrogens (Bouman *et al.*, 2005). The role of estrogens in inflammation has been assessed in sensitized mice with LPS. LPS elicits transcriptional activation of inflammatory genes in microglial cells. Their expression is inhibited in the brain from ovariectomized mice (Soucy *et al.*, 2005). On another hand, testosterone exerts a suppressive effect on monocytes and macrophages likely by decreasing the expression of TLR-4 (Rettew *et al.*, 2008). This is more ambiguous in vivo. Castration of male mice strikingly accelerates wound healing and dampens associated inflammatory response. Similarly, systemic treatment with flutamide depresses inflammatory response (Ashcroft & Mills, 2002). Other cells involved in the innate immune response are likely targeted by sex hormones. Indeed, the activity of neutrophils and natural killer cells is suppressed by estrogens (Fish, 2008). Estrogens regulate the differentiation of dendritic cells from bone marrow precursors to conventional dendritic cells producing IL-12. The treatment of mature splenic dendritic cells with estrogens leads to

The impact of sex hormones on adaptive immunity may explain the generally superior ability of females to deal with and to be protected from infections, but their effect on innate immunity clearly depends on hormone doses, explaining the differences between in vitro and in vivo data. This is related to the stress system that has potent action on inflammatory

Although it is difficult to separate biological factors from social and economic factors, the epidemiological studies have shown that the sexual dimorphism may explain the differences in the susceptibility to and/or the severity of bacterial infections between men and women. The use of experimental models of infection demonstrates the role of sex hormones in this sexual dimorphism. Sex hormones target the immune system known to be essential in the host response to infection and, in turn, can be modulated by infection. While estrogens induce efficient cell-mediated and humoral immune responses necessary to bacterial clearance, androgens are rather suppressive. The pregnancy is an excellent model of the interplay between hormonal and immune systems and it teaches us that hormonal control of immune responses varies with time. It would be essential in the future to examine the sex-based differences in immune responses in humans likely by using tissue bio-banks

Anderson, A. D.; Kruszon-Moran, D.; Loftis, A. D.; McQuillan, G.; Nicholson, W. L.;

*Tropical Medicine and Hygiene*, Vol.81, No.4, pp. 691-694, ISSN 1476-1645 Angele, M. K.; Wichmann, M. W.; Ayala, A.; Cioffi, W. G. & Chaudry, I. H. (1997).

Angstwurm, M. W. A.; Gaertner, R. & Schopohl, J. (2005). Outcome in elderly patients with

*Archives of Surgery,* Vol.132, No.11, pp. 1207-1214, ISSN 0004-0010

*Medicine*, Vol.33, No.12, pp. 2786-2793, ISSN 0090-3493

Priestley, R. A.; Candee, A. J.; Patterson, N. E. & Massung, R. F. (2009). Seroprevalence of Q fever in the United States, 2003-2004. *The American Journal of* 

Testosterone receptor blockade after hemorrhage in males. Restoration of the depressed immune functions and improved survival following subsequent sepsis.

severe infection is influenced by sex hormones but not gender. *Critical Care* 

the expansion of IFN-gamma-producing dendritic cells (Bengtsson *et al.*, 2004).

and immune responses (Chrousos, 2010).

and high throughput methods.

**7. References** 

**6. Conclusion** 

#### **5. How sex hormones affect bacterial infection?**

Although epidemiological analysis and experimental models of infection provide convincing evidence of the role of sex hormones in host susceptibility to bacterial pathogens, the mechanisms used by sex hormones to modulate the susceptibility of hosts to pathogens are poorly understood. It is likely that sex hormones target immune cells according to their critical role in host defense (Fish, 2008). It is known that estrogens, androgens and glucocorticoids influence a large proportion of cell transcriptome (Duma *et al.*, 2010) and interact with specific receptors on immune cells. Cell-mediated and humoral immune responses represent the adaptive part of the immunity and are usually associated with the clearance of intracellular pathogens. Females exhibit robust cell-mediated and humoral immune responses after infectious challenge or vaccination as compared with males (Bouman *et al.*, 2005). This is partly related to changes in T cell distribution. Women have higher CD4+ T cells number than men, which accounts for the robustness of adaptive immune responses. In addition, the numbers of regulatory T cells (Treg) that shape immune responses vary during the ovarian cycle: the Treg number increases during the follicular phase of menstrual cycle when estrogens are high and decreases during the luteal phase when estrogens are low (Arruvito *et al.*, 2007). The increase in Treg number during the pregnancy is essential for materno-fetal tolerance but favors the occurrence of infectious diseases due to intracellular pathogens (Belkaid & Tarbell, 2009). Hence, estrogens appear as regulators of CD4+ T cell subsets; they also affect the Th1/Th2 equilibrium known to be essential in the control of bacterial infections. Indeed, it is clearly demonstrated that estrogens and progesterone favor Th2 cell responses during the third trimester of pregnancy (Munoz-Suano *et al.*, 2011). Low doses of estrogens are associated with Th1 cell responses that support microbicidal responses via IFN-gamma production; the effect of estrogens seems to be related to increased expression of t-bet, a master regulator of Th1 differentiation. In contrast, high doses of estrogens promote Th2-cell responses known to interfere with antibacterial immunity (Fish, 2008). Estrogens affect antibody production via their action on B cells; they decrease the negative selection of immature B cells and increase the survival of autoreactive B cells and polyclonal activation of B cells (Grimaldi *et al.*, 2002; Verthelyi, 2001). This is consistent with increased levels of autoimmune diseases in women (McCombe *et al.*, 2009), but it is not demonstrated that estrogen-mediated increased humoral response to pathogens is due to a direct effect on B cells. In contrast to estrogens, the effects of androgens such as testosterone on adaptive immune response are suppressive, which accounts for decreased T- and B-cell proliferation, immunoglobulin and cytokine production (Fish, 2008). This may explain why men are more susceptible than women to infectious agents because of the inability to mount efficient adaptive immune response.

Sex hormones may also affect the innate immune response that is the first line of defense against pathogens and that is necessary to shape adaptive immune response. Monocytes and macrophages are cell effectors of innate anti-infectious immunity and support inflammatory responses. The number of circulating monocytes is higher in men and women after the menopause than in fertile women (Bouman *et al.*, 2004). The effects of estrogens on monocytes and macrophages are suppressive but have to be analyzed according to the context. They likely act on CD16 promoter, leading to downmodulated CD16 expression and decreased production of proinflammatory cytokines (Kramer *et al.*, 2004). Inflammatory cytokines such as TNF and IL-1 are modulated during the ovarian cycle: low doses of estrogens are associated with increased production of TNF and IL-1 as compared with high

Although epidemiological analysis and experimental models of infection provide convincing evidence of the role of sex hormones in host susceptibility to bacterial pathogens, the mechanisms used by sex hormones to modulate the susceptibility of hosts to pathogens are poorly understood. It is likely that sex hormones target immune cells according to their critical role in host defense (Fish, 2008). It is known that estrogens, androgens and glucocorticoids influence a large proportion of cell transcriptome (Duma *et al.*, 2010) and interact with specific receptors on immune cells. Cell-mediated and humoral immune responses represent the adaptive part of the immunity and are usually associated with the clearance of intracellular pathogens. Females exhibit robust cell-mediated and humoral immune responses after infectious challenge or vaccination as compared with males (Bouman *et al.*, 2005). This is partly related to changes in T cell distribution. Women have higher CD4+ T cells number than men, which accounts for the robustness of adaptive immune responses. In addition, the numbers of regulatory T cells (Treg) that shape immune responses vary during the ovarian cycle: the Treg number increases during the follicular phase of menstrual cycle when estrogens are high and decreases during the luteal phase when estrogens are low (Arruvito *et al.*, 2007). The increase in Treg number during the pregnancy is essential for materno-fetal tolerance but favors the occurrence of infectious diseases due to intracellular pathogens (Belkaid & Tarbell, 2009). Hence, estrogens appear as regulators of CD4+ T cell subsets; they also affect the Th1/Th2 equilibrium known to be essential in the control of bacterial infections. Indeed, it is clearly demonstrated that estrogens and progesterone favor Th2 cell responses during the third trimester of pregnancy (Munoz-Suano *et al.*, 2011). Low doses of estrogens are associated with Th1 cell responses that support microbicidal responses via IFN-gamma production; the effect of estrogens seems to be related to increased expression of t-bet, a master regulator of Th1 differentiation. In contrast, high doses of estrogens promote Th2-cell responses known to interfere with antibacterial immunity (Fish, 2008). Estrogens affect antibody production via their action on B cells; they decrease the negative selection of immature B cells and increase the survival of autoreactive B cells and polyclonal activation of B cells (Grimaldi *et al.*, 2002; Verthelyi, 2001). This is consistent with increased levels of autoimmune diseases in women (McCombe *et al.*, 2009), but it is not demonstrated that estrogen-mediated increased humoral response to pathogens is due to a direct effect on B cells. In contrast to estrogens, the effects of androgens such as testosterone on adaptive immune response are suppressive, which accounts for decreased T- and B-cell proliferation, immunoglobulin and cytokine production (Fish, 2008). This may explain why men are more susceptible than women to infectious

agents because of the inability to mount efficient adaptive immune response.

Sex hormones may also affect the innate immune response that is the first line of defense against pathogens and that is necessary to shape adaptive immune response. Monocytes and macrophages are cell effectors of innate anti-infectious immunity and support inflammatory responses. The number of circulating monocytes is higher in men and women after the menopause than in fertile women (Bouman *et al.*, 2004). The effects of estrogens on monocytes and macrophages are suppressive but have to be analyzed according to the context. They likely act on CD16 promoter, leading to downmodulated CD16 expression and decreased production of proinflammatory cytokines (Kramer *et al.*, 2004). Inflammatory cytokines such as TNF and IL-1 are modulated during the ovarian cycle: low doses of estrogens are associated with increased production of TNF and IL-1 as compared with high

**5. How sex hormones affect bacterial infection?** 

doses of estrogens (Bouman *et al.*, 2005). The role of estrogens in inflammation has been assessed in sensitized mice with LPS. LPS elicits transcriptional activation of inflammatory genes in microglial cells. Their expression is inhibited in the brain from ovariectomized mice (Soucy *et al.*, 2005). On another hand, testosterone exerts a suppressive effect on monocytes and macrophages likely by decreasing the expression of TLR-4 (Rettew *et al.*, 2008). This is more ambiguous in vivo. Castration of male mice strikingly accelerates wound healing and dampens associated inflammatory response. Similarly, systemic treatment with flutamide depresses inflammatory response (Ashcroft & Mills, 2002). Other cells involved in the innate immune response are likely targeted by sex hormones. Indeed, the activity of neutrophils and natural killer cells is suppressed by estrogens (Fish, 2008). Estrogens regulate the differentiation of dendritic cells from bone marrow precursors to conventional dendritic cells producing IL-12. The treatment of mature splenic dendritic cells with estrogens leads to the expansion of IFN-gamma-producing dendritic cells (Bengtsson *et al.*, 2004).

The impact of sex hormones on adaptive immunity may explain the generally superior ability of females to deal with and to be protected from infections, but their effect on innate immunity clearly depends on hormone doses, explaining the differences between in vitro and in vivo data. This is related to the stress system that has potent action on inflammatory and immune responses (Chrousos, 2010).

#### **6. Conclusion**

Although it is difficult to separate biological factors from social and economic factors, the epidemiological studies have shown that the sexual dimorphism may explain the differences in the susceptibility to and/or the severity of bacterial infections between men and women. The use of experimental models of infection demonstrates the role of sex hormones in this sexual dimorphism. Sex hormones target the immune system known to be essential in the host response to infection and, in turn, can be modulated by infection. While estrogens induce efficient cell-mediated and humoral immune responses necessary to bacterial clearance, androgens are rather suppressive. The pregnancy is an excellent model of the interplay between hormonal and immune systems and it teaches us that hormonal control of immune responses varies with time. It would be essential in the future to examine the sex-based differences in immune responses in humans likely by using tissue bio-banks and high throughput methods.

#### **7. References**


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**11** 

*Germany* 

**The Special Implication of Sex Hormones** 

*University of Würzburg, Department of Obstetrics and Gynaecology, Würzburg,* 

Pregnancy bears a great challenge to the immune system: simultaneously, immune cells have to protect the reproductive tract against imminent infections, while the developing conceptus has to be tolerated. To face this problem, a distinct composition of immune cells has to be present in the decidualized endometrium (Gomez-Lopez, Guilbert *et al.*, 2010). The predominant subsets amongst those represent uterine natural killer cells (uNK cells) and cells of the monocyte/macrophage lineage like monocytes, macrophages and dendritic cells (DC) (Loke and King, 1995). Among the latter ones, DC as antigen-presenting cells (APC)

DC represent a highly adaptive cell type, which can either be transformed into an immunostimulatory phenotype after exposure to inflammatory or infectious signals or into a tolerogenic phenotype preventing T cell activation when located in an adequate antiinflammatory microenvironment. Establishing contact with invading microorganisms or cells, DC acquire their antigens and process them into antigenic peptides in the context of MHC class I and class II molecules as ligands for antigen-specific T cell receptors. This action is accompanied by migration of DC to secondary lymphoid organs. Here, the processed antigens are presented together with co-stimulatory molecules like CD40, CD80 and CD86 as well as CD83 and MHC molecules (Inaba, Metlay *et al.*, 1990;Steinman, 2003) to

Apart from antigens, DC differentiation and maturation is considerably influenced by cytokines, hormones and other soluble factors. Lacking infectious/danger- signals or localiezed in a distinct anti-inflammatory micromilieu (e.g. TGF beta, IL-10), a specific phenotype of DC is generated which prevents T cell activation and which is supposed to

In human endometrium only a small number of fully matured DC are detected (Rieger, Honig *et al.*, 2004). Thereby, the amount of mature, CD83+ DC is generally low in both pregnant and non-pregnant endometrium with a slight peak in late secretory phase endometrium. During pregnancy, a distinct DC subtype expressing CD14, CD68, HLA-DR and DC-SIGN is found in increased levels in decidua (Bonifaz, Bonnyay *et al.*, 2002;Kammerer, Eggert *et al.*, 2003). These DC, presumably an intertype between immature DC (iDC) and macrophages, are supposed to represent a "pro-fetal" tolerogenic population which is able to induce a TH2 pre-dominant state (Miyazaki, Tsuda *et al.*, 2003) and to

**1. Introduction** 

are forming an important subgroup (Steinman, 2003).

select and activate naive T cells (Sallusto and Lanzavecchia, 1999).

protect the semi-allogenic fetus (Rutella, Bonanno *et al.*, 2006).

suppress the activation of T cells (Kammerer, Eggert *et al.*, 2003).

**on Dendritic Cells During Pregnancy** 

Sabine E. Segerer and Ulrike Kämmerer

German Society for Trauma Surgery. *Critical Care Medicine*, Vol.39, No.4, pp. 621- 628, ISSN 1530-0293


### **The Special Implication of Sex Hormones on Dendritic Cells During Pregnancy**

Sabine E. Segerer and Ulrike Kämmerer

*University of Würzburg, Department of Obstetrics and Gynaecology, Würzburg, Germany* 

#### **1. Introduction**

254 Sex Hormones

Wang, Y. ; Cela, E. ; Gagnon, S. & Sweezey, N. B. (2010). Estrogen aggravates inflammation

Yamamoto, Y.; Saito, H.; Setogawa, T. & Tomioka, H. (1991). Sex differences in host

628, ISSN 1530-0293

Vol.11, 166, ISSN 1465-993X

Vol.59, No.11, pp. 4089-4096, ISSN 0019-9567

German Society for Trauma Surgery. *Critical Care Medicine*, Vol.39, No.4, pp. 621-

in *Pseudomonas aeruginosa* pneumonia in cystic fibrosis mice. *Respiratory Research*,

resistance to *Mycobacterium marinum* infection in mice. *Infection and Immunity*,

Pregnancy bears a great challenge to the immune system: simultaneously, immune cells have to protect the reproductive tract against imminent infections, while the developing conceptus has to be tolerated. To face this problem, a distinct composition of immune cells has to be present in the decidualized endometrium (Gomez-Lopez, Guilbert *et al.*, 2010). The predominant subsets amongst those represent uterine natural killer cells (uNK cells) and cells of the monocyte/macrophage lineage like monocytes, macrophages and dendritic cells (DC) (Loke and King, 1995). Among the latter ones, DC as antigen-presenting cells (APC) are forming an important subgroup (Steinman, 2003).

DC represent a highly adaptive cell type, which can either be transformed into an immunostimulatory phenotype after exposure to inflammatory or infectious signals or into a tolerogenic phenotype preventing T cell activation when located in an adequate antiinflammatory microenvironment. Establishing contact with invading microorganisms or cells, DC acquire their antigens and process them into antigenic peptides in the context of MHC class I and class II molecules as ligands for antigen-specific T cell receptors. This action is accompanied by migration of DC to secondary lymphoid organs. Here, the processed antigens are presented together with co-stimulatory molecules like CD40, CD80 and CD86 as well as CD83 and MHC molecules (Inaba, Metlay *et al.*, 1990;Steinman, 2003) to select and activate naive T cells (Sallusto and Lanzavecchia, 1999).

Apart from antigens, DC differentiation and maturation is considerably influenced by cytokines, hormones and other soluble factors. Lacking infectious/danger- signals or localiezed in a distinct anti-inflammatory micromilieu (e.g. TGF beta, IL-10), a specific phenotype of DC is generated which prevents T cell activation and which is supposed to protect the semi-allogenic fetus (Rutella, Bonanno *et al.*, 2006).

In human endometrium only a small number of fully matured DC are detected (Rieger, Honig *et al.*, 2004). Thereby, the amount of mature, CD83+ DC is generally low in both pregnant and non-pregnant endometrium with a slight peak in late secretory phase endometrium. During pregnancy, a distinct DC subtype expressing CD14, CD68, HLA-DR and DC-SIGN is found in increased levels in decidua (Bonifaz, Bonnyay *et al.*, 2002;Kammerer, Eggert *et al.*, 2003). These DC, presumably an intertype between immature DC (iDC) and macrophages, are supposed to represent a "pro-fetal" tolerogenic population which is able to induce a TH2 pre-dominant state (Miyazaki, Tsuda *et al.*, 2003) and to suppress the activation of T cells (Kammerer, Eggert *et al.*, 2003).

The Special Implication of Sex Hormones on Dendritic Cells During Pregnancy 257

receptors confer to specific intracellular signaling pathways promoting genomic and nongenomic effects. Gene transcription and regulation is also mediated by nuclear ER acting as transcription factors and thus regulating long-term effects (Tamrazi, Carlson *et al.*, 2002;Biswas, Singh *et al.*, 2005). Recently, another estrogen receptor, GPR30, which represents an intracellular transmembrane G protein-coupled receptor was detected (Revankar, Cimino *et al*., 2005). This receptor is proposed to initiate rapid non-genomic signaling effects and was found to be expressed in human endometrium throughout

B and T lymphocytes were detected to be specific targets of estradiol (Peeva and Zouali, 2005;Nalbandian, Paharkova-Vatchkova *et al.*, 2005;Nalbandian and Kovats, 2005;Smithson, Couse *et al.*, 1998). Thereby, estrogen was able to modulate B lymphopoiesis and the production of immunoglobulins (Ig) where effects were mediated via ERα (Erlandsson, Jonsson *et al.*, 2003). T cell function was found to be modulated by estradiol changing the

Regarding DC, estradiol effects were mediated via ERα and β, both of which are expressed in DC. So far, the intracellular G-protein receptor GPR 30 has not been described in DC. Even though estradiol promotes the differentiation of DC (Paharkova-Vatchkova, Maldonado *et al.*, 2004;Segerer, Muller *et al.*, 2009) towards an immunostimulatory phenotype, expressing co-stimulatory molecules like CD40, CD83 and CD86, T-cell priming was significantly impaired in the presence of estradiol (Segerer, Muller *et al.*, 2009). In addition, investigations of the effects of E2 on murine spleen CD11c-positive dendritic cells revealed an increased stimulatory capacity of DCs and an elevated expression of the anti-

In autoimmune diseases, estrogens seem to have conflicting effects on immune cells. While a reduction of disease activity was seen during pregnancy in multiple sclerosis, it was reported that systemic lupus erythematodes (SLE) could frequently flare up during

It could be speculated, that the effect of estrogen on DC participates in the flare up of SLE during pregnancy: DC generated from monocytes which were isolated from patients suffering from SLE exhibited a matured, pro-inflammatory phenotype, expressing costimulatory molecules. In addition, these SLE-DC were very effective in activating T-cells (Ding, Mehta *et al.*, 2006). Perhaps, estrogen could even accelerate the maturation-process of

During the menstrual cycle, progesterone is produced by granulosa cells and the corpus luteum (Bachelot and Binart, 2005). In pregnancy, the corpus luteum is rescued 4-5 weeks after implantation. At that time, placental progesterone production becames sufficient to maintain pregnancy (Csapo and Pulkkinen, 1978). Several studies revealed that progesterone acts in an immunosuppressive way (Stites and Siiteri, 1983;Miyaura and Iwata, 2002b). Analysing the effects of progesterone on T lymphocytes, a direct and indirect inhibition of TH1 cell development was detected (Miyaura and Iwata, 2002a). DC cultured under the influence of progesterone changed their phenotype into an immunostimulatory maturated phenotype expressing co-stimulatory molecules (Ivanova, Kyurkchiev *et al.*, 2005;Segerer, Muller *et al.*, 2009). In contrast to effects seen for hCG and estradiol, the capacity of DC to stimulate T-cell proliferation was not significantly altered (Segerer, Muller *et al.*, 2009). However, progesterone had profound effects on rat mDC by suppressing the

menstrual cycle and in early pregnancy decidua (Kolkova, Noskova *et al.*, 2010).

cytokine profile (Karpuzoglu and Zouali, 2011;Pernis, 2007;Salem, 2004).

inflammatory cytokines like IL-10 (Yang, Hu *et al.*, 2006).

pregnancy and remit with menopause (Petri, Howard *et al.*, 1991).

SLE-DC and thus promote disease during ongoing pregnancy.

**4. Progesterone** 

There is evidence that female sex hormones contribute to the modulation of decidual immune cells into tolerogenic subtypes (for review see: (Kyurkchiev, Ivanova-Todorova *et al.*, 2010) and especially can impact on DC (Segerer, Muller *et al.*, 2009). Thereby, the "micromilieu" determines if the captured antigens are presented in a tolerogenic or immune activating way resulting also in different phenotypes of DC. In the following, we will highlight the impact of the characteristic pregnancy hormones on DC and their potent implication on the development of a tolerogenic subtype of DC.

#### **2. hCG**

Human chorionic gonadotropin (hCG) is a heterodimeric hormone of the glycoprotein hormone family and is composed by two subunits linked in a non-covalent way. While the alpha subunit does not differ from the other glycoprotein hormones, the beta subunit of hCG represents the biggest one of the glycoprotein hormones holding a large glycosylated domain which refers to its high stability. hCG represents the very early hormonal factor of pregnancy already produced by the trophoblast layer of the blastocyst before implantation (Bonduelle, Dodd *et al.*, 1988;Lopata and Hay, 1989) and after implantation by the syncytiotrophoblast in increasing amounts (Hoshina, Boothby *et al.*, 1985). The production of hCG reaches a peak between the 10th and 11th week of gestation and thereafter declines to a lower but constant level throughout pregnancy. Preventing luteolysis of the corpus luteum and stimulating the production of progesterone (Keay, Vatish *et al.*, 2004), hCG represents the essential factor to protect and to promote early pregnancy. Despite of these direct effects, hCG can also act as a paracrine factor modulating the proliferation of myometrial smooth muscle cells (Horiuchi, Nikaido *et al.*, 2000;Kornyei, Lei *et al.*, 1993) and increasing endometrial angiogenesis (Berndt, Blacher *et al.*, 2009).

There is also evidence that hCG contributes to maternal tolerance of the developing conceptus by influencing the surrounding immune cells. Thus, experiments on uNK cells revealed that hCG acts as a regulator of uNK cell proliferation (Kane, Kelly *et al.*, 2009). Despite of this direct effect on uNK cell proliferation, hCG can facilitate the adequate establishment and maintenance of pregnancy by inducing the secretion factors which promote angiogeneis by uNK cells and thus could even support placentation (Lash, Schiessl *et al.*, 2006).

Investigations on DC demonstrated hCG receptors to be constitutively expressed by these cells which allows a direct activation of DC via hCG (Yoshimura, Inaba *et al.*, 2003). Experiments on mouse DC revealed that hCG acts in an immunregulatory way by increasing the production of immunosuppressive factors like Interleukin-10 (IL-10) by DC and by reducing antigen-specific T-cell proliferation (Wan, Versnel *et al.*, 2008). In humans, hCG was also able to significantly decrease T-cell stimulatory capacity of DC (Huck, Steck *et al.*, 2005). In contrast, the phenotype of hCG-treated DC ressembeled that of matured DC expressing co-stimulatory molecules like CD 40, CD83 and CD 86 (Segerer, Muller *et al.*, 2009). Thus, hCG seems to be able to induce a tolerogenic subtype of DC even though costimulatory molecules of the mature subtype were expressed.

#### **3. Estrogen**

Estrogen receptors (ER) alpha and beta (ER α/β) are widely expressed in human endometrium but also in most immune cells (Lindzey, Wetsel *et al.*, 1998). Thereby, they are localized in different cellular compartments. The cytoplasmatic and membrane associated receptors confer to specific intracellular signaling pathways promoting genomic and nongenomic effects. Gene transcription and regulation is also mediated by nuclear ER acting as transcription factors and thus regulating long-term effects (Tamrazi, Carlson *et al.*, 2002;Biswas, Singh *et al.*, 2005). Recently, another estrogen receptor, GPR30, which represents an intracellular transmembrane G protein-coupled receptor was detected (Revankar, Cimino *et al*., 2005). This receptor is proposed to initiate rapid non-genomic signaling effects and was found to be expressed in human endometrium throughout menstrual cycle and in early pregnancy decidua (Kolkova, Noskova *et al.*, 2010).

B and T lymphocytes were detected to be specific targets of estradiol (Peeva and Zouali, 2005;Nalbandian, Paharkova-Vatchkova *et al.*, 2005;Nalbandian and Kovats, 2005;Smithson, Couse *et al.*, 1998). Thereby, estrogen was able to modulate B lymphopoiesis and the production of immunoglobulins (Ig) where effects were mediated via ERα (Erlandsson, Jonsson *et al.*, 2003). T cell function was found to be modulated by estradiol changing the cytokine profile (Karpuzoglu and Zouali, 2011;Pernis, 2007;Salem, 2004).

Regarding DC, estradiol effects were mediated via ERα and β, both of which are expressed in DC. So far, the intracellular G-protein receptor GPR 30 has not been described in DC. Even though estradiol promotes the differentiation of DC (Paharkova-Vatchkova, Maldonado *et al.*, 2004;Segerer, Muller *et al.*, 2009) towards an immunostimulatory phenotype, expressing co-stimulatory molecules like CD40, CD83 and CD86, T-cell priming was significantly impaired in the presence of estradiol (Segerer, Muller *et al.*, 2009). In addition, investigations of the effects of E2 on murine spleen CD11c-positive dendritic cells revealed an increased stimulatory capacity of DCs and an elevated expression of the antiinflammatory cytokines like IL-10 (Yang, Hu *et al.*, 2006).

In autoimmune diseases, estrogens seem to have conflicting effects on immune cells. While a reduction of disease activity was seen during pregnancy in multiple sclerosis, it was reported that systemic lupus erythematodes (SLE) could frequently flare up during pregnancy and remit with menopause (Petri, Howard *et al.*, 1991).

It could be speculated, that the effect of estrogen on DC participates in the flare up of SLE during pregnancy: DC generated from monocytes which were isolated from patients suffering from SLE exhibited a matured, pro-inflammatory phenotype, expressing costimulatory molecules. In addition, these SLE-DC were very effective in activating T-cells (Ding, Mehta *et al.*, 2006). Perhaps, estrogen could even accelerate the maturation-process of SLE-DC and thus promote disease during ongoing pregnancy.

### **4. Progesterone**

256 Sex Hormones

There is evidence that female sex hormones contribute to the modulation of decidual immune cells into tolerogenic subtypes (for review see: (Kyurkchiev, Ivanova-Todorova *et al.*, 2010) and especially can impact on DC (Segerer, Muller *et al.*, 2009). Thereby, the "micromilieu" determines if the captured antigens are presented in a tolerogenic or immune activating way resulting also in different phenotypes of DC. In the following, we will highlight the impact of the characteristic pregnancy hormones on DC and their potent

Human chorionic gonadotropin (hCG) is a heterodimeric hormone of the glycoprotein hormone family and is composed by two subunits linked in a non-covalent way. While the alpha subunit does not differ from the other glycoprotein hormones, the beta subunit of hCG represents the biggest one of the glycoprotein hormones holding a large glycosylated domain which refers to its high stability. hCG represents the very early hormonal factor of pregnancy already produced by the trophoblast layer of the blastocyst before implantation (Bonduelle, Dodd *et al.*, 1988;Lopata and Hay, 1989) and after implantation by the syncytiotrophoblast in increasing amounts (Hoshina, Boothby *et al.*, 1985). The production of hCG reaches a peak between the 10th and 11th week of gestation and thereafter declines to a lower but constant level throughout pregnancy. Preventing luteolysis of the corpus luteum and stimulating the production of progesterone (Keay, Vatish *et al.*, 2004), hCG represents the essential factor to protect and to promote early pregnancy. Despite of these direct effects, hCG can also act as a paracrine factor modulating the proliferation of myometrial smooth muscle cells (Horiuchi, Nikaido *et al.*, 2000;Kornyei, Lei *et al.*, 1993) and increasing

There is also evidence that hCG contributes to maternal tolerance of the developing conceptus by influencing the surrounding immune cells. Thus, experiments on uNK cells revealed that hCG acts as a regulator of uNK cell proliferation (Kane, Kelly *et al.*, 2009). Despite of this direct effect on uNK cell proliferation, hCG can facilitate the adequate establishment and maintenance of pregnancy by inducing the secretion factors which promote angiogeneis by

Investigations on DC demonstrated hCG receptors to be constitutively expressed by these cells which allows a direct activation of DC via hCG (Yoshimura, Inaba *et al.*, 2003). Experiments on mouse DC revealed that hCG acts in an immunregulatory way by increasing the production of immunosuppressive factors like Interleukin-10 (IL-10) by DC and by reducing antigen-specific T-cell proliferation (Wan, Versnel *et al.*, 2008). In humans, hCG was also able to significantly decrease T-cell stimulatory capacity of DC (Huck, Steck *et al.*, 2005). In contrast, the phenotype of hCG-treated DC ressembeled that of matured DC expressing co-stimulatory molecules like CD 40, CD83 and CD 86 (Segerer, Muller *et al.*, 2009). Thus, hCG seems to be able to induce a tolerogenic subtype of DC even though co-

Estrogen receptors (ER) alpha and beta (ER α/β) are widely expressed in human endometrium but also in most immune cells (Lindzey, Wetsel *et al.*, 1998). Thereby, they are localized in different cellular compartments. The cytoplasmatic and membrane associated

uNK cells and thus could even support placentation (Lash, Schiessl *et al.*, 2006).

implication on the development of a tolerogenic subtype of DC.

endometrial angiogenesis (Berndt, Blacher *et al.*, 2009).

stimulatory molecules of the mature subtype were expressed.

**2. hCG** 

**3. Estrogen** 

During the menstrual cycle, progesterone is produced by granulosa cells and the corpus luteum (Bachelot and Binart, 2005). In pregnancy, the corpus luteum is rescued 4-5 weeks after implantation. At that time, placental progesterone production becames sufficient to maintain pregnancy (Csapo and Pulkkinen, 1978). Several studies revealed that progesterone acts in an immunosuppressive way (Stites and Siiteri, 1983;Miyaura and Iwata, 2002b). Analysing the effects of progesterone on T lymphocytes, a direct and indirect inhibition of TH1 cell development was detected (Miyaura and Iwata, 2002a). DC cultured under the influence of progesterone changed their phenotype into an immunostimulatory maturated phenotype expressing co-stimulatory molecules (Ivanova, Kyurkchiev *et al.*, 2005;Segerer, Muller *et al.*, 2009). In contrast to effects seen for hCG and estradiol, the capacity of DC to stimulate T-cell proliferation was not significantly altered (Segerer, Muller *et al.*, 2009). However, progesterone had profound effects on rat mDC by suppressing the

The Special Implication of Sex Hormones on Dendritic Cells During Pregnancy 259

During pregnancy, the levels of the glycoprotein hormones activin A and inhibin A as well as female sex hormones (hCG, estradiol, progesterone) are substantially elevated in parallel with increased occurrence of cells of the monocyte/macrophage lineage like DC in the decidua. In vitro experiments revealed that all of them can induce a tolerogenic subtype of DC even though effects were found on different levels (reduction of the expression of co-stimulatory molecules, reduction of T-cell stimulation, tolerogenic cytokine-profile). Thus, this distinct mixture of hormonal factors seems to be indispensable to support the development of

[1] Bachelot A and Binart N (2005) Corpus luteum development: lessons from genetic

[2] Berndt S, Blacher S, Perrier dS, Thiry M, Tsampalas M, Cruz A, Pequeux C, Lorquet S,

[3] Birdsall M, Ledger W, Groome N, Abdalla H, and Muttukrishna S (1997) Inhibin A and

[4] Biswas DK, Singh S, Shi Q, Pardee AB, and Iglehart JD (2005) Crossroads of estrogen

[5] Bonduelle ML, Dodd R, Liebaers I, Van SA, Williamson R, and Akhurst R (1988) Chorionic

embryos derived from tripronucleate zygotes. Hum Reprod, 3, 909-914. [6] Bonifaz L, Bonnyay D, Mahnke K, Rivera M, Nussenzweig MC, and Steinman RM (2002)

and pericyte recruitment. J Clin Endocrinol Metab, 94, 4567-4574.

receptor and NF-kappaB signaling. Sci STKE, 2005, e27.

Munaut C, Noel A et al (2009) Chorionic gonadotropin stimulation of angiogenesis

activin A in the first trimester of human pregnancy. J Clin Endocrinol Metab, 82,

gonadotrophin-beta mRNA, a trophoblast marker, is expressed in human 8-cell

Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance. J Exp Med, 196, 1627-1638. [7] Butts CL, Shukair SA, Duncan KM, Bowers E, Horn C, Belyavskaya E, Tonelli L, and

Sternberg EM (2007) Progesterone inhibits mature rat dendritic cells in a receptor-

maintenance of early pregnancy. Luteectomy evidence. Obstet Gynecol Surv, 33,

function of myeloid dendritic cells in systemic lupus erythematosus. J Immunol,

differentiation factor is induced during human monocyte activation. J Exp Med,

receptor specificity in oestradiol-mediated effects on B lymphopoiesis and

[8] Chang H, Brown CW, and Matzuk MM (2002) Genetic analysis of the mammalian transforming growth factor-beta superfamily. Endocr Rev, 23, 787-823. [9] Csapo AI and Pulkkinen M (1978) Indispensability of the human corpus luteum in the

[10] Ding D, Mehta H, McCune WJ, and Kaplan MJ (2006) Aberrant phenotype and

[11] Eramaa M, Hurme M, Stenman UH, and Ritvos O (1992) Activin A/erythroid

[12] Erlandsson MC, Jonsson CA, Islander U, Ohlsson C, and Carlsten H (2003) Oestrogen

immunoglobulin production in male mice. Immunology, 108, 346-351.

tolerance-inducing DC which could play a key role in the acceptance of the fetus.

models in mice. Curr Top Dev Biol, 68, 49-84.

mediated fashion. Int Immunol, 19, 287-296.

**6. Summary** 

**7. References** 

1557-1560.

69-81.

177, 5878-5889.

176, 1449-1452.

production of pro-inflammatory cytokines like tumor necrosis factor alpha (TNF) and interleukin 1beta (IL-1) as well as by inhibiting the DC-stimulated proliferation of T-cells (Butts, Shukair *et al.*, 2007). Thus, even though the expression of co-stimulatory molecules and T-cell stimulatory capacity was not affected progesterone seems to modulate immune responses by changing the cytokine secretion prolife.

#### **5. Activin A and inhibin A**

Activin A represents a distinct growth factor and member of the TGF beta superfamily (Chang, Brown *et al.*, 2002) which is composed by two activin A subunits. Its counterpart inhibin A is formed by a dimer consisting of an activin subunit and a structurally different subunit (Phillips, Jones *et al.*, 2005). Initially, the function of these glycoprotein hormones was defined as feedback factors regulating the release of the follicle-stimulating hormone (FSH) (Tong, Wallace *et al.*, 2003). Later, it was observed that activin A and inhibin A are endowed by diverse functions acting as cytokines in an autocrine or paracrine manner, too. Thus, activin A was detected to be involved in inflammatory responses (Jones, de Kretser *et al.*, 2004) and acting as a chemoattractant for monocytes (Eramaa, Hurme *et al.*, 1992;Shao, Frigon, Jr. *et al.*, 1992).

During pregnancy, activin A and inhibin A are locally produced by human placenta, decidua and fetal membranes resulting in serum levels in the low ng/ml range peaking at 10 weeks of gestation (Lockwood, Ledger *et al.*, 1997). Both glycoprotein hormones seem to have some impact on implantation and early embryo development (Birdsall, Ledger *et al.*, 1997;Muttukrishna, Jauniaux *et al.*, 2004;Jones, Salamonsen *et al.*, 2002). Furthermore, they have been found to be useful diagnostic markers for pregnancy success. While decreasing activin A levels indicated both an ongoing miscarriage or ectopic pregnancy (Florio, Severi *et al.*, 2007), decreasing inhibin A levels were used as a specific marker to reveal preclinical abortions (Muttukrishna, Jauniaux *et al.*, 2004;Prakash, Laird *et al.*, 2005).

Previous studies revealed that activin A is able to modulate immune responses via type I and II activin receptors on DC (Robson, Phillips *et al.*, 2008). Even though the expression of co-stimulatory molecules was not significantly affected neither by activin A nor inhibin A, both glycoprotein hormones were able to decrease the T-cell stimulatory capacity of DC. Thus, we propose that activin A and inhibin A are distinct modulating factors that could promote the generation of tolerance-inducing DC by affecting their T-cell stimulatory capacity (Segerer, Muller *et al.*, 2008) .


Table 1.

#### **6. Summary**

258 Sex Hormones

production of pro-inflammatory cytokines like tumor necrosis factor alpha (TNF) and interleukin 1beta (IL-1) as well as by inhibiting the DC-stimulated proliferation of T-cells (Butts, Shukair *et al.*, 2007). Thus, even though the expression of co-stimulatory molecules and T-cell stimulatory capacity was not affected progesterone seems to modulate immune

Activin A represents a distinct growth factor and member of the TGF beta superfamily (Chang, Brown *et al.*, 2002) which is composed by two activin A subunits. Its counterpart inhibin A is formed by a dimer consisting of an activin subunit and a structurally different subunit (Phillips, Jones *et al.*, 2005). Initially, the function of these glycoprotein hormones was defined as feedback factors regulating the release of the follicle-stimulating hormone (FSH) (Tong, Wallace *et al.*, 2003). Later, it was observed that activin A and inhibin A are endowed by diverse functions acting as cytokines in an autocrine or paracrine manner, too. Thus, activin A was detected to be involved in inflammatory responses (Jones, de Kretser *et al.*, 2004) and acting as a chemoattractant for monocytes (Eramaa, Hurme *et al.*, 1992;Shao,

During pregnancy, activin A and inhibin A are locally produced by human placenta, decidua and fetal membranes resulting in serum levels in the low ng/ml range peaking at 10 weeks of gestation (Lockwood, Ledger *et al.*, 1997). Both glycoprotein hormones seem to have some impact on implantation and early embryo development (Birdsall, Ledger *et al.*, 1997;Muttukrishna, Jauniaux *et al.*, 2004;Jones, Salamonsen *et al.*, 2002). Furthermore, they have been found to be useful diagnostic markers for pregnancy success. While decreasing activin A levels indicated both an ongoing miscarriage or ectopic pregnancy (Florio, Severi *et al.*, 2007), decreasing inhibin A levels were used as a specific marker to reveal preclinical

Previous studies revealed that activin A is able to modulate immune responses via type I and II activin receptors on DC (Robson, Phillips *et al.*, 2008). Even though the expression of co-stimulatory molecules was not significantly affected neither by activin A nor inhibin A, both glycoprotein hormones were able to decrease the T-cell stimulatory capacity of DC. Thus, we propose that activin A and inhibin A are distinct modulating factors that could promote the generation of tolerance-inducing DC by affecting their T-cell stimulatory

**DC phenotype T-cell stimulatory** 

(CD40 ↑, CD83 ↑, CD86 ↑) ↓ IL-10 <sup>↑</sup>

(CD40 ↑, CD83 ↑, CD86 ↑) ↓ IL-10 <sup>↑</sup>

(CD40 ↑, CD83 ↑, CD86 ↑) <sup>↓</sup> no significant

↓ (in rats) → (in humans)

**capacity cytokines** 

TNF-α ↓, IL-1 β ↓ (in rats)

effects

abortions (Muttukrishna, Jauniaux *et al.*, 2004;Prakash, Laird *et al.*, 2005).

responses by changing the cytokine secretion prolife.

**5. Activin A and inhibin A** 

Frigon, Jr. *et al.*, 1992).

capacity (Segerer, Muller *et al.*, 2008) .

**hCG** immunostimulatory

**estrogen** immunostimulatory

**progesterone** immunostimulatory

**activin A and inhibin A** 

Table 1.

(CD40 ↑, CD83 ↑, CD86 ↑)

immunostimulatory

During pregnancy, the levels of the glycoprotein hormones activin A and inhibin A as well as female sex hormones (hCG, estradiol, progesterone) are substantially elevated in parallel with increased occurrence of cells of the monocyte/macrophage lineage like DC in the decidua. In vitro experiments revealed that all of them can induce a tolerogenic subtype of DC even though effects were found on different levels (reduction of the expression of co-stimulatory molecules, reduction of T-cell stimulation, tolerogenic cytokine-profile). Thus, this distinct mixture of hormonal factors seems to be indispensable to support the development of tolerance-inducing DC which could play a key role in the acceptance of the fetus.

#### **7. References**


The Special Implication of Sex Hormones on Dendritic Cells During Pregnancy 261

[29] Lash GE, Schiessl B, Kirkley M, Innes BA, Cooper A, Searle RF, Robson SC, and Bulmer

[30] Lindzey J, Wetsel WC, Couse JF, Stoker T, Cooper R, and Korach KS (1998) Effects of

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[32] Loke YW and King A (1995) Human Implantation. Cell Biology and Immunology.,

[33] Lopata A and Hay DL (1989) The potential of early human embryos to form blastocysts, hatch from their zona and secrete HCG in culture. Hum Reprod, 4, 87-94. [34] Miyaura H and Iwata M (2002) Direct and indirect inhibition of Th1 development by

[35] Miyazaki S, Tsuda H, Sakai M, Hori S, Sasaki Y, Futatani T, Miyawaki T, and Saito S

[36] Muttukrishna S, Jauniaux E, McGarrigle H, Groome N, and Rodeck CH (2004) In-vivo

[37] Nalbandian G and Kovats S (2005) Understanding sex biases in immunity: effects of

[38] Nalbandian G, Paharkova-Vatchkova V, Mao A, Nale S, and Kovats S (2005) The

[39] Paharkova-Vatchkova V, Maldonado R, and Kovats S (2004) Estrogen preferentially

[40] Peeva E and Zouali M (2005) Spotlight on the role of hormonal factors in the emergence of autoreactive B-lymphocytes. Immunol Lett, 101, 123-143. [41] Pernis AB (2007) Estrogen and CD4+ T cells. Curr Opin Rheumatol, 19, 414-420.

[42] Petri M, Howard D, and Repke J (1991) Frequency of lupus flare in pregnancy. The Hopkins Lupus Pregnancy Center experience. Arthritis Rheum, 34, 1538-1545. [43] Phillips DJ, Jones KL, Clarke IJ, Scheerlinck JP, and de Kretser DM (2005) Activin A:

[44] Prakash A, Laird S, Tuckerman E, Li TC, and Ledger WL (2005) Inhibin A and activin

[45] Revankar CM, Cimino DF, Sklar LA, Arterburn JB, Prossnitz ER (2005) A

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**1. Introduction** 

**12** 

*Iran* 

Rose Fouladi

*Tehran University, Tehran,* 

**Sex Hormones and** 

**Neuromuscular Control System** 

There is evidence that females sustain more exercise- related musculoskeletal injuries than males. Sex differences in injury rates are apparent in some connective tissues such as ligaments. Although girls and boys have an equal chance of ligament injuries prior to adolescence, girls have a higher rate immediately after maturation (Tursz et al., 1986). Female athletes participating in cutting, jumping and pivoting sports have a 4-6 times

Ligament, tendon, bone and endometrium contain estrogen receptors responsive to female sex hormones. Estrogen has direct effect on soft tissue strength, muscle function and collagen metabolism and behavior. It is demonstrated the influence of female endocrinology on knee joint behavior, as male and females differ substantially in the type, level and periodic exposure of circulating sex hormones after puberty. While hormone levels remain fairly constant in males, females are exposed to rhythmic fluctuations in endogenous hormones during the course of menstrual cycle. The absolute levels of estrogen and progesterone varying considerably during the course of a female's menstrual cycle and there are some variations in the hormonal levels. At the beginning of the menstrual cycle, estrogen (E) and progesterone (P) remain close to their minimum levels. Toward the middle of the cycle, estrogen level rises and

Also, estrogen indirectly influences the female neuromuscular system. Neuromuscular patterns in males and females differ during maturation. Males demonstrate power, strength and coordination increasing correlate with their age and maturational stage, whereas girls show little change throughout maturation (Kellis et al., 1999; Beunen et al., 1988). In females, quadriceps strength increases and you know it can increase the tibia anterior translation and subsequently the rate of ACL injury. The reliance of males on a more hamstring dominant strategy may be more protective of the ACL because the hamstring and ACL are agonist to prevent anterior displacement of the tibia on the femur. Also, females have shown more impaired proprioception assessing knee motion into extension than males (Rozzi et al., 1999). Significant slowing of muscle relaxation also occurs during the ovulation (estrogen surge) of the menstrual cycle. Serum estrogen concentrations fluctuate radically throughout the cycle and estrogen has measurable effects on muscle function and tendon and ligament strength. Moreover Estrogen has effects on the central nervous system. It has demonstrated differences in skill performance in females during different phases of the menstrual cycle, also a decrease in motor skills in the premenstrual phase at the late luteal. These data

greater chance of ACL tearing than their male counterparts (Arendt et al., 1999).

in the middle of the luteal phase both E and P levels increase.


### **Sex Hormones and Neuromuscular Control System**

Rose Fouladi *Tehran University, Tehran, Iran* 

#### **1. Introduction**

262 Sex Hormones

[46] Rieger L, Honig A, Sutterlin M, Kapp M, Dietl J, Ruck P, and Kammerer U (2004)

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[54] Smithson G, Couse JF, Lubahn DB, Korach KS, and Kincade PW (1998) The role of

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[55] Steinman RM (2003) Some interfaces of dendritic cell biology. APMIS, 111, 675-697. [56] Stites DP and Siiteri PK (1983) Steroids as immunosuppressants in pregnancy.

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Antigen-presenting cells in human endometrium during the menstrual cycle

N, Zanker D, Wilson K et al (2008) Activin-A: a novel dendritic cell-derived cytokine that potently attenuates CD40 ligand-specific cytokine and chemokine

Pandolfi S, Natoni F et al (2006) Hepatocyte growth factor favors monocyte differentiation into regulatory interleukin (IL)-10++IL-12low/neg accessory cells

mediated inflammations by differential regulation of TH1/TH2 cytokine

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Khan NA, Benner R, and Kiekens RC (2008) Chorionic gonadotropin induces dendritic cells to express a tolerogenic phenotype. J Leukoc Biol, 83, 894-901. [60] Yang L, Hu Y, and Hou Y (2006) Effects of 17beta-estradiol on the maturation, nuclear

factor kappa B p65 and functions of murine spleen CD11c-positive dendritic cells.

Ikehara S (2003) Analyses of dendritic cell subsets in pregnancy. Am J Reprod

There is evidence that females sustain more exercise- related musculoskeletal injuries than males. Sex differences in injury rates are apparent in some connective tissues such as ligaments. Although girls and boys have an equal chance of ligament injuries prior to adolescence, girls have a higher rate immediately after maturation (Tursz et al., 1986). Female athletes participating in cutting, jumping and pivoting sports have a 4-6 times greater chance of ACL tearing than their male counterparts (Arendt et al., 1999).

Ligament, tendon, bone and endometrium contain estrogen receptors responsive to female sex hormones. Estrogen has direct effect on soft tissue strength, muscle function and collagen metabolism and behavior. It is demonstrated the influence of female endocrinology on knee joint behavior, as male and females differ substantially in the type, level and periodic exposure of circulating sex hormones after puberty. While hormone levels remain fairly constant in males, females are exposed to rhythmic fluctuations in endogenous hormones during the course of menstrual cycle. The absolute levels of estrogen and progesterone varying considerably during the course of a female's menstrual cycle and there are some variations in the hormonal levels. At the beginning of the menstrual cycle, estrogen (E) and progesterone (P) remain close to their minimum levels. Toward the middle of the cycle, estrogen level rises and in the middle of the luteal phase both E and P levels increase.

Also, estrogen indirectly influences the female neuromuscular system. Neuromuscular patterns in males and females differ during maturation. Males demonstrate power, strength and coordination increasing correlate with their age and maturational stage, whereas girls show little change throughout maturation (Kellis et al., 1999; Beunen et al., 1988). In females, quadriceps strength increases and you know it can increase the tibia anterior translation and subsequently the rate of ACL injury. The reliance of males on a more hamstring dominant strategy may be more protective of the ACL because the hamstring and ACL are agonist to prevent anterior displacement of the tibia on the femur. Also, females have shown more impaired proprioception assessing knee motion into extension than males (Rozzi et al., 1999). Significant slowing of muscle relaxation also occurs during the ovulation (estrogen surge) of the menstrual cycle. Serum estrogen concentrations fluctuate radically throughout the cycle and estrogen has measurable effects on muscle function and tendon and ligament strength.

Moreover Estrogen has effects on the central nervous system. It has demonstrated differences in skill performance in females during different phases of the menstrual cycle, also a decrease in motor skills in the premenstrual phase at the late luteal. These data

Sex Hormones and Neuromuscular Control System 265

Proprioception which refers specially to conscious and unconscious appreciation of joint position, kinesthesia (the sensation of joint motion or acceleration) and the perception of force (an ability to estimate joint loads). These signals are transmitted to the spinal cord via afferent (sensory) pathways. Conscious awareness of joint motion, position and force is essential for motor learning and the anticipation of movements in sport, while unconscious proprioception modulates muscle function and initiates reflex joint stabilization. The efferent (motor) response to sensory information is termed neuromuscular control (Prentice 2011). The deficit of neuromuscular control of the knee joint may be responsible for the high

The central nervous system (CNS) is the primary mediator for the perception and execution of musculoskeletal control and movement. Perception and sensation of joint movement are monitored by tree main subsystems: 1) the somatosensory system, 2) the vestibular system, and 3) the visual system. The somatosensory system often referred to as proprioception, receives input from peripheral articular and musculotendinous receptors concerning changes in muscle length and tension, in addition to information regarding joint position and motion. The vestibular system receives information from the vestibules and semicircular canals of the ear, which aid in keeping the body in balance, while the visual system provides the body with visual cues, contributing to balance by providing reference points for orientation. Information gathered by the somatosensory system, in addition to that from vestibular and visual systems, is processed at three distinct levels of motor control: the spinal level, the brain stem and the higher centers such as the motor cortex, basal ganglia, and cerebellum.

rate of knee injury in female athletes (Hewett et al., 1996; Huston et al., 1996).

Fig. 1. Neuromuscular control pathway (reproduced from Lephart 1998)

Proprioception and neuromuscular control may be assessed clinically through evaluation of the afferent and efferent neuromuscular pathways. The afferent pathway is qualified through the examination of joint kinesthesia and joint position sensibility, which provide the researcher with a measurement of conscious appreciation of joint motion and position sensibility. Joint kinesthesia is determined clinically by establishing the threshold to detection of passive motion, an assessment of the ability to detect relatively slow passive joint motion. Testing joint position sense, another method of assessing the afferent pathway, determines the ability of the subject to comprehend a presented joint angle and then once

indicate that estrogen can be effective on neuromuscular function which may facilitate the potential for neuromuscular imbalances to develop in female athletes. There is also evidence that estrogen influences electrical activity of neurons both centrally and peripherally (Lee et al., 2002; Papka et al., 2001; Rozzi et al., 1999). Furthermore, postural control impairments have been demonstrated in females with premenstrual symptoms in the mid-luteal phase (Friden et al., 2003). These findings support the hypothesis that lower extremity neuromuscular performance may be influenced by circulating sex hormones.

Because of existing estrogen and progesterone receptors on the human ACL fibroblasts, females' sex hormones may directly influence the structure and composition of the ACL. Some researchers have indicated different cycle phases for increased incidence of ACL injury. Some of them found that females have a greater risk of ACL injury during the ovulatory phase of the menstrual cycle in which estrogen surge is present (Wojtys et al., 1998). However, the others implicated a significant greater number of ACL injuries occurred onset of the cycle (days 1 and 2) (Slauterbeck et al., 2002). They believed cyclic changes in estrogen and progesterone may change expression of genes encoding tissue-remodeling enzymes and proteins, which in turn could favor either net tissue degradation or repair at specific times during the menstrual cycle.

In some studies, researchers have suggested that the large number of non-contact ACL injuries in female athletes may be related to knee laxity, which might be influenced by hormones. In normal menstruating females, significant increases in knee laxity have been noted in the pre ovulatory and mid luteal phases of the menstrual cycle compared to menses. This is believed to coincide with elevated levels of estrogen, and estrogen and progesterone respectively. Although hormonal changes might result in increased injury risk for female athletes, the effects of hormonal level on occurrence of injury in these athletes are not fully understood.

In addition to sex hormones receptors, there are mechanoreceptors in human ACL. These mechanoreceptors are referred to as Ruffini receptors, Pacini receptors, Golgy tendon organlike receptors and free nerve endings that they may have a proprioceptive function (Adachi et al., 2002). Proprioception is an important part of neuromuscular performance, and can be defined as the individual's awareness of his or her extremities' position and motion in space. It involves sensory activities of the tendons, ligaments, capsules and muscles, and can be internal peripheral areas of the body that contribute to postural control, joint stability and conscious sensation of movement. Position sense at the knee joint is influenced by central and peripheral mechanisms, such as muscles, tendons, articulate, cutaneous and ACL receptors (Shultz et al., 2005). The ACL have two complementary functions: mechanical and sensory (proprioception) (Ekenros et al., 2010). It has been implicated that sensory information from the ACL assists in providing functional stability to the knee joint by contributing to neuromuscular control (Riemann et al., 2002).

Since the relationship between ACL injury and female hormone concentration and also, the relationship between ACL injury and neuromuscular control (proprioception) deficit is supported by recent studies. This chapter is going to investigate the relationship between female hormonal level and proprioception, by measuring the knee joint position sense (JPS) and serum estrogen and progesterone concentrations throughout the menstrual cycle.

#### **2. Neuromuscular control and proprioception**

Neuromuscular control of the knee is defined as the unconscious response to an afferent signal concerning dynamic knee joint stability. These afferent signals are produced by

indicate that estrogen can be effective on neuromuscular function which may facilitate the potential for neuromuscular imbalances to develop in female athletes. There is also evidence that estrogen influences electrical activity of neurons both centrally and peripherally (Lee et al., 2002; Papka et al., 2001; Rozzi et al., 1999). Furthermore, postural control impairments have been demonstrated in females with premenstrual symptoms in the mid-luteal phase (Friden et al., 2003). These findings support the hypothesis that lower extremity

Because of existing estrogen and progesterone receptors on the human ACL fibroblasts, females' sex hormones may directly influence the structure and composition of the ACL. Some researchers have indicated different cycle phases for increased incidence of ACL injury. Some of them found that females have a greater risk of ACL injury during the ovulatory phase of the menstrual cycle in which estrogen surge is present (Wojtys et al., 1998). However, the others implicated a significant greater number of ACL injuries occurred onset of the cycle (days 1 and 2) (Slauterbeck et al., 2002). They believed cyclic changes in estrogen and progesterone may change expression of genes encoding tissue-remodeling enzymes and proteins, which in turn could favor either net tissue degradation or repair at

In some studies, researchers have suggested that the large number of non-contact ACL injuries in female athletes may be related to knee laxity, which might be influenced by hormones. In normal menstruating females, significant increases in knee laxity have been noted in the pre ovulatory and mid luteal phases of the menstrual cycle compared to menses. This is believed to coincide with elevated levels of estrogen, and estrogen and progesterone respectively. Although hormonal changes might result in increased injury risk for female athletes, the effects of hormonal level on occurrence of injury in these athletes are not fully understood. In addition to sex hormones receptors, there are mechanoreceptors in human ACL. These mechanoreceptors are referred to as Ruffini receptors, Pacini receptors, Golgy tendon organlike receptors and free nerve endings that they may have a proprioceptive function (Adachi et al., 2002). Proprioception is an important part of neuromuscular performance, and can be defined as the individual's awareness of his or her extremities' position and motion in space. It involves sensory activities of the tendons, ligaments, capsules and muscles, and can be internal peripheral areas of the body that contribute to postural control, joint stability and conscious sensation of movement. Position sense at the knee joint is influenced by central and peripheral mechanisms, such as muscles, tendons, articulate, cutaneous and ACL receptors (Shultz et al., 2005). The ACL have two complementary functions: mechanical and sensory (proprioception) (Ekenros et al., 2010). It has been implicated that sensory information from the ACL assists in providing functional stability to the knee joint by

Since the relationship between ACL injury and female hormone concentration and also, the relationship between ACL injury and neuromuscular control (proprioception) deficit is supported by recent studies. This chapter is going to investigate the relationship between female hormonal level and proprioception, by measuring the knee joint position sense (JPS) and serum estrogen and progesterone concentrations throughout the menstrual cycle.

Neuromuscular control of the knee is defined as the unconscious response to an afferent signal concerning dynamic knee joint stability. These afferent signals are produced by

neuromuscular performance may be influenced by circulating sex hormones.

specific times during the menstrual cycle.

contributing to neuromuscular control (Riemann et al., 2002).

**2. Neuromuscular control and proprioception** 

Proprioception which refers specially to conscious and unconscious appreciation of joint position, kinesthesia (the sensation of joint motion or acceleration) and the perception of force (an ability to estimate joint loads). These signals are transmitted to the spinal cord via afferent (sensory) pathways. Conscious awareness of joint motion, position and force is essential for motor learning and the anticipation of movements in sport, while unconscious proprioception modulates muscle function and initiates reflex joint stabilization. The efferent (motor) response to sensory information is termed neuromuscular control (Prentice 2011). The deficit of neuromuscular control of the knee joint may be responsible for the high rate of knee injury in female athletes (Hewett et al., 1996; Huston et al., 1996).

The central nervous system (CNS) is the primary mediator for the perception and execution of musculoskeletal control and movement. Perception and sensation of joint movement are monitored by tree main subsystems: 1) the somatosensory system, 2) the vestibular system, and 3) the visual system. The somatosensory system often referred to as proprioception, receives input from peripheral articular and musculotendinous receptors concerning changes in muscle length and tension, in addition to information regarding joint position and motion.

The vestibular system receives information from the vestibules and semicircular canals of the ear, which aid in keeping the body in balance, while the visual system provides the body with visual cues, contributing to balance by providing reference points for orientation.

Information gathered by the somatosensory system, in addition to that from vestibular and visual systems, is processed at three distinct levels of motor control: the spinal level, the brain stem and the higher centers such as the motor cortex, basal ganglia, and cerebellum.

Fig. 1. Neuromuscular control pathway (reproduced from Lephart 1998)

Proprioception and neuromuscular control may be assessed clinically through evaluation of the afferent and efferent neuromuscular pathways. The afferent pathway is qualified through the examination of joint kinesthesia and joint position sensibility, which provide the researcher with a measurement of conscious appreciation of joint motion and position sensibility. Joint kinesthesia is determined clinically by establishing the threshold to detection of passive motion, an assessment of the ability to detect relatively slow passive joint motion. Testing joint position sense, another method of assessing the afferent pathway, determines the ability of the subject to comprehend a presented joint angle and then once

Sex Hormones and Neuromuscular Control System 267

Neuromuscular control of the knee can be defined as the unconscious to an afferent signal concerning dynamic knee joint stability. The absents of neuromuscular control of the knee joint may be responsible for the increased rate of knee injury in females, but it is not normally measured with the 3 dimensional kinematic system described below and were

Changes in neuromuscular control of the knee in adolescent athletes are documented. Several studies have documented a substantial increase in neuromuscular strength and coordination

Normal menstrual cycle in women includes fluctuation of sex hormones' levels in a regular duration that usually takes 21-35 days. Ovarian cycle of the normal menstrual cycle has 2 phases, follicular and luteal. Follicular phase is started by menstruation with 2-4 days

Estrogen and progesterone levels are in the lowest level at menses but little by little estrogen increases and it goes up to its highest level just before the ovulation, at the middle of the cycle. Duration of the estrogen surge is so limit and it just takes 24 to 48 hours. After ovulation, both estrogen and progesterone levels go up slowly and they are in their highest

following the growth spurt in adolescent boys but not in the average adolescent girls.

recorded during the same laboratory evaluation (Lephart et al., 2000).

**7. Normal menstrual cycle** 

level at the middle of luteal phas (Berek et al., 2002).

Fig. 2. Menstrual cycle phases (reproduced from Berek, 2002)

bleeding.

removed; actively or passively reproduce the joint angle. Assessment of the efferent pathway is conducted through measurements of balance and muscle activity, which provide a direct determination of the efferent response to afferent stimulation. (Lephart 2000)

#### **3. Hormonal effects on ACL**

Sex hormone fluctuations have been associated with tissue alterations and an increased incidence of non-contact ACL injuries among female athletes (Wojtys et al., 2002). Estrogen and progesterone receptors have been detected within the ACL (Liu et al., 1996). Several researchers have suggested the relationship between estrogen peak levels and increased ACL laxity (Shultz et al., 2004; Slauterbeck et al., 2001). This associated change in tissue tolerance may predispose the ACL to failure at lower tensile loads and/or alter the protective muscle reflex actions associated with ACL tissue receptor stimulation (Raunest et al., 1996).

#### **4. Hormonal effects on tissue**

Estrogen receptors alpha and beta have been reported in skeletal muscle thereby providing a plausible tissue-based mechanism for influencing neuromuscular control and force transmission pathways (Huijing et al 2005; Wilk et al., 2005). In addition, research has not completely described the influence of sex hormone receptors in skeletal muscles on tissue mechanisms that can alter neuromuscular control (Dedrick et al., 2008).

#### **5. Neuromuscular control mechanisms between sexes**

Similar neuromuscular control strategies have been reported between Men and women during landing up until puberty. There is a link between hormonal fluctuations and changes in neuromuscular control, since alterations in hormonal levels constitute a primary change in development after puberty. Neuromuscular control strategies incorporated during a landing sequence appear to change in adult females, where increased knee valgus alignment places the ACL at greater risk of injury (Hewett et al., 2004).

#### **6. Hormonal effects on neuromuscular control**

Neuromuscular control patterns, such as fine motor activity and reaction time performance have been reported to fluctuate over the course of the menstrual cycle (Posthuma et al., 1987), with more consistent performance in women using oral contraceptive (OC). It was discovered that there is an increase in postural sway during single limb stance and threshold for detection of passive knee motion in the mid-luteal phase of the cycle (Friden et al., 2003, 2005). Improved neuromuscular coordination may occur in women taking OC with a reduced number of premenstrual symptoms. However, the relationship between fluctuations in ovarian sex hormone levels and neuromuscular strategies has not been fully described (Dedrick et al., 2006).

Neuromuscular patterns in males and females change substantially during maturation. Males demonstrate improve in power, straight and coordination with age that correlate with their maturational stage, whereas, girls show little change throughout maturation.

For example, in study by kelis et al vertical jump height demonstrated by boys increased steadily during maturation, but in girls it did not (Kellis et al., 1999; Beunen et al., 1988).

Neuromuscular control of the knee can be defined as the unconscious to an afferent signal concerning dynamic knee joint stability. The absents of neuromuscular control of the knee joint may be responsible for the increased rate of knee injury in females, but it is not normally measured with the 3 dimensional kinematic system described below and were recorded during the same laboratory evaluation (Lephart et al., 2000).

Changes in neuromuscular control of the knee in adolescent athletes are documented. Several studies have documented a substantial increase in neuromuscular strength and coordination following the growth spurt in adolescent boys but not in the average adolescent girls.

#### **7. Normal menstrual cycle**

266 Sex Hormones

removed; actively or passively reproduce the joint angle. Assessment of the efferent pathway is conducted through measurements of balance and muscle activity, which provide

Sex hormone fluctuations have been associated with tissue alterations and an increased incidence of non-contact ACL injuries among female athletes (Wojtys et al., 2002). Estrogen and progesterone receptors have been detected within the ACL (Liu et al., 1996). Several researchers have suggested the relationship between estrogen peak levels and increased ACL laxity (Shultz et al., 2004; Slauterbeck et al., 2001). This associated change in tissue tolerance may predispose the ACL to failure at lower tensile loads and/or alter the protective muscle

Estrogen receptors alpha and beta have been reported in skeletal muscle thereby providing a plausible tissue-based mechanism for influencing neuromuscular control and force transmission pathways (Huijing et al 2005; Wilk et al., 2005). In addition, research has not completely described the influence of sex hormone receptors in skeletal muscles on tissue

Similar neuromuscular control strategies have been reported between Men and women during landing up until puberty. There is a link between hormonal fluctuations and changes in neuromuscular control, since alterations in hormonal levels constitute a primary change in development after puberty. Neuromuscular control strategies incorporated during a landing sequence appear to change in adult females, where increased knee valgus alignment

Neuromuscular control patterns, such as fine motor activity and reaction time performance have been reported to fluctuate over the course of the menstrual cycle (Posthuma et al., 1987), with more consistent performance in women using oral contraceptive (OC). It was discovered that there is an increase in postural sway during single limb stance and threshold for detection of passive knee motion in the mid-luteal phase of the cycle (Friden et al., 2003, 2005). Improved neuromuscular coordination may occur in women taking OC with a reduced number of premenstrual symptoms. However, the relationship between fluctuations in ovarian sex hormone levels and neuromuscular strategies has not been fully

Neuromuscular patterns in males and females change substantially during maturation. Males demonstrate improve in power, straight and coordination with age that correlate with

For example, in study by kelis et al vertical jump height demonstrated by boys increased steadily during maturation, but in girls it did not (Kellis et al., 1999; Beunen et al., 1988).

their maturational stage, whereas, girls show little change throughout maturation.

a direct determination of the efferent response to afferent stimulation. (Lephart 2000)

reflex actions associated with ACL tissue receptor stimulation (Raunest et al., 1996).

mechanisms that can alter neuromuscular control (Dedrick et al., 2008).

**5. Neuromuscular control mechanisms between sexes** 

places the ACL at greater risk of injury (Hewett et al., 2004).

**6. Hormonal effects on neuromuscular control** 

**3. Hormonal effects on ACL** 

**4. Hormonal effects on tissue** 

described (Dedrick et al., 2006).

Normal menstrual cycle in women includes fluctuation of sex hormones' levels in a regular duration that usually takes 21-35 days. Ovarian cycle of the normal menstrual cycle has 2 phases, follicular and luteal. Follicular phase is started by menstruation with 2-4 days bleeding.

Estrogen and progesterone levels are in the lowest level at menses but little by little estrogen increases and it goes up to its highest level just before the ovulation, at the middle of the cycle. Duration of the estrogen surge is so limit and it just takes 24 to 48 hours. After ovulation, both estrogen and progesterone levels go up slowly and they are in their highest level at the middle of luteal phas (Berek et al., 2002).

Fig. 2. Menstrual cycle phases (reproduced from Berek, 2002)

Sex Hormones and Neuromuscular Control System 269

The movement capture system comprised of digital photography, skin markers and AutoCAD software was utilized for measuring joint angles during the joint position sense test. A digital video camera was located 185cm away from the subject and elevated 65cm from the ground. Photos were taken during movement and holding moments of the leg, respectively. After completing the procedure, the test and replicated angles were measured

To measure JPS in the knee joint, participants moved their limbs into flexion. Before the tests, each subject watched a video tape showing the squatting movement demonstrated by a trained person performing the movement at a controlled velocity and the subjects were asked to squat as shown in the film. We tried to control velocity approximately, because the JPS accuracy could be influenced by high or low velocity (Stillman et al., 2001). The starting position was knee straightening (0°). The subject stood with eyes closed, and was instructed to: (1) lift the unexamined foot (left limb) from the floor; (2) slowly flex the weight bearing (WB) limb (right limb) until told to stop (~30°); (3) identify the knee position while isometrically holding the test position for about 5 seconds; (4) return to the erect bilateral WB stance (for 7 seconds); and (5) reproduce the previous unilateral flexed position, concentrating on the knee. The holding times used in this study were based on previous studies (Hopper et al., 2003; Marks et al., 1994; Marks et al., 1993). Measurement of knee JPS was repeated three times and

In some previous studies JPS was measured in NWB position that is not functional. For example, Aydog et al (2005) evaluated knee JPS throughout the three different phases of the menstrual cycle but they used Biodex System 3 dynamometer to measure knee JPS in a semi-horizontal position. Their method also stimulated cutaneous mechanoreceptors which

In our study, JPS of the knee joint was evaluated in WB position, as it potentially provides

At the end of each session, venous blood samples (5 ml) were taken from a superficial forearm vein. The blood sample was allowed to clot at room temperature and then the serum was separated and stored to be analyzed. Serum estrogen and progesterone concentrations were measured with Elisa method by Elisa Reader (SLT model) in Endocrine Research Center lab. Data was collected throughout the 3 phases of menstrual cycle i.e. early follicular phase (onset of menses), mid-follicular phase (the 7th to 9th days of the cycle) and mid-luteal phase

Estrogen concentration was found 22.81 (16.75) pg/ml at menses, 125.65 (84.82) at midfollicular and 179.5 (94.35) at mid-luteal phase. Fig. 4 depicts estrogen concentrations across the menstrual cycle phases. Serum estrogen concentration was significantly higher during the mid-follicular and mid-luteal phases as compared with the early-follicular (menses) phase (P=0.0001). There was no significant difference between peak estrogen concentrations

the average of the three measurements were calculated in each phase.

have an important role on the knee JPS that may change the test accuracy.

**9. Joint position sense (JPS) measurement** 

using the AutoCAD software.

more functional information.

(20th to 23th days of the cycle).

in the mid-follicular versus the mid-luteal phase.

**11. Results 11.1 Hormones** 

**10. Blood sampling** 

#### **8. Procedure**

Although, joint proprioception includes some parts, most of the studies have measured JPS as an important part of it. There are different methods for knee JPS evaluation. Some researchers have measured JPS with isokinetic dynamometer- Biodex in non weight bearing position (Aydog et al., 2005; Hertel et al., 2006). In the other studies, JPS was measured by reproduction of the target angle in standing position by using a system comprised of skin markers and digital photography (Stillman BC et al., 2001;Shultz et al., 2003; Shultz et al., 2005; Mir et al., 2008).

In our study the JPS was measured 3 times a month with different level of sex hormones. It was evaluated by reproduction of the target angle (30° flexion) in standing position and absolute angular error (AAE) was considered as a dependent variable.

Testing procedure was completed in an isolated room; the participants were asked to wear loose-fitting shorts. Since previous studies had shown there is no significant difference between the bilateral knee joints' position sense (Herington 2005), we chose the right leg as the tested limb. In all tests, visual cues were eliminated by a blindfold. Each participant was asked to lie down in supine position on the treatment plinth and four circular markers (4 cm in diameter) were attached to their leg at three locations: (1) proximal to a quarter of the distance along a line joining the greater trochanter to the lateral knee joint line, (2) over the fibular neck, (3) over the proximal part of the lateral malleolus. Then each subject asked to sit down (with hip and knee 90° flex), and the fourth marker was attached over the illiotibial tract adjacent to the superior border of the patella. The choice of marker locations was based on previous studies (Deie et al., 2002; Lamoreux et al., 1996). The participant then stood with their feet-shoulder width apart. The left foot was lifted from the floor. The right hand was placed over the chest, and the subject was allowed to use minimum contact of the fingertips in the left hand for balance. One goniometer was adhered onto the wall, out of sight of subject, at angle of 30° as an indicator.

Fig. 3. Measuring the knee joint angle in weight bearing position

Although, joint proprioception includes some parts, most of the studies have measured JPS as an important part of it. There are different methods for knee JPS evaluation. Some researchers have measured JPS with isokinetic dynamometer- Biodex in non weight bearing position (Aydog et al., 2005; Hertel et al., 2006). In the other studies, JPS was measured by reproduction of the target angle in standing position by using a system comprised of skin markers and digital photography (Stillman BC et al., 2001;Shultz et al., 2003; Shultz et al.,

In our study the JPS was measured 3 times a month with different level of sex hormones. It was evaluated by reproduction of the target angle (30° flexion) in standing position and

Testing procedure was completed in an isolated room; the participants were asked to wear loose-fitting shorts. Since previous studies had shown there is no significant difference between the bilateral knee joints' position sense (Herington 2005), we chose the right leg as the tested limb. In all tests, visual cues were eliminated by a blindfold. Each participant was asked to lie down in supine position on the treatment plinth and four circular markers (4 cm in diameter) were attached to their leg at three locations: (1) proximal to a quarter of the distance along a line joining the greater trochanter to the lateral knee joint line, (2) over the fibular neck, (3) over the proximal part of the lateral malleolus. Then each subject asked to sit down (with hip and knee 90° flex), and the fourth marker was attached over the illiotibial tract adjacent to the superior border of the patella. The choice of marker locations was based on previous studies (Deie et al., 2002; Lamoreux et al., 1996). The participant then stood with their feet-shoulder width apart. The left foot was lifted from the floor. The right hand was placed over the chest, and the subject was allowed to use minimum contact of the fingertips in the left hand for balance. One goniometer was adhered onto the wall, out of sight of

absolute angular error (AAE) was considered as a dependent variable.

Fig. 3. Measuring the knee joint angle in weight bearing position

**8. Procedure** 

2005; Mir et al., 2008).

subject, at angle of 30° as an indicator.

#### **9. Joint position sense (JPS) measurement**

The movement capture system comprised of digital photography, skin markers and AutoCAD software was utilized for measuring joint angles during the joint position sense test. A digital video camera was located 185cm away from the subject and elevated 65cm from the ground. Photos were taken during movement and holding moments of the leg, respectively. After completing the procedure, the test and replicated angles were measured using the AutoCAD software.

To measure JPS in the knee joint, participants moved their limbs into flexion. Before the tests, each subject watched a video tape showing the squatting movement demonstrated by a trained person performing the movement at a controlled velocity and the subjects were asked to squat as shown in the film. We tried to control velocity approximately, because the JPS accuracy could be influenced by high or low velocity (Stillman et al., 2001). The starting position was knee straightening (0°). The subject stood with eyes closed, and was instructed to: (1) lift the unexamined foot (left limb) from the floor; (2) slowly flex the weight bearing (WB) limb (right limb) until told to stop (~30°); (3) identify the knee position while isometrically holding the test position for about 5 seconds; (4) return to the erect bilateral WB stance (for 7 seconds); and (5) reproduce the previous unilateral flexed position, concentrating on the knee. The holding times used in this study were based on previous studies (Hopper et al., 2003; Marks et al., 1994; Marks et al., 1993). Measurement of knee JPS was repeated three times and the average of the three measurements were calculated in each phase.

In some previous studies JPS was measured in NWB position that is not functional. For example, Aydog et al (2005) evaluated knee JPS throughout the three different phases of the menstrual cycle but they used Biodex System 3 dynamometer to measure knee JPS in a semi-horizontal position. Their method also stimulated cutaneous mechanoreceptors which have an important role on the knee JPS that may change the test accuracy.

In our study, JPS of the knee joint was evaluated in WB position, as it potentially provides more functional information.

#### **10. Blood sampling**

At the end of each session, venous blood samples (5 ml) were taken from a superficial forearm vein. The blood sample was allowed to clot at room temperature and then the serum was separated and stored to be analyzed. Serum estrogen and progesterone concentrations were measured with Elisa method by Elisa Reader (SLT model) in Endocrine Research Center lab. Data was collected throughout the 3 phases of menstrual cycle i.e. early follicular phase

(onset of menses), mid-follicular phase (the 7th to 9th days of the cycle) and mid-luteal phase (20th to 23th days of the cycle).

#### **11. Results**

#### **11.1 Hormones**

Estrogen concentration was found 22.81 (16.75) pg/ml at menses, 125.65 (84.82) at midfollicular and 179.5 (94.35) at mid-luteal phase. Fig. 4 depicts estrogen concentrations across the menstrual cycle phases. Serum estrogen concentration was significantly higher during the mid-follicular and mid-luteal phases as compared with the early-follicular (menses) phase (P=0.0001). There was no significant difference between peak estrogen concentrations in the mid-follicular versus the mid-luteal phase.

Sex Hormones and Neuromuscular Control System 271

Pearson correlation test showed a negative correlation between AAE and estrogen level with no significant relationship (r= -0.275, p= .058). But, spearman correlation test indicated a negative correlation between AAE and progesterone level with a significant relationship (r= -0.370, p= 0.010). Therefore, the highest level of JPS error was related to the lowest level of estrogen and progesterone. Also, the lowest level of JPS error was related to the highest level of sex hormones. While hormones' levels increase, the knee JPS error decreases. Because of the significant relationship between progesterone and AAE, it seems this

estrogen+AAE

estrogen+AAE

progesterone+ AAE

0246 **AAE**

0246 **AAE**

Fig. 7. The linear correlation between progesterone and absolute angular error

progesterone+AAE

Fig. 6. The linear correlation between estrogen and absolute angular error

 menses Mid-follicular Mid-luteal AAE means 4.18 (2.13) 3.65 (2.78) 2.51 (1.66)

Table 1. The value of absolute angular error across the menstrual cycle

hormone has the main effect on the knee JPS accuracy.

**PROGESTERONE**

**ESTROGEN**

Fig. 4. Estrogen concentrations across three phases of the menstrual cycle

Serum progesterone concentration was found 0.58 (0.62) at menses, 0.51(0.71) at midfollicular and 7.35(5.87) at mid-luteal phase. These results show that serum progesterone concentration was significantly higher during the mid-luteal phase as compared with the menses and mid-follicular phases (P=0.0001). However there was no significant difference between peak progesterone concentrations in the menses versus mid-follicular phase. Fig. 5 depicts progesterone concentrations across the menstrual cycle phases.

Fig. 5. Progestrone concentrations across the menstrual cycle

#### **12. Joint Position Sense (absolute error of repositioning)**

We found females' knee JPS accuracy changes in different phases of menstrual cycle. The greatest amount of mean (SD) value of absolute error was at menses and the least amount of it, was at mid-luteal phase. This finding is also in agreement with previous study that indicated the active knee JPS was significantly reduced during menstruation (Aydog et al., 2005).

Fig. 4. Estrogen concentrations across three phases of the menstrual cycle

depicts progesterone concentrations across the menstrual cycle phases.

Fig. 5. Progestrone concentrations across the menstrual cycle

2005).

**12. Joint Position Sense (absolute error of repositioning)** 

We found females' knee JPS accuracy changes in different phases of menstrual cycle. The greatest amount of mean (SD) value of absolute error was at menses and the least amount of it, was at mid-luteal phase. This finding is also in agreement with previous study that indicated the active knee JPS was significantly reduced during menstruation (Aydog et al.,

Serum progesterone concentration was found 0.58 (0.62) at menses, 0.51(0.71) at midfollicular and 7.35(5.87) at mid-luteal phase. These results show that serum progesterone concentration was significantly higher during the mid-luteal phase as compared with the menses and mid-follicular phases (P=0.0001). However there was no significant difference between peak progesterone concentrations in the menses versus mid-follicular phase. Fig. 5


Table 1. The value of absolute angular error across the menstrual cycle

Pearson correlation test showed a negative correlation between AAE and estrogen level with no significant relationship (r= -0.275, p= .058). But, spearman correlation test indicated a negative correlation between AAE and progesterone level with a significant relationship (r= -0.370, p= 0.010). Therefore, the highest level of JPS error was related to the lowest level of estrogen and progesterone. Also, the lowest level of JPS error was related to the highest level of sex hormones. While hormones' levels increase, the knee JPS error decreases. Because of the significant relationship between progesterone and AAE, it seems this hormone has the main effect on the knee JPS accuracy.

Fig. 6. The linear correlation between estrogen and absolute angular error

Fig. 7. The linear correlation between progesterone and absolute angular error

Sex Hormones and Neuromuscular Control System 273

However, some other researchers didn't find the highest level of laxity in luteal phase, and suggested antagonistic role for progesterone and estrogen. But they found no significant

Therefore, according to progesterone effect on knee laxity in the mentioned studies and our finding about its influence on JPS, we suggest progesterone and estrogen are synergies in knee JPS accuracy. Hence we suppose that elevation of both these hormones in luteal phase is the reason of knee joint absolute angular error decreasing and knee JPS accuracy

The findings of our study are strengthened by controlling many confounding variables such as age, menstrual regularity, previous injuries and length of daytime because of its influence on females' sex hormones (Speroff et al., 2005). We collected all our sampling in one season with almost the same day length during the study. Moreover, we started our study with every participant being in their follicular phase (the menses) and we used the same sequence for all the participants. None of our subjects wasn't pregnant and didn't use oral contraceptive in their 3 recent months because of its effects on their knee joint performance (Lebrun et al., 1994) and it could influence on their postural control (Ekenros et al., 2010). We used skin marker to reduce the skin stimulation and we also used AutoCAD software to measure the knee angles as it has high test-retest reliability. Therefore our JPS test can be considered highly accurate. In the current study, JPS of the knee joint was evaluated in WB

position which potentially provides more functional information (Baker et al., 2002).

It is concluded that sex hormones, especially progesterone can influence on accuracy of the knee JPS in healthy female athletes. Their reduction at menstruation can reduce the knee JPS accuracy and increase the knee joint injury probability. Knee JPS accuracy decreases in menses, when circulating sex- hormones levels are low and after a time when both were

As such, Female athletes are at risk of ACL tearing during menstruation. Further studies are needed to investigate the effect of some devices or some ways to protect the knee joint

[1] Adachi N, Ochi M, Uchio Y et al (2002) Mechanoreceptors in the anterior cruciate ligament contribute to the joint position sense. Acta Orthop Scand 73:330-334 [2] Arendt EA, Agel J, Dick RW(1999) Anterior cruciate ligament injury patterns among

[3] Arendt E, Dick R (1999) Knee injury patterns among men and women in collegiate

[4] Aydog ST, Hascelik Z, Demirel HA, Tetik O, Aydog E, Doral MN (2005). The effects of

[5] Baker V, Bannel K, Stillman B, et al (2002) Abnormal knee joint position sense in individuals with patello femoral pain syndrome. J Orthop Res 20:208-14

basketball and soccer. NCAA data and review of literature. Am J Sports Med

menstrual cycle on the knee joint position sense:preliminary study. Knee Surg

collegiate men and women. J Athl Train 34:86-92

Sports Troumatol Arthrosc 13:649-653.

difference of laxity between mid-follicular and luteal phases (Park et al., 2007).

increasing.

**14. Conclusion** 

during this high risk time.

23,694-701

**15. References** 

elevated.

#### **13. Discussion**

Previous authors report different results regarding changes in knee joint proprioception across the menstrual cycle. The main observation of our study was that higher value of reposition error was found in the menses and the participants made less error in reproduction of joint position sense in two other phases and they were more accurate during the luteal phase. While sex hormones' levels increase, the knee JPS error decreases. The significant relationship was between progesterone and absolute angular error and it seems this hormone has the main effect on the knee JPS accuracy.

These results are due to measurable effects of estrogen on muscle, tendon and ligament strength and function (Sarwer et al., 1996). Estrogen also has influence on the central nervous system and females have different skill performances during different phases of the menstrual cycle (Lebrun et al., 1994). Some researchers demonstrated a decrease in motor skills around the menstruation and this showed that estrogen may influence on neuromuscular function (Posthuma et al., 1987). It is indicated that ACL injuries happened most frequently on day 1 and 2 of menses and it isn't random but occurs usually around the time of menses, when circulating sex- hormones levels decrease and after both estrogen and progesterone elevation (Slauterbeck et al., 2002). In addition, it is believed that proprioception can be influenced by emotional and environmental conditions and because of females' behavioral and emotional character changing in early menstruation increasing in error of knee JPS in menses can be described. Aydog et al (2005) also indicated the active knee JPS was significantly reduced in the menstruation compared to follicular and early luteal phases. They believed that changes in proprioception might be a consequence of changes in distal latency or excitability of the mechanoreceptors.

On the other hand, it is observed that knee laxity exists more in mid-follicular and luteal phases of the females' menstrual cycle. Females with increased knee laxity are less sensitive to joint displacement or loading, and are more reliant on active control of the gastrocnemius and biceps femoris muscles to potentially compensate for reduced passive instability (Shultz et al., 2004; Park et al., 2007).

In another study, some researchers demonstrated increased knee laxity was observed during ovulation (after estrogen serge) but no significant changes in knee mechanics corresponding to menstrual phases were found. They also found knee laxity correlates positively with knee joint loads. They suggested that increased knee joint laxity during menstrual cycle leads to greater knee joint loads in selected high risk movements in healthy young females (Park et al., 2009).

Although, some researchers said knee joint laxity may not explain the higher incidence of females' ACL injury, they suggested that muscle strength and dynamic stability are more important (Bowerman et al., 2006). Some others measured knee JPS with isokinetic dynamometer in sitting position with high skin stimulations and suggested passive joint position sense and joint laxity don't change across the menstrual cycle (Hertel et al., 2006). It is also indicated that ligament laxity does not affect the proprioceptive function of the knee, and it may compensate with muscle contraction (Adachi et al., 2002). These findings are consistent with Johanson's Final Common Input Theory. Based on this theory, due to the joint-tendon-muscle relationship, muscle spindles act with joint afferent information and then send final common signal. As the highest knee joint laxity has been observed in the luteal phase, in the current study the most JPS accuracy can be described.

However, some other researchers didn't find the highest level of laxity in luteal phase, and suggested antagonistic role for progesterone and estrogen. But they found no significant difference of laxity between mid-follicular and luteal phases (Park et al., 2007).

Therefore, according to progesterone effect on knee laxity in the mentioned studies and our finding about its influence on JPS, we suggest progesterone and estrogen are synergies in knee JPS accuracy. Hence we suppose that elevation of both these hormones in luteal phase is the reason of knee joint absolute angular error decreasing and knee JPS accuracy increasing.

The findings of our study are strengthened by controlling many confounding variables such as age, menstrual regularity, previous injuries and length of daytime because of its influence on females' sex hormones (Speroff et al., 2005). We collected all our sampling in one season with almost the same day length during the study. Moreover, we started our study with every participant being in their follicular phase (the menses) and we used the same sequence for all the participants. None of our subjects wasn't pregnant and didn't use oral contraceptive in their 3 recent months because of its effects on their knee joint performance (Lebrun et al., 1994) and it could influence on their postural control (Ekenros et al., 2010). We used skin marker to reduce the skin stimulation and we also used AutoCAD software to measure the knee angles as it has high test-retest reliability. Therefore our JPS test can be considered highly accurate. In the current study, JPS of the knee joint was evaluated in WB position which potentially provides more functional information (Baker et al., 2002).

#### **14. Conclusion**

272 Sex Hormones

Previous authors report different results regarding changes in knee joint proprioception across the menstrual cycle. The main observation of our study was that higher value of reposition error was found in the menses and the participants made less error in reproduction of joint position sense in two other phases and they were more accurate during the luteal phase. While sex hormones' levels increase, the knee JPS error decreases. The significant relationship was between progesterone and absolute angular error and it seems

These results are due to measurable effects of estrogen on muscle, tendon and ligament strength and function (Sarwer et al., 1996). Estrogen also has influence on the central nervous system and females have different skill performances during different phases of the menstrual cycle (Lebrun et al., 1994). Some researchers demonstrated a decrease in motor skills around the menstruation and this showed that estrogen may influence on neuromuscular function (Posthuma et al., 1987). It is indicated that ACL injuries happened most frequently on day 1 and 2 of menses and it isn't random but occurs usually around the time of menses, when circulating sex- hormones levels decrease and after both estrogen and progesterone elevation (Slauterbeck et al., 2002). In addition, it is believed that proprioception can be influenced by emotional and environmental conditions and because of females' behavioral and emotional character changing in early menstruation increasing in error of knee JPS in menses can be described. Aydog et al (2005) also indicated the active knee JPS was significantly reduced in the menstruation compared to follicular and early luteal phases. They believed that changes in proprioception might be a consequence of

On the other hand, it is observed that knee laxity exists more in mid-follicular and luteal phases of the females' menstrual cycle. Females with increased knee laxity are less sensitive to joint displacement or loading, and are more reliant on active control of the gastrocnemius and biceps femoris muscles to potentially compensate for reduced passive instability (Shultz

In another study, some researchers demonstrated increased knee laxity was observed during ovulation (after estrogen serge) but no significant changes in knee mechanics corresponding to menstrual phases were found. They also found knee laxity correlates positively with knee joint loads. They suggested that increased knee joint laxity during menstrual cycle leads to greater knee joint loads in selected high risk movements in healthy

Although, some researchers said knee joint laxity may not explain the higher incidence of females' ACL injury, they suggested that muscle strength and dynamic stability are more important (Bowerman et al., 2006). Some others measured knee JPS with isokinetic dynamometer in sitting position with high skin stimulations and suggested passive joint position sense and joint laxity don't change across the menstrual cycle (Hertel et al., 2006). It is also indicated that ligament laxity does not affect the proprioceptive function of the knee, and it may compensate with muscle contraction (Adachi et al., 2002). These findings are consistent with Johanson's Final Common Input Theory. Based on this theory, due to the joint-tendon-muscle relationship, muscle spindles act with joint afferent information and then send final common signal. As the highest knee joint laxity has been observed in the

luteal phase, in the current study the most JPS accuracy can be described.

this hormone has the main effect on the knee JPS accuracy.

changes in distal latency or excitability of the mechanoreceptors.

et al., 2004; Park et al., 2007).

young females (Park et al., 2009).

**13. Discussion** 

It is concluded that sex hormones, especially progesterone can influence on accuracy of the knee JPS in healthy female athletes. Their reduction at menstruation can reduce the knee JPS accuracy and increase the knee joint injury probability. Knee JPS accuracy decreases in menses, when circulating sex- hormones levels are low and after a time when both were elevated.

As such, Female athletes are at risk of ACL tearing during menstruation. Further studies are needed to investigate the effect of some devices or some ways to protect the knee joint during this high risk time.

#### **15. References**


Sex Hormones and Neuromuscular Control System 275

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[28] Lebrun CM (1994) The effect of the phase of the menstrual cycle and the birth control

[29] Lee DY, Chai YG, Lee EB, Kim KW et al (2002). Beta-estradiol inhibits high-voltage-

[30] Lephart SM, Pincivero DM, Giraldo Jl et al (1997) The role of proprioception in the management and rehabilitation of athletic injuries. Am J Sports Med 25:130-7 [31] Lephart SM, Pincivero DM, Rozzi SL (1998) . Proprioception of the ankle and knee.

[32] Liu S H, Al-Shaikh RA, Panossian V et al (1996) Primary immunolocalization of

[33] Marks R (1994) The reliability of knee position sense measurements in healthy women.

[34] Marks R, Quinney HA, Wessel J (1993) Proprioceptive sensibility in women with

[35] Mir SM, Hadian MR, Talebian S, Naseri N (2008) Functional assessment of knee joint

[36] Moller-Neilsen J, Hammar M (1989) Women's soccer injury in relation to the menstrual

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[39] Park SK, Stefanyshyn DJ, Hart DA et al (2007) Influence of hormones on knee joint

[40] Park SK, Stefanyshyn DJ, Ramage B (2009) Relationship between knee joint laxity and knee joint mechanics during the menstrual cycle. Br J Sports Med 43:174-179. [41] Park SK, Stefanyshyn DJ, Ramage B, Hart DA, Ronsky JL (2009) Alterations in knee

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7

Orthop 23, 573-578


**13** 

*Brazil* 

**Acute and Chronic Testosterone** 

*Exercise Laboratory Research, Physical Education School,* 

*Federal University of Rio Grande do Sul* 

Eduardo Lusa Cadore and Luiz Fernando Martins Kruel

**Responses to Physical Exercise and Training** 

High-intensity physical training is a powerful stimulus to acute increases in blood steroid hormone levels (Ahtiainen et al., 2005; Cadore et al., 2008a, 2008b, 2009a; Häkkinen & Pakarinen, 1994a, 1995; Staron et al., 1994). Moreover, strength training (ST) has been shown to stimulate greater increases in testosterone levels when compared to aerobic training (Copeland, et al., 2002; Tremblay et al., 2003), which can be explained by the powerful influence of the anaerobic glycolytic pathway in stimulating acute hormonal increases in response to exercise (Kraemer & Ratamess, 2005). This stimulus features control mechanisms independent from luteinising hormone (LH) stimulation (Fahrner & Hackney, 1998; Lu et al., 1997), and some factors related to the training session are directly associated with this response (Cadore et al., 2008c; Häkkinen et al., 1988; Häkkinen & Pakarinen, 1995,

Despite the well-known acute hormonal response to physical exercise (Kraemer et al., 1990; Hansen et al., 2001; Cadore et al., 2009c), data on resting concentrations remain controversial. Some studies demonstrate increased resting testosterone levels following ST (Ahtiainen et al., 2003; Häkkinen et al., 1998; Izquierdo et al., 2006; Kraemer et al., 1993; Kraemer et al., 1999; Marx et al., 2001; Staron et al., 1994), leading authors to suggest this type of training as a form of intervention for maintaining testosterone levels during ageing (Kraemer et al., 1999). However, increases in resting testosterone levels were not observed in middle-aged (Cadore et al. 2008a) or elderly people (Häkkinen et al., 2000, 2001a; Kraemer et al., 1999). Chronic adaptations to ST apparently occur at the level of cellular androgen receptors (ARs), given that ARs present on muscle cells seem to increase in number in response to this type of training (Inoue et al., 1994; Willoughby & Taylor, 2004), and this adaptation may result in improved

Conversely, reductions in testosterone levels associated with increases in cortisol levels have been observed in response to aerobic training in athletes subjected to high-volume training (Maïmoun et al., 2003). These alterations may be associated with the overtraining process and consequent suppression of the hypothalamic-pituitary-gonadal and adrenocortical axis (Bell et al., 2001; Hu et al., 1999; Kraemer et al., 1995). Nevertheless, reductions in testosterone levels may occur without overtraining. This alteration may be transient, reflecting the variation in volume and training intensity, and it may be explained by changes

hormone-receptor interaction (Bamman et al., 2001; Willoughby & Taylor, 2004).

in plasma volume (Hu et al., 1999; Kraemer & Ratamess, 2005; Maïmoun et al., 2003).

**1. Introduction** 

Kraemer et al., 1993; Smilios et al., 2003, 2006).


### **Acute and Chronic Testosterone Responses to Physical Exercise and Training**

Eduardo Lusa Cadore and Luiz Fernando Martins Kruel *Exercise Laboratory Research, Physical Education School, Federal University of Rio Grande do Sul Brazil* 

#### **1. Introduction**

276 Sex Hormones

[43] Prentice WE (2011) Rehabilitation techniques: For sports medicine and athletic training. 5th edn. The McGraw-Hill companies, New York, pp: 122-139. [44] Raunest J, Sager M, Burgener E (1996) Proprioceptive mechanisms in the cruciate

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[48] Sarwer R, Beltran NB, Rutherford OM (1996) Changes in muscle strength, relaxation rate and fatiguability during the human menstrual cycle. J Physiol 493:267-272 [49] Sciore P, Smith S, Frank CB (1997) Detection of receptors for estrogen and

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[52] Shultz SJ, Sander TC, Kirk SE et al (2005) Sex differences in knee joint laxity change across the female menstrual cycle. J Sports Med Phys Fitness 45:594-603 [53] Shultz SJ, Sander TC, Johnson KM et al (2004) Relationship between sex hormones and anterior knee laxity across the menstrual cycle. Med Sci Sports Exerc 36:1165-1174 [54] Speroff L, Fritz MA 2005 Clinical gynecologic endocrinology and infertility. Lippincott W illiams E Wilkins. 7th ed., A wolters kluwer company. pp:187-225 [55] Stillman BC, McMeeken JM (2001) The role of weight bearing in the clinical assessment

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[57] Wilk A, Gustafsson T, Esbjornsson M et al (2005) Expression of oestrogen receptor α

[58] Wojtys EM, Huston LJ, Lindenfeld TN et al (1998) Association between the menstrual

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anterior cruciate ligament injury. J Athl Train 37:275-280

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27:312-319

26:614-9

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Trans Orthop Res Soc 22:51

ligaments: and electromugraphic study on reflex activity in the thigh muscles. J

characteristics of male and female soccer and basketball players. Am J Sports Med

progesterone in human ligaments and rabbit ligaments and tendons by RT-PCR.

High-intensity physical training is a powerful stimulus to acute increases in blood steroid hormone levels (Ahtiainen et al., 2005; Cadore et al., 2008a, 2008b, 2009a; Häkkinen & Pakarinen, 1994a, 1995; Staron et al., 1994). Moreover, strength training (ST) has been shown to stimulate greater increases in testosterone levels when compared to aerobic training (Copeland, et al., 2002; Tremblay et al., 2003), which can be explained by the powerful influence of the anaerobic glycolytic pathway in stimulating acute hormonal increases in response to exercise (Kraemer & Ratamess, 2005). This stimulus features control mechanisms independent from luteinising hormone (LH) stimulation (Fahrner & Hackney, 1998; Lu et al., 1997), and some factors related to the training session are directly associated with this response (Cadore et al., 2008c; Häkkinen et al., 1988; Häkkinen & Pakarinen, 1995, Kraemer et al., 1993; Smilios et al., 2003, 2006).

Despite the well-known acute hormonal response to physical exercise (Kraemer et al., 1990; Hansen et al., 2001; Cadore et al., 2009c), data on resting concentrations remain controversial. Some studies demonstrate increased resting testosterone levels following ST (Ahtiainen et al., 2003; Häkkinen et al., 1998; Izquierdo et al., 2006; Kraemer et al., 1993; Kraemer et al., 1999; Marx et al., 2001; Staron et al., 1994), leading authors to suggest this type of training as a form of intervention for maintaining testosterone levels during ageing (Kraemer et al., 1999). However, increases in resting testosterone levels were not observed in middle-aged (Cadore et al. 2008a) or elderly people (Häkkinen et al., 2000, 2001a; Kraemer et al., 1999). Chronic adaptations to ST apparently occur at the level of cellular androgen receptors (ARs), given that ARs present on muscle cells seem to increase in number in response to this type of training (Inoue et al., 1994; Willoughby & Taylor, 2004), and this adaptation may result in improved hormone-receptor interaction (Bamman et al., 2001; Willoughby & Taylor, 2004).

Conversely, reductions in testosterone levels associated with increases in cortisol levels have been observed in response to aerobic training in athletes subjected to high-volume training (Maïmoun et al., 2003). These alterations may be associated with the overtraining process and consequent suppression of the hypothalamic-pituitary-gonadal and adrenocortical axis (Bell et al., 2001; Hu et al., 1999; Kraemer et al., 1995). Nevertheless, reductions in testosterone levels may occur without overtraining. This alteration may be transient, reflecting the variation in volume and training intensity, and it may be explained by changes in plasma volume (Hu et al., 1999; Kraemer & Ratamess, 2005; Maïmoun et al., 2003).

Acute and Chronic Testosterone Responses to Physical Exercise and Training 279

0.77 and r = 0.84, P < 0.05). In an investigation of the relationship between the endocrine system and strength production, Häkkinen & Pakarinen (1993a) observed a correlation between maximum strength and testosterone levels as well as testosterone:SHBG ratio (r = 0.62 and 0.68, respectively, P < 0.01). A study by Izquierdo et al. (2001) revealed that individuals with greater increases in isometric strength following ST also showed higher total (r = 0.78, P < 0.01) and free (r = 0.71, P < 0.05) testosterone levels. The same trend was observed in data obtained by Ahtiainen et al. (2003) when evaluating highly trained men subjected to a 21-week ST program, where positive correlations were observed between the changes in isometric strength and total testosterone (r = 0.84, P < 0.01), the testosterone:cortisol ratio (r = 0.88, P < 0.01) and the isometric strength development and free testosterone (pre-training values: r = 0.78, P < 0.05 and post-training values: r = 0.82, P < 0.05). A study conducted in our laboratory by Cadore et al. (2008a) showed significant correlations between testosterone:SHBG ratios and DHEA concentrations as well as strength production in bench press, leg press and squat exercises (r = 0.55 to 0.82, P < 0.05 to P < 0.001) in trained and untrained middle-aged men. In another study, Cadore et al. (2010) showed significant correlations between increases in the strength of knee extensors and average basal total testosterone levels throughout the training period (3 measurements in 12 weeks of training) (r = 0.94, P < 0.01) and the average total testosterone:cortisol ratio (r = 0.93, P < 0.01). Table 1 shows the results obtained from studies where correlations between hormonal parameters and variables related to muscle strength were identified. One aspect that must be emphasised is that other structural factors, such as pennation angle and fibre type composition, may interfere with strength production (Ramos et al., 1998), just as the volume and intensity of ST largely influence the increase in strength

**Author Strength performance vs. sex hormonal parameters** 

MVC after ST with FT post training (r=0.82, p<0.05)

Cadore et al., 2010 <sup>↑</sup> knee extension 1 RM values after ST with TT and TT:COR ratio (r=0.94

<sup>2001</sup>↑ MVC after ST with TT and FT (r = 0.78 and 0.71, respectively, p<0.01).

Table 1. Relatioship between sex hormonal parameters and strength performance. TT: total testosterone; FT: free testosterone; COR: cortisol; DHEA, dehidroepiandrosterone; SHBG: sex hormone binding globuline; ↑: increases; MVC: maximal voluntary contraction (maximal isometric strength); and, 1 RM: one-maximum repetition (maximal dynamic strength); ST:

Pakarinen, 1993a TT and TT:SHBG with MVC and RFD (r = 0.66 to 0.69, p<0.01)

and 0.93, respectively, p<0.01)

↑ MVC after ST with TT and TT:COR ratio (r=0.84 and 0.88, P<0.01), and

Squat 1 RM values with TT:SHBG and DHEA before (r=0.71 and 0.65, respectively) and after ST bout (r=0.76 and 0.82, respectively) (p<0.01 to 0.05).

↑ MVC after ST with TT and TT:COR (r=0.57 and 0.61, respectively,

average of TT:SHBG ratio and ↑ RFD after ST (r=0.84, p<0.05)

Annual average of TT:COR ratio and MVC (r=0.77, p<0.05); and, annual

↑ 1 RM values after ST with FT and TT (r=0.55 and 0.43, respectively,

resulting from training (Marx et al., 2001).

p<0.05)

p<0.05)

Ahtianinen *et al*.,

Cadore *et al*., 2008a

Häkkinen &

Häkkinen & Pakarinen, 1993b

1988

2000

Häkkinen *et al*.,

Häkkinen *et al*.,

Izquierdo *et al*.,

strength training.

2003

It has been suggested that the neuromuscular adaptations observed during ST (Häkkinen et al., 2000, 2001b; Tsolakiis et al., 2004) are partly mediated by acute responses to circulating testosterone levels resulting from the training session (Kraemer & Ratamess, 2005) as well as modifications in the cellular receptors present on muscle cells (Ahtiainen et al., 2011; Kadi et al., 2000). Besides the known effects exerted by these hormones on muscle metabolism (Bhasin et al., 2001), the magnitude of increase in muscular strength in individuals subjected to strength training has been associated with testosterone concentration and other hormonerelated parameters (i.e., the testosterone:cortisol ratio and testosterone:sex hormone-binding globulin (SHBG)) (Cadore et al., 2010; Häkkinen et al., 1988, 2000; Häkkinen & Pakarinen, 1993a, 1993b; Izquierdo et al., 2001). The figure 1 shows a Schematic diagram of the mechanism of training adaptations: anabolic process as adaptation to strength training and chronic catabolic process resulting from excessive volume of both strength and aerobic training.

Due to the possible importance of acute hormone responses, as well as chronic adaptation of androgen receptors due to strength training, it may be important to determine which aspects of training influence these responses to establish an optimum anabolic environment during a training session or period. Therefore, the objective of this chapter is to review existing data on the influence of testosterone levels on physical training and to determine which factors related to the training session are associated with the acute and chronic hormone responses the endocrine system to exercise.

#### **2. Testosterone and strength trainability**

Testosterone is a powerful stimulator of protein synthesis, specifically in the context of muscle metabolism (Griggs et al., 1989). Its effects are exerted through the interaction between the hormone and its specific receptor located on the muscle cell (Bamman et al., 2001). The main mechanism through which testosterone induces protein synthesis is the activation and induction of the proliferation of satellite cells, which subsequently incorporate into muscle fibres, resulting in an increase in myonuclear number (Kadi et al., 2000). Moreover, this hormone is capable of influencing strength production by stimulating the transition of type II fibres to a more glycolytic profile (Ramos et al., 1998), increasing the secretion of insulin-like growth factor I (IGF-I), mediated by its influence on the amplitude of growth hormone (GH) pulses (Bross et al., 1999) as well as its influence on the production of neurotransmitters that are important for muscle contraction (Kraemer et al., 1999).

Several studies demonstrate that among individuals subjected to the same volume and intensity of ST, those presenting higher testosterone levels achieve greater muscular strength and/or power following training (Ahtiainen et al., 2003; Cadore et al., 2010; Häkkinen et al., 1988; Häkkinen & Pakarinen, 1993a). This suggests that the trainability of individuals is related to testosterone and hormonal parameters associated with this hormone, such as the testosterone:sex hormone-binding globulin (SHBG) ratio and the testosterone:cortisol ratio, as demonstrated by results obtained in published studies (Ahtiainen et al., 2003; Cadore et al., 2010; Häkkinen et al., 1988; Häkkinen & Pakarinen, 1993a). Furthermore, in transversal studies that investigated middle-aged and elderly individuals, strength production was correlated to serum testosterone levels (Häkkinen & Pakarinen, 1993a; Cadore et al., 2008a).

Häkkinen et al. (1988) investigated male weightlifting athletes (22.3 ± 2.1 years) and observed a correlation between testosterone:cortisol and testosterone:SHBG ratios and the variations in maximum strength and rate of force development (RFD), respectively, as a result of ST (r =

It has been suggested that the neuromuscular adaptations observed during ST (Häkkinen et al., 2000, 2001b; Tsolakiis et al., 2004) are partly mediated by acute responses to circulating testosterone levels resulting from the training session (Kraemer & Ratamess, 2005) as well as modifications in the cellular receptors present on muscle cells (Ahtiainen et al., 2011; Kadi et al., 2000). Besides the known effects exerted by these hormones on muscle metabolism (Bhasin et al., 2001), the magnitude of increase in muscular strength in individuals subjected to strength training has been associated with testosterone concentration and other hormonerelated parameters (i.e., the testosterone:cortisol ratio and testosterone:sex hormone-binding globulin (SHBG)) (Cadore et al., 2010; Häkkinen et al., 1988, 2000; Häkkinen & Pakarinen, 1993a, 1993b; Izquierdo et al., 2001). The figure 1 shows a Schematic diagram of the mechanism of training adaptations: anabolic process as adaptation to strength training and chronic catabolic process resulting from excessive volume of both strength and aerobic training. Due to the possible importance of acute hormone responses, as well as chronic adaptation of androgen receptors due to strength training, it may be important to determine which aspects of training influence these responses to establish an optimum anabolic environment during a training session or period. Therefore, the objective of this chapter is to review existing data on the influence of testosterone levels on physical training and to determine which factors related to the training session are associated with the acute and chronic

Testosterone is a powerful stimulator of protein synthesis, specifically in the context of muscle metabolism (Griggs et al., 1989). Its effects are exerted through the interaction between the hormone and its specific receptor located on the muscle cell (Bamman et al., 2001). The main mechanism through which testosterone induces protein synthesis is the activation and induction of the proliferation of satellite cells, which subsequently incorporate into muscle fibres, resulting in an increase in myonuclear number (Kadi et al., 2000). Moreover, this hormone is capable of influencing strength production by stimulating the transition of type II fibres to a more glycolytic profile (Ramos et al., 1998), increasing the secretion of insulin-like growth factor I (IGF-I), mediated by its influence on the amplitude of growth hormone (GH) pulses (Bross et al., 1999) as well as its influence on the production

of neurotransmitters that are important for muscle contraction (Kraemer et al., 1999).

Several studies demonstrate that among individuals subjected to the same volume and intensity of ST, those presenting higher testosterone levels achieve greater muscular strength and/or power following training (Ahtiainen et al., 2003; Cadore et al., 2010; Häkkinen et al., 1988; Häkkinen & Pakarinen, 1993a). This suggests that the trainability of individuals is related to testosterone and hormonal parameters associated with this hormone, such as the testosterone:sex hormone-binding globulin (SHBG) ratio and the testosterone:cortisol ratio, as demonstrated by results obtained in published studies (Ahtiainen et al., 2003; Cadore et al., 2010; Häkkinen et al., 1988; Häkkinen & Pakarinen, 1993a). Furthermore, in transversal studies that investigated middle-aged and elderly individuals, strength production was correlated to serum testosterone levels (Häkkinen &

Häkkinen et al. (1988) investigated male weightlifting athletes (22.3 ± 2.1 years) and observed a correlation between testosterone:cortisol and testosterone:SHBG ratios and the variations in maximum strength and rate of force development (RFD), respectively, as a result of ST (r =

hormone responses the endocrine system to exercise.

**2. Testosterone and strength trainability** 

Pakarinen, 1993a; Cadore et al., 2008a).

0.77 and r = 0.84, P < 0.05). In an investigation of the relationship between the endocrine system and strength production, Häkkinen & Pakarinen (1993a) observed a correlation between maximum strength and testosterone levels as well as testosterone:SHBG ratio (r = 0.62 and 0.68, respectively, P < 0.01). A study by Izquierdo et al. (2001) revealed that individuals with greater increases in isometric strength following ST also showed higher total (r = 0.78, P < 0.01) and free (r = 0.71, P < 0.05) testosterone levels. The same trend was observed in data obtained by Ahtiainen et al. (2003) when evaluating highly trained men subjected to a 21-week ST program, where positive correlations were observed between the changes in isometric strength and total testosterone (r = 0.84, P < 0.01), the testosterone:cortisol ratio (r = 0.88, P < 0.01) and the isometric strength development and free testosterone (pre-training values: r = 0.78, P < 0.05 and post-training values: r = 0.82, P < 0.05). A study conducted in our laboratory by Cadore et al. (2008a) showed significant correlations between testosterone:SHBG ratios and DHEA concentrations as well as strength production in bench press, leg press and squat exercises (r = 0.55 to 0.82, P < 0.05 to P < 0.001) in trained and untrained middle-aged men. In another study, Cadore et al. (2010) showed significant correlations between increases in the strength of knee extensors and average basal total testosterone levels throughout the training period (3 measurements in 12 weeks of training) (r = 0.94, P < 0.01) and the average total testosterone:cortisol ratio (r = 0.93, P < 0.01). Table 1 shows the results obtained from studies where correlations between hormonal parameters and variables related to muscle strength were identified. One aspect that must be emphasised is that other structural factors, such as pennation angle and fibre type composition, may interfere with strength production (Ramos et al., 1998), just as the volume and intensity of ST largely influence the increase in strength resulting from training (Marx et al., 2001).


Table 1. Relatioship between sex hormonal parameters and strength performance. TT: total testosterone; FT: free testosterone; COR: cortisol; DHEA, dehidroepiandrosterone; SHBG: sex hormone binding globuline; ↑: increases; MVC: maximal voluntary contraction (maximal isometric strength); and, 1 RM: one-maximum repetition (maximal dynamic strength); ST: strength training.

Acute and Chronic Testosterone Responses to Physical Exercise and Training 281

(2003) suggest that these mechanisms may also be mediators of testosterone increase in

It may be stated that the hormonal response to exercise is connected to certain characteristics inherent to the training session, such as the number of sets and repetitions, the relative intensity (percentage of 1 maximum repetition - 1 RM) and time intervals (McCaulley et al., 2009). The amount of work done during ST may be a determining factor in the acute hormone response, leading to an optimum combination of anabolic and catabolic hormone stimulation. This, in turn, may result in a more favourable environment for neuromuscular adaptations to

The preponderant influence of volume on the hormonal response to different training methods was observed by Häkkinen and Pakarinen (1993b), who compared the hormonal response to a session involving 20 sets of 1 RM with a session composed by 10 sets of 10 repetitions at 70% of 1 RM; both ST bout were conducted with 3-minute intervals between sets. The authors observed a significant increase (P<0.05) in total (22%) and free (23%) testosterone in response to the high-volume training and no increase in the training session that included a higher load and fewer repetitions. Smilios et al. (2003) showed that the hormone response observed in young men increased as the number of sets in each session increased, approaching maximum strength, muscle hypertrophy and resistance. These authors observed that when the number of sets was increased from 4 to 6, the anabolic hormone levels stabilised, while cortisol levels continued to increase. Their results suggest that modifying the volume of a ST session causes alterations in the balance between anabolic and catabolic hormones. When considering different training methods, McCaulley et al. (2009) observed a higher total testosterone response to muscle hypertrophy protocols when compared to protocols that aimed at developing maximum strength and power, despite equalisation of the total work load for each session (load x sets x repetitions). When investigating the influence of the total muscle mass involved in training, Häkkinen et al. (1998b) demonstrated a greater testosterone response in young and elderly men using protocols involving simultaneous use of the lower and upper limbs (27%). However, an increase in hormone levels was also observed for protocols that involved the upper and lower limbs separately (P<0.01), indicating that the greater the amount of muscle mass

Regarding the influence of resting intervals on the acute hormone response, the smaller the interval between sets, the greater the stimulus (Kraemer et al., 1990). Nevertheless, when the sets are performed with maximum repetitions, the interval appears to have no influence within a certain intensity range, as demonstrated by Ahtiainen et al. (2005), who showed that there was no difference in acute hormone response between two protocols of 10 RM with 2- and 5-minute intervals. Notwithstanding, sessions with moderate to high intensity that involve multiple sets and short time intervals, during which energy is derived mainly from glycolytic lactate metabolism, appear to be the greatest stimulus for the steroid

Little is known about the influence of combined aerobic and strength training (i.e., concurrent training) on the acute testosterone response. A study by Goto et al. (2005) demonstrated that the GH response to strength training was found to be suppressed by prior aerobic training. However, no differences were observed in strength training-induced

**3.2 The influence of strength training variables on testosterone responses** 

training, resulting in increases in muscle strength and mass (Smilios et al*.,* 2003).

involved, the greater the acute total testosterone response.

hormone response to ST.

response to this type of training.

Fig. 1. Schematic diagram of the mechanism of training adaptations: anabolic process as adaptation to strength training and chronic catabolic process resulting from excessive volume of both strength and aerobic training. AR, androgen receptor; CSA, cross-sectional area.

#### **3. Acute testosterone responses to physical exercise**

Acute testosterone responses to ST exhibit plasticity, and their pattern depends on factors related to the training session, such as volume, intensity, method (i.e., single or multiple sets) (Cadore et al., 2009a; Häkkinen & Pakarinen, 1993a; McCaulley et al., 2009), type of muscle contraction (Durand et al., 2003; Kraemer et al., 2006) and muscle mass involved (Häkkinen et al., 1998), as well as factors such as age (Kraemer et al., 1999; Cadore et al., 2009a) and the individual's level of training (Ahtiainen et al., 2003; Cadore et al., 2008a, 2009; Kraemer et al., 1999). The response of testosterone levels to ST may expose the skeletal musculature to an elevated peripheral hormonal concentration, which may improve the interaction between the hormone and its cellular receptors (Hoffman et al., 2003, Willoughby & Taylor, 2004). Regarding aerobic training, even though the importance of the anabolic hormone response remains unclear, it seems that testosterone is more responsive to higher intensity exercises (Enea et al., 2009) and a longer duration of exercises (Harris et al., 1989; Trembley et al., 2005).

#### **3.1 Possibles physiological mechanisms for the stimulus**

The response of testosterone levels to exercise sessions may reflect certain regulatory mechanisms in addition to the processes that regulate the secretion of this hormone at rest (Fahrner and Hackney, 1998; Lu et al., 1997). A study conducted by Lu et al. (1997) demonstrated that exercise-induced increase in testosterone levels in male rats correlated with an increase in blood lactate levels. Following this observation, the authors proceeded to conduct an *in vitro* study where lactate was infused into the rats' testes, and a dosedependent increase in testosterone was observed. Methods of ST aimed at achieving muscle hypertrophy or resistance have been shown to cause high lactate production (McCaulley et al., 2009; Smilios et al., 2003), suggesting a strong relationship between the mechanism of testosterone increase and lactate stimulation in the testes (Lu et al., 1997).

Other mechanisms may be responsible for the exercise-induced increase in testosterone levels, among which are increased sympathetic activity in response to exercise (Fahrner and Hackney, 1998) and blood flow and vasodilation related to the release of nitric oxide, which increases hormone secretion (Meskaitis et al., 1997). Even though various studies have used different strength training exercise protocols, Kraemer et al. (1999) and Ahtiainen et al.

Fig. 1. Schematic diagram of the mechanism of training adaptations: anabolic process as adaptation to strength training and chronic catabolic process resulting from excessive volume of both strength and aerobic training. AR, androgen receptor; CSA, cross-sectional area.

Acute testosterone responses to ST exhibit plasticity, and their pattern depends on factors related to the training session, such as volume, intensity, method (i.e., single or multiple sets) (Cadore et al., 2009a; Häkkinen & Pakarinen, 1993a; McCaulley et al., 2009), type of muscle contraction (Durand et al., 2003; Kraemer et al., 2006) and muscle mass involved (Häkkinen et al., 1998), as well as factors such as age (Kraemer et al., 1999; Cadore et al., 2009a) and the individual's level of training (Ahtiainen et al., 2003; Cadore et al., 2008a, 2009; Kraemer et al., 1999). The response of testosterone levels to ST may expose the skeletal musculature to an elevated peripheral hormonal concentration, which may improve the interaction between the hormone and its cellular receptors (Hoffman et al., 2003, Willoughby & Taylor, 2004). Regarding aerobic training, even though the importance of the anabolic hormone response remains unclear, it seems that testosterone is more responsive to higher intensity exercises (Enea et al., 2009) and a longer duration of exercises (Harris et al.,

The response of testosterone levels to exercise sessions may reflect certain regulatory mechanisms in addition to the processes that regulate the secretion of this hormone at rest (Fahrner and Hackney, 1998; Lu et al., 1997). A study conducted by Lu et al. (1997) demonstrated that exercise-induced increase in testosterone levels in male rats correlated with an increase in blood lactate levels. Following this observation, the authors proceeded to conduct an *in vitro* study where lactate was infused into the rats' testes, and a dosedependent increase in testosterone was observed. Methods of ST aimed at achieving muscle hypertrophy or resistance have been shown to cause high lactate production (McCaulley et al., 2009; Smilios et al., 2003), suggesting a strong relationship between the mechanism of

Other mechanisms may be responsible for the exercise-induced increase in testosterone levels, among which are increased sympathetic activity in response to exercise (Fahrner and Hackney, 1998) and blood flow and vasodilation related to the release of nitric oxide, which increases hormone secretion (Meskaitis et al., 1997). Even though various studies have used different strength training exercise protocols, Kraemer et al. (1999) and Ahtiainen et al.

**3. Acute testosterone responses to physical exercise** 

**3.1 Possibles physiological mechanisms for the stimulus** 

testosterone increase and lactate stimulation in the testes (Lu et al., 1997).

1989; Trembley et al., 2005).

(2003) suggest that these mechanisms may also be mediators of testosterone increase in response to this type of training.

#### **3.2 The influence of strength training variables on testosterone responses**

It may be stated that the hormonal response to exercise is connected to certain characteristics inherent to the training session, such as the number of sets and repetitions, the relative intensity (percentage of 1 maximum repetition - 1 RM) and time intervals (McCaulley et al., 2009). The amount of work done during ST may be a determining factor in the acute hormone response, leading to an optimum combination of anabolic and catabolic hormone stimulation. This, in turn, may result in a more favourable environment for neuromuscular adaptations to training, resulting in increases in muscle strength and mass (Smilios et al*.,* 2003).

The preponderant influence of volume on the hormonal response to different training methods was observed by Häkkinen and Pakarinen (1993b), who compared the hormonal response to a session involving 20 sets of 1 RM with a session composed by 10 sets of 10 repetitions at 70% of 1 RM; both ST bout were conducted with 3-minute intervals between sets. The authors observed a significant increase (P<0.05) in total (22%) and free (23%) testosterone in response to the high-volume training and no increase in the training session that included a higher load and fewer repetitions. Smilios et al. (2003) showed that the hormone response observed in young men increased as the number of sets in each session increased, approaching maximum strength, muscle hypertrophy and resistance. These authors observed that when the number of sets was increased from 4 to 6, the anabolic hormone levels stabilised, while cortisol levels continued to increase. Their results suggest that modifying the volume of a ST session causes alterations in the balance between anabolic and catabolic hormones. When considering different training methods, McCaulley et al. (2009) observed a higher total testosterone response to muscle hypertrophy protocols when compared to protocols that aimed at developing maximum strength and power, despite equalisation of the total work load for each session (load x sets x repetitions). When investigating the influence of the total muscle mass involved in training, Häkkinen et al. (1998b) demonstrated a greater testosterone response in young and elderly men using protocols involving simultaneous use of the lower and upper limbs (27%). However, an increase in hormone levels was also observed for protocols that involved the upper and lower limbs separately (P<0.01), indicating that the greater the amount of muscle mass involved, the greater the acute total testosterone response.

Regarding the influence of resting intervals on the acute hormone response, the smaller the interval between sets, the greater the stimulus (Kraemer et al., 1990). Nevertheless, when the sets are performed with maximum repetitions, the interval appears to have no influence within a certain intensity range, as demonstrated by Ahtiainen et al. (2005), who showed that there was no difference in acute hormone response between two protocols of 10 RM with 2- and 5-minute intervals. Notwithstanding, sessions with moderate to high intensity that involve multiple sets and short time intervals, during which energy is derived mainly from glycolytic lactate metabolism, appear to be the greatest stimulus for the steroid hormone response to ST.

Little is known about the influence of combined aerobic and strength training (i.e., concurrent training) on the acute testosterone response. A study by Goto et al. (2005) demonstrated that the GH response to strength training was found to be suppressed by prior aerobic training. However, no differences were observed in strength training-induced

Acute and Chronic Testosterone Responses to Physical Exercise and Training 283

However, the influence of the training status on the testosterone response to ST was not found in many studies. When submitting untrained and previously strength-trained individuals to a 21-week strength training protocol, Ahtiainen et al. (2003) observed similar alterations in total and free testosterone for both groups before and after training. This discrepancy may be due to factors such as sample profile, exercise protocol or potential changes in plasma volume. Moreover, testosterone responses to this type of exercise may be influenced by the relationship of the hormone with its cellular receptors, given that this interaction appears to be greater in trained individuals and that they thus may not require the same magnitude of an acute response in order to obtain an optimum hormone-receptor

While some studies demonstrate an increase in the resting levels of testosterone as an adjustment to ST (Izquierdo et al., 2006; Kraemer et al., 1995; Nicklas et al., 1995; Raastad et al., 2003; Tsolakis et al., 2004), other studies have observed no significant differences in this parameter (Ahtiainen et al., 2003; Hansen et al., 2001; Hickson et al., 1994). So far, available data indicate that only young individuals are capable of altering their resting hormone concentrations (Häkkinen et al., 1988; Staron et al., 1994; Tsolakis et al., 2004), whereas middle-aged and elderly individuals show no significant changes in such parameters (Häkkinen & Pakarinen, 1994; Häkkinen et al., 2000; 2001a; Izquierdo et al., 2006; Ryan et al., 1994). Increases in resting levels of testosterone seem to occur during periods of highvolume (Kramer et al., 1995; Marx et al., 2001) and high-intensity training (Staron et al., 1994; Kraemer et al., 1998; Raastad et al., 2003). These changes may occur in men (Häkkinen et al., 1988) and women (Marx et al., 2001) in response to long (Häkkinen et al., 1988; Marx et al.,

The influence of training volume on chronic adaptations of basal testosterone was described in a study by Marx et al. (2001), which 34 women (22 ± 5 years) were evaluated before and after performing a 24-week ST protocol. In this study, resting levels of testosterone were measured in order to compare groups of ST performing single vs. multiple sets. The results showed an increase in testosterone in both training groups, and the first adaptations took place after 12 weeks of training. However, after 24 weeks of training, only the multiple sets group had further increases in their resting testosterone levels, which was higher in this point than after 12 weeks, and higher than the single set ST group. Even though the performance of high-volume ST sessions may induce higher acute increases in catabolic hormones (Smilios et al., 2003), the study developed by Marx et al. (2001) demonstrated that high-volume and high-intensity ST may lead to higher chronic increases in anabolic hormones compared to low-volume ST. This may contribute to the greater strength production observed in individuals who trained with multiple sets when compared to those

The Table 2 shows subjects characteristics, training protocol and results from some of the studies that have investigated resting hormone adaptations to ST. Nevertheless, modifications in resting concentrations appear to be transient, resulting from the increase or decrease in intensity and, mainly, in volume (Ahtiainen et al., 2003). However, the precise role of resting testosterone concentrations in neuromuscular adaptation to training is yet to

interaction (Ahtiainen et al., 2011; Willoughby & Taylor, 2004).

**4. Chronic endocrine adaptations to physical training** 

**4.1 Basal testosterone adaptations induced by strength training** 

2001) or short training periods (Staron et al., 1994; Kraemer et al., 1998).

who trained with simple sets (Kemmler et al., 2004).

be determined.

testosterone concentrations, with or without prior aerobic training, possibly due to the lowvolume protocol used in the study. However, unpublished data from our laboratory show that the manipulation of the order of modalities (strength and aerobic) may, in fact, influence the testosterone response produced by concurrent training sessions. A significant increase was observed in both protocols following the first modality, though levels remained high only at the end of the training session when the protocol involved aerobic training followed by strength training; the same response was not observed when strength training was performed before aerobic training. When comparing acute testosterone responses to strength and aerobic training, some studies show that strength training appears to stimulate greater increases in testosterone levels when compared to aerobic training (Copeland et al., 2002, Tremblay et al., 2003). In a previous study (Cadore et al., 2009a), we showed significantly higher salivary free testosterone responses to water-based resistance exercise compared with water-based aerobic exercise in both young and elderly healthy men. These results can be explained by the powerful influence of the anaerobic glycolytic pathway on acute hormonal increases in response to exercise (Kraemer & Ratamess, 2005).

#### **3.3 The Influence of age and training status**

The profile of the population subjected to training sessions is one of the factors that influence hormone response to ST. Studies aimed at investigating this response in different age groups have mainly observed lower responses in elderly individuals as demonstrated by Kraemer et al. (1999). For instance, when comparing acute total and free testosterone responses in groups of men aged 30 ± 5 and 62 ± 3 years, Kraemer et al. (1999) demonstrated that even though both groups showed an increase in free testosterone, a lower testosterone response was observed in the elderly group. According to these authors, the reduced response is associated with andropause, which is characterised by a smaller number and decreased secretory capacity of Leydig cells due to ageing. Similar results were observed by Cadore et al. (2009a), where elderly individuals showed significantly lower free-testosterone responses to water-based resistance exercise than young men.

The individual's training status may also influence the response of the endocrine system to ST (Cadore et al., 2008a, 2009b; Kraemer et al., 1992), given that different anabolic responses may occur before and after a period of ST. Kraemer et al. (1992) investigated weightlifting male athletes aged 17 ± 2 years and observed that individuals with more than 2 years of training presented higher acute testosterone responses. A study by Cadore et al. (2008a) observed different patterns of hormone response in trained and untrained middle-aged men (40 ± 4 years) after a ST protocol. Significant increases in free testosterone (27%) were observed in members of the trained group, whereas significant increases in both total (28%) and free (22%) testosterone as well as DHEA (127%) were observed in the untrained group (P<0.05). These results may suggest a higher capacity for testosterone dissociation from carrier proteins, increasing the bioactivity of the hormone without the need for an increase in production. A study by Kraemer et al. (1999) showed higher free testosterone responses in young and elderly individuals following 10 weeks of periodic strength training. Some results suggest the existence of specific responses to certain types of training, as demonstrated in a study developed by Tremblay et al. (2003). The study showed greater increases in anabolic hormone levels in response to strength training in strength-trained subjects when compared to aerobically trained individuals, whereas aerobically trained subjects produced higher hormonal responses to aerobic exercise when compared to strength-trained individuals.

testosterone concentrations, with or without prior aerobic training, possibly due to the lowvolume protocol used in the study. However, unpublished data from our laboratory show that the manipulation of the order of modalities (strength and aerobic) may, in fact, influence the testosterone response produced by concurrent training sessions. A significant increase was observed in both protocols following the first modality, though levels remained high only at the end of the training session when the protocol involved aerobic training followed by strength training; the same response was not observed when strength training was performed before aerobic training. When comparing acute testosterone responses to strength and aerobic training, some studies show that strength training appears to stimulate greater increases in testosterone levels when compared to aerobic training (Copeland et al., 2002, Tremblay et al., 2003). In a previous study (Cadore et al., 2009a), we showed significantly higher salivary free testosterone responses to water-based resistance exercise compared with water-based aerobic exercise in both young and elderly healthy men. These results can be explained by the powerful influence of the anaerobic glycolytic pathway on acute hormonal increases in response to exercise (Kraemer & Ratamess, 2005).

The profile of the population subjected to training sessions is one of the factors that influence hormone response to ST. Studies aimed at investigating this response in different age groups have mainly observed lower responses in elderly individuals as demonstrated by Kraemer et al. (1999). For instance, when comparing acute total and free testosterone responses in groups of men aged 30 ± 5 and 62 ± 3 years, Kraemer et al. (1999) demonstrated that even though both groups showed an increase in free testosterone, a lower testosterone response was observed in the elderly group. According to these authors, the reduced response is associated with andropause, which is characterised by a smaller number and decreased secretory capacity of Leydig cells due to ageing. Similar results were observed by Cadore et al. (2009a), where elderly individuals showed significantly lower free-testosterone

The individual's training status may also influence the response of the endocrine system to ST (Cadore et al., 2008a, 2009b; Kraemer et al., 1992), given that different anabolic responses may occur before and after a period of ST. Kraemer et al. (1992) investigated weightlifting male athletes aged 17 ± 2 years and observed that individuals with more than 2 years of training presented higher acute testosterone responses. A study by Cadore et al. (2008a) observed different patterns of hormone response in trained and untrained middle-aged men (40 ± 4 years) after a ST protocol. Significant increases in free testosterone (27%) were observed in members of the trained group, whereas significant increases in both total (28%) and free (22%) testosterone as well as DHEA (127%) were observed in the untrained group (P<0.05). These results may suggest a higher capacity for testosterone dissociation from carrier proteins, increasing the bioactivity of the hormone without the need for an increase in production. A study by Kraemer et al. (1999) showed higher free testosterone responses in young and elderly individuals following 10 weeks of periodic strength training. Some results suggest the existence of specific responses to certain types of training, as demonstrated in a study developed by Tremblay et al. (2003). The study showed greater increases in anabolic hormone levels in response to strength training in strength-trained subjects when compared to aerobically trained individuals, whereas aerobically trained subjects produced higher hormonal responses to aerobic exercise when compared to strength-trained individuals.

**3.3 The Influence of age and training status** 

responses to water-based resistance exercise than young men.

However, the influence of the training status on the testosterone response to ST was not found in many studies. When submitting untrained and previously strength-trained individuals to a 21-week strength training protocol, Ahtiainen et al. (2003) observed similar alterations in total and free testosterone for both groups before and after training. This discrepancy may be due to factors such as sample profile, exercise protocol or potential changes in plasma volume. Moreover, testosterone responses to this type of exercise may be influenced by the relationship of the hormone with its cellular receptors, given that this interaction appears to be greater in trained individuals and that they thus may not require the same magnitude of an acute response in order to obtain an optimum hormone-receptor interaction (Ahtiainen et al., 2011; Willoughby & Taylor, 2004).

#### **4. Chronic endocrine adaptations to physical training**

#### **4.1 Basal testosterone adaptations induced by strength training**

While some studies demonstrate an increase in the resting levels of testosterone as an adjustment to ST (Izquierdo et al., 2006; Kraemer et al., 1995; Nicklas et al., 1995; Raastad et al., 2003; Tsolakis et al., 2004), other studies have observed no significant differences in this parameter (Ahtiainen et al., 2003; Hansen et al., 2001; Hickson et al., 1994). So far, available data indicate that only young individuals are capable of altering their resting hormone concentrations (Häkkinen et al., 1988; Staron et al., 1994; Tsolakis et al., 2004), whereas middle-aged and elderly individuals show no significant changes in such parameters (Häkkinen & Pakarinen, 1994; Häkkinen et al., 2000; 2001a; Izquierdo et al., 2006; Ryan et al., 1994). Increases in resting levels of testosterone seem to occur during periods of highvolume (Kramer et al., 1995; Marx et al., 2001) and high-intensity training (Staron et al., 1994; Kraemer et al., 1998; Raastad et al., 2003). These changes may occur in men (Häkkinen et al., 1988) and women (Marx et al., 2001) in response to long (Häkkinen et al., 1988; Marx et al., 2001) or short training periods (Staron et al., 1994; Kraemer et al., 1998).

The influence of training volume on chronic adaptations of basal testosterone was described in a study by Marx et al. (2001), which 34 women (22 ± 5 years) were evaluated before and after performing a 24-week ST protocol. In this study, resting levels of testosterone were measured in order to compare groups of ST performing single vs. multiple sets. The results showed an increase in testosterone in both training groups, and the first adaptations took place after 12 weeks of training. However, after 24 weeks of training, only the multiple sets group had further increases in their resting testosterone levels, which was higher in this point than after 12 weeks, and higher than the single set ST group. Even though the performance of high-volume ST sessions may induce higher acute increases in catabolic hormones (Smilios et al., 2003), the study developed by Marx et al. (2001) demonstrated that high-volume and high-intensity ST may lead to higher chronic increases in anabolic hormones compared to low-volume ST. This may contribute to the greater strength production observed in individuals who trained with multiple sets when compared to those who trained with simple sets (Kemmler et al., 2004).

The Table 2 shows subjects characteristics, training protocol and results from some of the studies that have investigated resting hormone adaptations to ST. Nevertheless, modifications in resting concentrations appear to be transient, resulting from the increase or decrease in intensity and, mainly, in volume (Ahtiainen et al., 2003). However, the precise role of resting testosterone concentrations in neuromuscular adaptation to training is yet to be determined.

Acute and Chronic Testosterone Responses to Physical Exercise and Training 285

cycle ergometer 3 times per week with intensity varying between 55 and 85% of aerobic power (9.7 ± 2.8 vs. 7.9 ± 3.0 pg/mL, P < 0.01). Possible discrepancies between the results of different studies may reflect the different intensities and volumes used, given that the intensity used by Strüder et al. (1999), for instance, was lower than the intensity used in our study (Cadore et al., 2010). However, in animal models, Hu et al. (1999) observed a significant reduction in testosterone levels in rats submitted to continuous swimming for 3 weeks. Levels were restored to normal following 6 weeks of training, suggesting an adjustment to training on LH secretion in the endocrine system that was associated with negative feedback. Even though testosterone reduction during aerobic training has not been clearly demonstrated, it is possible that a certain amount of time is necessary for the endocrine system to adapt to the volume and intensity of training when these factors exceed a certain stimulus threshold (Calbet et al., 1993;

Though high-volume physical training may result in the suppression of testosterone via direct inhibition due to the effect of cortisol on the testes (Brownlee et al., 2005), this does not completely explain the occurrence of testosterone reduction with aerobic training, given that increases in basal cortisol levels and the consequent testicular suppression are most commonly related to overtraining. In fact, testosterone levels have been shown to be reduced in endurance athletes with no alterations in cortisol levels (Maïmoun et al., 2003) as well as in non-athletes subjected to aerobic training (Cadore et al. 2010). Furthermore, other mechanisms, such as hypervolemia, increased utilisation of the hormone by muscle tissue and increased hepatic degradation of the hormone, may be responsible for the decrease in

Evidence shows that cell adjustments may be key factors for training-induced hypertrophy (Ahtiainen et al., 2011; Deschenes et al., 1994; Inoue et al., 1993, 1994; Kadi et al., 2000; Bamman et al., 2001; Willoughby & Taylor, 2004; Ratamess et al., 2005). Some of these adjustments correspond to an increase in the number of androgen receptors (AR) in the muscle, and they are apparently dependent on the pattern of acute testosterone response to exercise (Willoughby & Taylor, 2004). A greater number of ARs and an increased sensitivity of these receptors to the hormone may improve the trophic effects of testosterone on target cells (Kadi et al., 2000). Inoue et al. (1993) subjected male rats to training using electrical stimulation and demonstrated that muscle hypertrophy occurred in parallel with a significant increase in cellular ARs. In a different study, Inoue et al. (1994) observed that the suppression of androgen receptors by receptor antagonists reduced the increase in muscle

Kadi et al. (2000) measured the number of ARs per area of muscle fibre in the superior trapezius and the *vastus lateralis* muscle of high-performance weightlifting men. The sample was composed of trained individuals with (31 ± 3 years) and without (28 ± 8 years) the use of exogenous anabolic steroids as well as untrained individuals (23 ± 3 years). Results showed higher numbers of ARs per area of muscle fibre in the superior trapezius of individuals from both trained groups when compared with untrained individuals. Moreover, individuals using exogenous anabolic steroids presented higher values than those that were only training (P<0.05). Surprisingly, these differences were observed only in the superior trapezius. The proportion between the different types of muscle fibres present in each of the evaluated muscle groups (i.e., type I and type II) may have influenced the different behaviours of the

testosterone levels that result from endurance training (Izquierdo et al., 2004).

Kraemer & Ratamess, 2005; Maïmoun et al., 2003).

**4.3 Changes in muscle cell androgen receptors** 

mass obtained with electrical stimulation.


Table 2. Testosterone modifications at rest after strength training: TT: total testosterone; FT: free testosterone; ↑: increases; RM: maximal repetitions.

#### **4.2 Changes in circulating testosterone in response to aerobic training**

With regards to aerobic training, studies have demonstrated that endurance athletes have lower testosterone levels when compared to sedentary individuals (Strüder et al., 1998; Maïmoun et al., 2003). However, Strüder et al. (1998) showed that although testosterone levels were indeed lower in elderly male runners compared to age-matched sedentary subjects, the same was not true for previously sedentary subjects who performed aerobic training for 20 weeks 3 times per week with an intensity of 50 to 65% of aerobic power. A previous study conducted in our laboratory (Cadore et al., 2010) demonstrated a significant reduction in free testosterone in elderly men following 12 weeks of aerobic training on a

21 weeks, 2

Two years, 5

12 weeks, 3

16 weeks, 3

12 weeks, 4

training

exercise

Table 2. Testosterone modifications at rest after strength training: TT: total testosterone; FT:

With regards to aerobic training, studies have demonstrated that endurance athletes have lower testosterone levels when compared to sedentary individuals (Strüder et al., 1998; Maïmoun et al., 2003). However, Strüder et al. (1998) showed that although testosterone levels were indeed lower in elderly male runners compared to age-matched sedentary subjects, the same was not true for previously sedentary subjects who performed aerobic training for 20 weeks 3 times per week with an intensity of 50 to 65% of aerobic power. A previous study conducted in our laboratory (Cadore et al., 2010) demonstrated a significant reduction in free testosterone in elderly men following 12 weeks of aerobic training on a

**4.2 Changes in circulating testosterone in response to aerobic training** 

16 weeks, 3

1 RM

1 RM

times/week, 8-10 RM

10 ± 5 years of strength training, 4 times a week, 8-12 RM

times/week ↑ TT

times/week, 40-80% of

times/week, 50-80% of

times/week, 3-10 RM, strength vs. concurrent

24 weeks, 3-15 RM, single vs. 3 sets per

6-12 RM ↑ TT in men

times/week, 3-15 RM ↑ FT jovens

times/week, 5-15 RM No modifications

times/week, 5-15 RM No modifications

↑ FT in trained after 14 weeks; no modifications

No difference at rest, different responses to

No modifications

No modifications

training

↑ TT after concurrent

↑ TT in both groups after 12 weeks, higher after 3 sets training group

after 21 weeks

exercise

RM No modifications

**Author Subjects Training protocol Results** 

Young previously trained and untrained

Elite powerlifters young men

Middle-aged and elderly men and

<sup>1999</sup>Young and elderly men 10 weeks, 3

Middle-aged and elderly men

1994 Elderly men 16 weeks, 3

free testosterone; ↑: increases; RM: maximal repetitions.

women

*<sup>2001</sup>*Young men and women

<sup>1995</sup>Military young men

<sup>2001</sup>Young women

Middle-aged long-term trained and untrained

*al*., 2001a Elderly men 21 weeks, 40-80% of 1

<sup>1994</sup> Young men and women 8 weeks, 3 times/week,

men

men

Ahtiainen *et al*., 2003

Cadore *et al*., 2008a

Häkkinen *et* 

Häkkinen *et al*., 1988

Staron et al.,

Häkkinen & Pakarinen, 1994

Izquierdo *et al.,* 

Kraemer *et al*.,

Kraemer *et al*.,

Marx *et al*.,

Nicklas *et al*.,

Ryan *et al*.,

1995

cycle ergometer 3 times per week with intensity varying between 55 and 85% of aerobic power (9.7 ± 2.8 vs. 7.9 ± 3.0 pg/mL, P < 0.01). Possible discrepancies between the results of different studies may reflect the different intensities and volumes used, given that the intensity used by Strüder et al. (1999), for instance, was lower than the intensity used in our study (Cadore et al., 2010). However, in animal models, Hu et al. (1999) observed a significant reduction in testosterone levels in rats submitted to continuous swimming for 3 weeks. Levels were restored to normal following 6 weeks of training, suggesting an adjustment to training on LH secretion in the endocrine system that was associated with negative feedback. Even though testosterone reduction during aerobic training has not been clearly demonstrated, it is possible that a certain amount of time is necessary for the endocrine system to adapt to the volume and intensity of training when these factors exceed a certain stimulus threshold (Calbet et al., 1993; Kraemer & Ratamess, 2005; Maïmoun et al., 2003).

Though high-volume physical training may result in the suppression of testosterone via direct inhibition due to the effect of cortisol on the testes (Brownlee et al., 2005), this does not completely explain the occurrence of testosterone reduction with aerobic training, given that increases in basal cortisol levels and the consequent testicular suppression are most commonly related to overtraining. In fact, testosterone levels have been shown to be reduced in endurance athletes with no alterations in cortisol levels (Maïmoun et al., 2003) as well as in non-athletes subjected to aerobic training (Cadore et al. 2010). Furthermore, other mechanisms, such as hypervolemia, increased utilisation of the hormone by muscle tissue and increased hepatic degradation of the hormone, may be responsible for the decrease in testosterone levels that result from endurance training (Izquierdo et al., 2004).

#### **4.3 Changes in muscle cell androgen receptors**

Evidence shows that cell adjustments may be key factors for training-induced hypertrophy (Ahtiainen et al., 2011; Deschenes et al., 1994; Inoue et al., 1993, 1994; Kadi et al., 2000; Bamman et al., 2001; Willoughby & Taylor, 2004; Ratamess et al., 2005). Some of these adjustments correspond to an increase in the number of androgen receptors (AR) in the muscle, and they are apparently dependent on the pattern of acute testosterone response to exercise (Willoughby & Taylor, 2004). A greater number of ARs and an increased sensitivity of these receptors to the hormone may improve the trophic effects of testosterone on target cells (Kadi et al., 2000). Inoue et al. (1993) subjected male rats to training using electrical stimulation and demonstrated that muscle hypertrophy occurred in parallel with a significant increase in cellular ARs. In a different study, Inoue et al. (1994) observed that the suppression of androgen receptors by receptor antagonists reduced the increase in muscle mass obtained with electrical stimulation.

Kadi et al. (2000) measured the number of ARs per area of muscle fibre in the superior trapezius and the *vastus lateralis* muscle of high-performance weightlifting men. The sample was composed of trained individuals with (31 ± 3 years) and without (28 ± 8 years) the use of exogenous anabolic steroids as well as untrained individuals (23 ± 3 years). Results showed higher numbers of ARs per area of muscle fibre in the superior trapezius of individuals from both trained groups when compared with untrained individuals. Moreover, individuals using exogenous anabolic steroids presented higher values than those that were only training (P<0.05). Surprisingly, these differences were observed only in the superior trapezius. The proportion between the different types of muscle fibres present in each of the evaluated muscle groups (i.e., type I and type II) may have influenced the different behaviours of the

Acute and Chronic Testosterone Responses to Physical Exercise and Training 287

Ahtiainen J.P.; Hulme, J.J.; Kraemer, W.J.; Lehti, M.; Nyman, K.; Selanne, H.; Alen, M.;

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androgen receptors in response to training. In fact, while studying rats subjected to strength training, Deschenes et al. (1994) were only able to observe an increase in the number of ARs in muscles with a predominance of fast glycolytic fibres, whereas muscles with a predominance of slow oxidative fibres showed decreased numbers of ARs.

Willoughby & Taylor (2004) conducted a study where 18 young men were subjected to 3 sessions of ST with 3 sets of 8 to 10 RM. Results showed a significant increase in protein synthesis, AR expression and AR messenger RNA following the training sessions, where values increased up to 202% 48 hours after the third session. Furthermore, the study revealed a correlation between testosterone increase (significant following all sessions) and an increase in the number of receptors (r = 0.89, P < 0.05). These results suggest that the hormone-receptor complex constitutes an important element in the mechanism responsible for mediating the adjustments to strength training, such as an increase in muscle strength and exercise-induced hypertrophy (Inoue et al., 1993; Kadi et al., 2000; Willoughby & Taylor 2004).

#### **5. Conclusion**

As noted in the studies presented in this chapter, there is a connection between the trainability of individuals subjected to ST and their levels of circulating testosterone. However, factors related to the training sessions and population profile seem to influence acute and chronic hormone responses, which result in a plasticity in the pattern of testosterone response to physical exercise, particularly strength training. Among the types of strength training sessions, high-volume protocols with moderate to high intensity (70- 85% 1 RM), which are typically used to achieve muscle hypertrophy and predominantly rely on the glycolytic lactate metabolic pathway, appear to stimulate greater responses. Moreover, the increase in the number of ARs appears to have a key role in muscle hypertrophy observed with ST. However, determining which factors might be related to the hormone response to ST may be of great importance for the prescription of a training session and determination of the optimum period that will optimise the anabolic environment determined by testosterone and thus maximise the neuromuscular adaptations resulting from strength training.

#### **6. Acknowledgements**

We thank specially to CAPES and CNPq government associations for their support to this project.

#### **7. References**


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Willoughby & Taylor (2004) conducted a study where 18 young men were subjected to 3 sessions of ST with 3 sets of 8 to 10 RM. Results showed a significant increase in protein synthesis, AR expression and AR messenger RNA following the training sessions, where values increased up to 202% 48 hours after the third session. Furthermore, the study revealed a correlation between testosterone increase (significant following all sessions) and an increase in the number of receptors (r = 0.89, P < 0.05). These results suggest that the hormone-receptor complex constitutes an important element in the mechanism responsible for mediating the adjustments to strength training, such as an increase in muscle strength and exercise-induced

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We thank specially to CAPES and CNPq government associations for their support to this

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**5. Conclusion** 

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**6. Acknowledgements** 

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**14** 

**Hand Grip Strength in Relation to** 

**Fluctuating Asymmetry and Sexual** 

**Behaviour in Males And Females**

Stefan Van Dongen and Ellen Sprengers

*Antwerp University* 

*Belgium* 

**Morphological Measures of Masculinity,** 

Human evolutionary history is, as that of any other species, characterized by phenotypic and genetic changes as a result of natural and/or sexual selection. In spite of the fact that we live in relatively unnatural environments (especially in our Western culture), signals of this evolutionary history are tractable and allow gaining insights in ancestral processes of selection. One important aspect that has received a lot of attention is the process of mate selection and attractiveness (e.g., Thornhill & Gangestad, 1999). The central working hypothesis is that particular morphological features correlate with 'genetic' quality or mate value. Selection would then favour preferences for these features (i.e., evaluated to be attractive) such that choosing a mate baring these features would increase reproductive success. This has driven research in evolutionary psychology, and the main focus has been on attractiveness, secondary sex characteristics as hormone markers, hormone levels and

Overall, there is growing evidence that (especially) women evaluate sex-typical characteristics of the face, body and voice in men and that their preference may vary across the menstrual cycle. Also in women, typically feminine characteristics are judged to be more attractive. Although the adaptive value of these preferences is much more difficult to study, it appears reasonable to assume that more masculine characteristics in males correlate with increased circulating testosterone levels which in turn positively associate with dominance and physical performance while feminine characteristics reflect oestrogen levels which associate with increased fertility. Thus, there is evidence that these hormone-mediated characteristics bare information that, at least ancestrally, are important for mate selection and expected fitness. An interesting and open question then is why the sexual dimorphism in humans has not evolved to be more extreme. Most likely, some cost is associated to develop more masculine for males and/or more feminine for females. Alternatively, there is some evidence of an intra locus sexual conflict affecting fitness of siblings (Garver-Apgar et al., 2011). Most attention in the literature has been devoted to the association between masculinity and other measures of health, like fluctuating asymmetry (FA). Three possible outcomes have been proposed. More masculine or feminine features are (assumed to be) associated to higher testosterone or oestrogen levels, respectively, which, in turn, may act as

fluctuating asymmetry (e.g., Thornhill & Gangestad, 1999).

**1. Introduction** 

and elderly males. *International Journal of Sports Medicine,* Vol. 28, No. 5, (May 2007), pp. 401-406, ISSN 0172-4622


### **Hand Grip Strength in Relation to Morphological Measures of Masculinity, Fluctuating Asymmetry and Sexual Behaviour in Males And Females**

Stefan Van Dongen and Ellen Sprengers *Antwerp University Belgium* 

#### **1. Introduction**

292 Sex Hormones

Smilios, I.; Pilianidis, T.; Karamouzis, M. & Tokmakidis, S. Hormonal responses after

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and elderly males. *International Journal of Sports Medicine,* Vol. 28, No. 5, (May 2007),

various resistance exercise protocols. *Medicine and Science in Sports and Exercise,* Vol

Hagerman, F.C. & Hikida, R.S. (1994). Skeletal muscle adaptations during early phase of heavy-resistance training in men and women. *Journal of Applied Physiology,* 

(1999). Neuroendocrine system and mental function in sedentary and endurancetrained elderly males. *International Journal of Sports Medicine,* Vol. 20, No. 3 (Apr

exercise mode on endogenous steroid hormones in men. *Journal of Applied* 

post-exercise steroid hormone responses in trained males. *European Journal of* 

responses to resistance training and detraining in preadolescent males. *Journal of Strength and Conditioning Research,* Vol. 18, No. 3, (Aug 2004), pp.625–629, ISSN

androgen receptor expression *Medicine and Science in Sports and Exercise,* Vol. 36,

Human evolutionary history is, as that of any other species, characterized by phenotypic and genetic changes as a result of natural and/or sexual selection. In spite of the fact that we live in relatively unnatural environments (especially in our Western culture), signals of this evolutionary history are tractable and allow gaining insights in ancestral processes of selection. One important aspect that has received a lot of attention is the process of mate selection and attractiveness (e.g., Thornhill & Gangestad, 1999). The central working hypothesis is that particular morphological features correlate with 'genetic' quality or mate value. Selection would then favour preferences for these features (i.e., evaluated to be attractive) such that choosing a mate baring these features would increase reproductive success. This has driven research in evolutionary psychology, and the main focus has been on attractiveness, secondary sex characteristics as hormone markers, hormone levels and fluctuating asymmetry (e.g., Thornhill & Gangestad, 1999).

Overall, there is growing evidence that (especially) women evaluate sex-typical characteristics of the face, body and voice in men and that their preference may vary across the menstrual cycle. Also in women, typically feminine characteristics are judged to be more attractive. Although the adaptive value of these preferences is much more difficult to study, it appears reasonable to assume that more masculine characteristics in males correlate with increased circulating testosterone levels which in turn positively associate with dominance and physical performance while feminine characteristics reflect oestrogen levels which associate with increased fertility. Thus, there is evidence that these hormone-mediated characteristics bare information that, at least ancestrally, are important for mate selection and expected fitness. An interesting and open question then is why the sexual dimorphism in humans has not evolved to be more extreme. Most likely, some cost is associated to develop more masculine for males and/or more feminine for females. Alternatively, there is some evidence of an intra locus sexual conflict affecting fitness of siblings (Garver-Apgar et al., 2011). Most attention in the literature has been devoted to the association between masculinity and other measures of health, like fluctuating asymmetry (FA). Three possible outcomes have been proposed. More masculine or feminine features are (assumed to be) associated to higher testosterone or oestrogen levels, respectively, which, in turn, may act as

Hand Grip Strength in Relation to Morphological Measures of

HGS related significantly to 2D:4D in two

HGS related positively to rated masculinity,

first sexual intercourse and promiscuity

HGS related to SHR, aggressive behavior, age at

HGS related to facial attractiveness and SHR, but not to number of partners and age at first

HGS related to victimization, popularity but not

HGS did not relate to perceived aggressiveness,

HGS related to perceived aggressiveness, dominance and health, but not with

attractiveness, dominance and health

**2. Materials and methods** 

HGS did not relate to 2D:4D m 82 HGS did not relate to any of the above f 61

dominance and attractiveness

sexual intercourse

aggression

attractiveness

**2.1 Study design** 

data.

samples

Masculinity, Fluctuating Asymmetry and Sexual Behaviour in Males And Females 295

In this study we present data on relationships between HGS and i) objective measures of facial masculinity/femininity; ii) fluctuating asymmetry; iii) attractiveness and vi) sexual behaviour in a population of young males and females. The dataset presented here was not very large (total sample size of 100). Therefore, it cannot provide strong conclusions. Although the study of associations with HGS in this context is relatively new, quite some estimates have been published. It is therefore timely to review these results and combine them with the newly presented data here to come to more robust conclusions and suggestions for further research. We therefore also present a meta-analysis of all available

Hypothesis tested gender N Reference

HGS related to perception of dance ability m 40 Hugill et al. 2009

Table 1. Overview of results of relationships between hand-grip strength (HGS) and

We measured bilateral asymmetry and masculinity/femininity from scans (HP scanjet G4050, 4800\*9600 DPI) of hands and photographs (Nikon D70, 6 megapixel) of faces of 52 men and 48 women with an average age of 22.6 (SD = 2.66) and 22.3 (SD = 1.87) years respectively. The degree of handedness was also self-evaluated on a scale of 0 (extreme lefthanded) to 10 (extreme right-handed). Hand-grip strength (HGS) was determined using a Biometrics precision dynamometer. For each participant, the strength was determined twice

measures of masculinity and sexual behavior in previous studies.

m 88+52 Fink et al 2006

m 32 Fink et al 2007

m 82 Gallup et al 2007

m 38 Shoup & Gallup, 2008

m/f 255 Gallup et al., 2010

m 69

f 93

an immunosuppressant (Little et al., 2008). It has, therefore, often been argued that larger secondary sexual characteristics should be related to a healthier immune system because only healthy individuals can afford the high sex hormone handicap (Little et al., 2008). On the other hand, Getty (2002) and Kokko et al. (2002) have noted that, because of trade-offs between investment in reproductive traits and somatic investment (e.g., immune defences), high quality individuals may, under intense sexual selection, be 'forced' to invest in reproduction to such a large degree that they actually have worse health and poorer survival prospects than individuals of low quality. Thus, if both symmetry and masculinity/femininity signal quality, both should be positively correlated where high quality males can grow symmetric and masculine and high quality females can grow symmetric and feminine (e.g., Little et al., 2008). However, if sexual selection drives high quality individuals to display extreme masculine/feminine features, these may come at the expense of health. Under such a scenario, high quality individuals preferred for mating, with high circulating sex hormones (testosterone or estrogens) and/or associated morphological expression of masculinity/femininity would develop a less symmetric body relative to individuals with lower circulating hormones and less extreme morphological expression of masculinity/femininity. Recently, Puts (2010) argued that androgendependent masculine traits may be produced in proportion to inherited immunocompetence, so that good-gene males end up little healthier than average. The regulation of androgen levels and the response to them may thus have evolved as a means of producing sexually selected traits in proportion to a male's ability to safely bare them. If so, little or no relationship between sexual dimorphism and FA is expected, as individuals would trade inherited immunocompetence for sexual competitiveness (masculinity in males, femininity in females). A recent review found little evidence for an association between FA and sexual dimorphism, supporting Puts (2010) view, but further research was clearly recommended (Van Dongen submitted manuscript). Clearly, there is a need for an integrative approach studying associations between masculinity/femininity, health and sexual behaviour simultaneously. Furthermore, the associations between different measures of masculinity/femininity are not well understood.

Indeed, measures of masculinity and femininity can be obtained in many different ways. Body masculinity is often measured objectively by the shoulder-to-hip ratio (SHR), while femininity is reflected by the well known waist-to-hip ratio (WHR). Facial masculinity/femininity can be expressed by facial shape (Little et al., 2008) and recently one study suggested that the eye-mouth-eye (EME) angle would also reliably reflect masculinity/femininity (Danel & Pawlowski, 2007). Next to these objective measurements, masculinity/femininity is often studied through ratings of pictures by the opposite sex. To evaluate the fitness-relevance of variation in masculinity/femininity, some have studied associations with sexual behaviour, like age of first sexual contact and number of sexual partners (or promiscuity). Others have studies associations with dominance. More recently, associations with physical stress have been of interest and more specifically, hand-grip strength (HGS) appears to be very relevant in this context. Physical strength, and its closely related HGS, may play an important role in male-male competition, and also appears to be, albeit weakly, related to survival (e.g., Gallup et al., 2008). Results from several recent studies are summarized in Table 1, and suggest that HSG is a promising measure of masculinity, yet, also shows some heterogeneity. In addition, there seems to be a lack of studies investigating associations between HGS and other objective measures of masculinity/femininity.

In this study we present data on relationships between HGS and i) objective measures of facial masculinity/femininity; ii) fluctuating asymmetry; iii) attractiveness and vi) sexual behaviour in a population of young males and females. The dataset presented here was not very large (total sample size of 100). Therefore, it cannot provide strong conclusions. Although the study of associations with HGS in this context is relatively new, quite some estimates have been published. It is therefore timely to review these results and combine them with the newly presented data here to come to more robust conclusions and suggestions for further research. We therefore also present a meta-analysis of all available data.


Table 1. Overview of results of relationships between hand-grip strength (HGS) and measures of masculinity and sexual behavior in previous studies.

### **2. Materials and methods**

#### **2.1 Study design**

294 Sex Hormones

an immunosuppressant (Little et al., 2008). It has, therefore, often been argued that larger secondary sexual characteristics should be related to a healthier immune system because only healthy individuals can afford the high sex hormone handicap (Little et al., 2008). On the other hand, Getty (2002) and Kokko et al. (2002) have noted that, because of trade-offs between investment in reproductive traits and somatic investment (e.g., immune defences), high quality individuals may, under intense sexual selection, be 'forced' to invest in reproduction to such a large degree that they actually have worse health and poorer survival prospects than individuals of low quality. Thus, if both symmetry and masculinity/femininity signal quality, both should be positively correlated where high quality males can grow symmetric and masculine and high quality females can grow symmetric and feminine (e.g., Little et al., 2008). However, if sexual selection drives high quality individuals to display extreme masculine/feminine features, these may come at the expense of health. Under such a scenario, high quality individuals preferred for mating, with high circulating sex hormones (testosterone or estrogens) and/or associated morphological expression of masculinity/femininity would develop a less symmetric body relative to individuals with lower circulating hormones and less extreme morphological expression of masculinity/femininity. Recently, Puts (2010) argued that androgendependent masculine traits may be produced in proportion to inherited immunocompetence, so that good-gene males end up little healthier than average. The regulation of androgen levels and the response to them may thus have evolved as a means of producing sexually selected traits in proportion to a male's ability to safely bare them. If so, little or no relationship between sexual dimorphism and FA is expected, as individuals would trade inherited immunocompetence for sexual competitiveness (masculinity in males, femininity in females). A recent review found little evidence for an association between FA and sexual dimorphism, supporting Puts (2010) view, but further research was clearly recommended (Van Dongen submitted manuscript). Clearly, there is a need for an integrative approach studying associations between masculinity/femininity, health and sexual behaviour simultaneously. Furthermore, the associations between different measures

Indeed, measures of masculinity and femininity can be obtained in many different ways. Body masculinity is often measured objectively by the shoulder-to-hip ratio (SHR), while femininity is reflected by the well known waist-to-hip ratio (WHR). Facial masculinity/femininity can be expressed by facial shape (Little et al., 2008) and recently one study suggested that the eye-mouth-eye (EME) angle would also reliably reflect masculinity/femininity (Danel & Pawlowski, 2007). Next to these objective measurements, masculinity/femininity is often studied through ratings of pictures by the opposite sex. To evaluate the fitness-relevance of variation in masculinity/femininity, some have studied associations with sexual behaviour, like age of first sexual contact and number of sexual partners (or promiscuity). Others have studies associations with dominance. More recently, associations with physical stress have been of interest and more specifically, hand-grip strength (HGS) appears to be very relevant in this context. Physical strength, and its closely related HGS, may play an important role in male-male competition, and also appears to be, albeit weakly, related to survival (e.g., Gallup et al., 2008). Results from several recent studies are summarized in Table 1, and suggest that HSG is a promising measure of masculinity, yet, also shows some heterogeneity. In addition, there seems to be a lack of studies investigating associations between HGS and other objective measures of

of masculinity/femininity are not well understood.

masculinity/femininity.

We measured bilateral asymmetry and masculinity/femininity from scans (HP scanjet G4050, 4800\*9600 DPI) of hands and photographs (Nikon D70, 6 megapixel) of faces of 52 men and 48 women with an average age of 22.6 (SD = 2.66) and 22.3 (SD = 1.87) years respectively. The degree of handedness was also self-evaluated on a scale of 0 (extreme lefthanded) to 10 (extreme right-handed). Hand-grip strength (HGS) was determined using a Biometrics precision dynamometer. For each participant, the strength was determined twice

Hand Grip Strength in Relation to Morphological Measures of

Masculinity, Fluctuating Asymmetry and Sexual Behaviour in Males And Females 297

Fig. 1. Top: Scan of right hand with indication of four digit lengths (D2 to D5) and width of hand palm (P) (left) and Landmarks located at both sides of facial photographs, and linear distances derived: eye width (EW: P2-P3), distance from cheekbone to corner of mouth (CM: P1- P6), distance between pupil and most lateral side of nostril (PN: P4-P5) (right). Bottom: Position of landmarks placed on pictures of all faces (see also Little et al., 2008). Sexual dimorphism was calculated by four measures: Cheekbone prominence (D1/D2); Face width/lower face height (D1/D3); Jaw height / lower face height (D4/D3) and Lower face

height / Face height (D3/D5).

on each side and the maximum value was obtained as HGS. All participants also completed a questionnaire asking for their age of first sexual contact and their total lifetime number of sexual partners. Each photograph was rated for its attractiveness by 10 to 30 opposite sex raters. As the repeatability of these ratings was about 30%, reliable estimates of attractiveness were obtained.

For each participant the length of the left and right 2nd (D2), 3rd (D3), 4th (D4) and 5th digit (D5) as well as the width of the palm of the hand (P) were independently measured 3 times and averaged (Fig.1). On each photograph, initially 7 landmarks were placed on each side of the face to obtain measures of facial asymmetry: i.e.; the width of the eye (EW), the distance between the pupil of the eye and the widest point at the side of the nostrils (EN), and the distance between the cheek bone and the corner of the mouth (CM) (Fig.1). Landmarks were placed in 3 independent sessions (i.e., on three separate days) and distances were averaged across sessions to reduce measurement error. In addition, since traits within hands and face showed correlations in the signed FA, traits were averaged within hands and faces to obtain two composite estimates (handFA and faceFA1, see Van Dongen et al., 2009 for details). The relative lengths of the second to fourth digit (2D:4D ratio) was also calculated (see also Van Dongen, 2009).

In addition, 19 landmarks were placed (Fig. 1) and based on these landmarks, a procrustes analysis was performed in MorphoJ (available at: http://www.flywings.org.uk/ MorphoJ\_page.htm; Klingenberg, 2011) to extract an overall measure of facial FA (faceFA2). In addition, facial masculinity was obtained as outlined in Little et al. (2008) and the EME angle was also calculated (Daniel & Pawlowski, 2007). Masculinity was also obtained from the procrustes analysis in MorphoJ by performing a canonical variate analysis for sexual dimorphism. This will allow to visualize the sexual dimorphism and to correlate the canonical variate with the measure obtained following Little et al (2008) and as outlined in Fig.1. An average measure of facial masculinity was obtained from the four individual measures after standardisation. All measurements were performed in ImageJ, freely available at http://rsb.info.nih.gov/ij/. First we tested if measures of masculinity differed between males and females using t-tests. The correlations among the masculinity measures (facial, EME angle, HGS, 2D:4D) were also graphically explored using a biplot from a principal component analysis. Next, correlations with FA, sexual behaviour and attractiveness were also investigated.

#### **2.2 Literature search and meta-analysis**

Studies investigating associations between HGS, attractiveness, FA, other forms of masculinity/femininity, sexual behaviour and dominance were obtained from Web of Science and PubMed. Six papers were found of which results are summarized in Table 1. Effect sizes (Pearson's correlations) from these studies as well as the results presented here were grouped in 5 different categories: masculinity measures (objective measurements on body or face); digit ratios; ratings (of masculinity, dominance, popularity); sexual behaviour (age of first contact, promiscuity) and attractiveness. Effect sizes in these categories and for males and females were presented in a funnel plot (i.e., in relation with sample size) to explore problems of publication bias. Effect sizes were then compared among the 5 categories and between males and females by a mixed model ANOVA with reference as random effect.

on each side and the maximum value was obtained as HGS. All participants also completed a questionnaire asking for their age of first sexual contact and their total lifetime number of sexual partners. Each photograph was rated for its attractiveness by 10 to 30 opposite sex raters. As the repeatability of these ratings was about 30%, reliable estimates of

For each participant the length of the left and right 2nd (D2), 3rd (D3), 4th (D4) and 5th digit (D5) as well as the width of the palm of the hand (P) were independently measured 3 times and averaged (Fig.1). On each photograph, initially 7 landmarks were placed on each side of the face to obtain measures of facial asymmetry: i.e.; the width of the eye (EW), the distance between the pupil of the eye and the widest point at the side of the nostrils (EN), and the distance between the cheek bone and the corner of the mouth (CM) (Fig.1). Landmarks were placed in 3 independent sessions (i.e., on three separate days) and distances were averaged across sessions to reduce measurement error. In addition, since traits within hands and face showed correlations in the signed FA, traits were averaged within hands and faces to obtain two composite estimates (handFA and faceFA1, see Van Dongen et al., 2009 for details). The relative lengths of the second to fourth digit (2D:4D ratio) was also calculated (see also Van

In addition, 19 landmarks were placed (Fig. 1) and based on these landmarks, a procrustes analysis was performed in MorphoJ (available at: http://www.flywings.org.uk/ MorphoJ\_page.htm; Klingenberg, 2011) to extract an overall measure of facial FA (faceFA2). In addition, facial masculinity was obtained as outlined in Little et al. (2008) and the EME angle was also calculated (Daniel & Pawlowski, 2007). Masculinity was also obtained from the procrustes analysis in MorphoJ by performing a canonical variate analysis for sexual dimorphism. This will allow to visualize the sexual dimorphism and to correlate the canonical variate with the measure obtained following Little et al (2008) and as outlined in Fig.1. An average measure of facial masculinity was obtained from the four individual measures after standardisation. All measurements were performed in ImageJ, freely available at http://rsb.info.nih.gov/ij/. First we tested if measures of masculinity differed between males and females using t-tests. The correlations among the masculinity measures (facial, EME angle, HGS, 2D:4D) were also graphically explored using a biplot from a principal component analysis. Next, correlations with FA, sexual behaviour and

Studies investigating associations between HGS, attractiveness, FA, other forms of masculinity/femininity, sexual behaviour and dominance were obtained from Web of Science and PubMed. Six papers were found of which results are summarized in Table 1. Effect sizes (Pearson's correlations) from these studies as well as the results presented here were grouped in 5 different categories: masculinity measures (objective measurements on body or face); digit ratios; ratings (of masculinity, dominance, popularity); sexual behaviour (age of first contact, promiscuity) and attractiveness. Effect sizes in these categories and for males and females were presented in a funnel plot (i.e., in relation with sample size) to explore problems of publication bias. Effect sizes were then compared among the 5 categories and between males and females by a mixed model ANOVA with reference as

attractiveness were obtained.

Dongen, 2009).

random effect.

attractiveness were also investigated.

**2.2 Literature search and meta-analysis** 

Fig. 1. Top: Scan of right hand with indication of four digit lengths (D2 to D5) and width of hand palm (P) (left) and Landmarks located at both sides of facial photographs, and linear distances derived: eye width (EW: P2-P3), distance from cheekbone to corner of mouth (CM: P1- P6), distance between pupil and most lateral side of nostril (PN: P4-P5) (right). Bottom: Position of landmarks placed on pictures of all faces (see also Little et al., 2008). Sexual dimorphism was calculated by four measures: Cheekbone prominence (D1/D2); Face width/lower face height (D1/D3); Jaw height / lower face height (D4/D3) and Lower face height / Face height (D3/D5).

Hand Grip Strength in Relation to Morphological Measures of

approach (procrustes masculinity).

F

F

By sex (males above diagonal/females below diagonal)

females separately (bottom table).

Across males and females:


Procrustes masculinity

Masculinity, Fluctuating Asymmetry and Sexual Behaviour in Males And Females 299

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Facial masculinity

 FAhand FAface1 FAface2 2D:4D masc\_face HGS angle #partners AFC attract FAhand - - - - - - FAface1 0.05 - - - - - - - - - FAface2 0.08 **0.42\*\*\*** - - - - - - - - 2D:4D -0.08 0.14 0.13 - - - - - - masc\_face -0.06 0.13 0.08 0.07 - - - - - - HGS 0.05 -0.01 -0.16 -0.05 **0.42\*\*\*** - - - - angle -0.12 -0.15 **-0.22\*** 0.02 -0.16 -0.06 - - - - #partners **-0.28\*\* -0.22\*** -0.01 -0.06 -0.07 -0.05 0.12 - - - AFC 0.17 **0.31\*\* 0.20** 0.02 -0.02 0.08 -0.04 -0.64\*\*\* - attract. -0.04 **0.21\*** 0.02 0.03 -0.08 **-0.25\*** 0.06 0.06 -0.14 -

 FAhand FAface1 FAface2 2D:4D masc\_face HGS angle #partners AFC attract FAhand - -0.13 -0.10 -0.10 -0.20 0.07 -0.04 -0.21 **0.29\*** 0.01 FAface1 0.26 - **0.40\*\*** 0.08 0.21 -0.03 0.12 -0.23 **0.33\*** -0.13 FAface2 **0.35 0.46\*\*** - 0.19 0.27 -0.11 -0.08 **-0.32\*** 0.20 0.08 2D:4D -0.01 0.24 0.00 - 0.27 0.04 0.10 -0.14 0.02 0.09 masc\_face 0.04 0.04 0.01 0.06 - 0.12 0.09 0.04 -0.17 0.26 HGS -0.08 -0.13 -0.24 0.17 0.25 - -0.03 0.09 -0.04 0.04 angle -0.27 **-0.33\* -0.40\*\*** -0.11 **-0.34\*** 0.11 - 0.17 0.01 0.06 #partners **-0.36\*** -0.21 0.03 0.00 -0.02 0.05 0.04 - **-0.65\*\*\*** -0.04 AFC -0.04 **0.29 0.24\*** -0.10 -0.05 -0.09 -0.08 **-0.62\*\*\*** - -0.07 attract -0.07 -0.26 -0.07 --0.11 -0.04 -0.18 0.01 0.04 -0.16 - Table 3. Correlation coefficients and statistical significance (\*: p<0.05; \*\*: p<0.01; \*\*\*: p<0.001, indicated in bold) of associations among fluctuating asymmetry (FA) values (hand and face), measures of masculinity (face, hand grip strength (HGS), eye-mouth-eye angle and digit ratio (2D:4D)), sexual behavior (number of partners and age of first sexual contact (AFC)) and attractiveness. Correlations are given across both sexes (top table) and for males and

masculinity) and the canonical variate obtained from the geometrics morphometrics

#### **3. Results**

#### **3.1 Measures of masculinity/femininity and sexual dimorphism**

Each of the four measures of masculinity (based on the landmarks in Fig. 1) showed a statistically significant sexual dimorphism (Table 2). Therefore, an average measure was obtained after standardisation (further called facial masculinity or masc\_face).


Table 2. Tests of facial sexual dimorphism in the four individual measures (Fig. 1).

Facial shape also differed significantly between males and females based on the geometric morphometrics approach (p<0.0001). The shape differences are given in Figure 2.

Fig. 2. Shape differences between males (light blue) and females (dark blue) from the canonical covariate analysis on the procrustes coordinates.

The canonical variate of the shape difference between males and females correlated strongly with facial masculinity as calculated above (see Table 2 for details). Thus, facial masculinity as measured by the relative proportions of different distances in the face (Table 2; Little et al., 2008) closely reflects the sexual dimorphism present in the landmarks used. We, therefore, used facial masculinity based on the relative proportions of the distances in Figure 1 for comparability with other studies.

Each of the four measures of masculinity (based on the landmarks in Fig. 1) showed a statistically significant sexual dimorphism (Table 2). Therefore, an average measure was

Cheekbone prominence 1.13 1.17 t97=4.25 <0.0001 Face width / lower face height 1.17 1.23 t97=4.50 <0.0001 Jaw height / lower face height 0.42 0.41 t97=-2.72 0.008 Lower face height / face height 0.59 0.57 t97=-3.14 0.002

Facial shape also differed significantly between males and females based on the geometric

19

<sup>1</sup> <sup>2</sup> 3 4 <sup>5</sup> <sup>6</sup> 7 8

9 10

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Table 2. Tests of facial sexual dimorphism in the four individual measures (Fig. 1).

morphometrics approach (p<0.0001). The shape differences are given in Figure 2.

Fig. 2. Shape differences between males (light blue) and females (dark blue) from the

The canonical variate of the shape difference between males and females correlated strongly with facial masculinity as calculated above (see Table 2 for details). Thus, facial masculinity as measured by the relative proportions of different distances in the face (Table 2; Little et al., 2008) closely reflects the sexual dimorphism present in the landmarks used. We, therefore, used facial masculinity based on the relative proportions of the distances in Figure

18

canonical covariate analysis on the procrustes coordinates.

1 for comparability with other studies.

males females t-statistic p-value

**3.1 Measures of masculinity/femininity and sexual dimorphism** 

obtained after standardisation (further called facial masculinity or masc\_face).

**3. Results** 

Fig. 3. Association between facial masculinity as obtained following Little et al. (2008) (facial masculinity) and the canonical variate obtained from the geometrics morphometrics approach (procrustes masculinity).


Table 3. Correlation coefficients and statistical significance (\*: p<0.05; \*\*: p<0.01; \*\*\*: p<0.001, indicated in bold) of associations among fluctuating asymmetry (FA) values (hand and face), measures of masculinity (face, hand grip strength (HGS), eye-mouth-eye angle and digit ratio (2D:4D)), sexual behavior (number of partners and age of first sexual contact (AFC)) and attractiveness. Correlations are given across both sexes (top table) and for males and females separately (bottom table).

Hand Grip Strength in Relation to Morphological Measures of

variation across two variables.

**3.2 Asymmetry measurements** 

for details).

PC3 (24%)


(F1798,1426=2.28, p<0.0001).

Masculinity, Fluctuating Asymmetry and Sexual Behaviour in Males And Females 301

masculinity traits confirmed the lack of strong correlations. The first principal component explained 37% of the total variation and was determined by HGS and facial masculinity. The second and third each explained about 25% of the total variation and each reflected one other variable, 2D:4D and EME angle respectively (Fig.6). Thus, in order to capture a large amount of the variation, three components were required, one of which only combined

The degree of measurement error (ME) of FA (i.e., the percentage of variance due to ME relative to the total variance (FA+ME)) due to scanning and placing landmarks, were the following: D2: 16%; D3: 20%; D4: 18%; D5: 13%. None of the hand measurements showed significant directional asymmetry (all p > 0.05). After standardization, asymmetries of handtraits were averaged into a single measure of asymmetry per individual (FAhand). Both handedness and asymmetry in power between right and left hand were significantly correlated with the signed asymmetry of the hand (handedness: r = 0.33, p = 0.001; power: r = 0.30, p = 0.001) (see also Van Dongen et al., 2009). For the three facial characteristics measurements were less accurate (EW: 57%; EN: 14%; CM: 73%). Facial FA showed significant directional asymmetry (all p > 0.05), two facial traits (EW: t99 = 3.2, p = 0.002 and

Fig. 6. 3D biplot of associations among the 4 measures of masculinity/Femininity (see text

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Because the three measurements of facial FA did not show high accuracy (though did show associations with sexual behavior, see Van Dongen et al., 2009 and below) we decided to take additional measurement on the face in the form of 19 landmarks. Procrustes ANOVA showed significant directional asymmetry (F31,1798=2.42, p<0.0001) and significant FA

PN: t99 = 1.98, p = 0.05) showed larger values on the right side, on average.


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Next to the facial masculinity studied here (which was significantly dimorphic: t97=5.69, p<0.0001), only one other measure of masculinity/femininity also showed a significant sexual dimorphism in our sample. Males showed significantly higher HGS (t97=10.1, p<0.0001), but no differences were observed for 2D:4D (t94=1.54, p=0.12) and the EME angle (t97=0.92, p=0.36) (see also Fig. 4). Across males and females, only HGS and facial masculinity showed a significant positive correlation (Table 3), a pattern that appeared consistent among both sexes (Fig.5), albeit not significantly so within sexes (Table 3). In woman, the EME angle and facial masculinity were negatively correlated, yet, unexpectedly, slightly positively in males (Table 3, Fig. 5). A principal component analysis of the

Fig. 4. Boxplots of the differences in measures of potentially sexually dimorphic traits: from top left to bottom right: facial masculinity based on the 19 landmarks, eye-mouth-eye angle, handgrip strength and digit ratio)

Fig. 5. Associations between measures of masculinity that were statistically significant (Table 3).

masculinity traits confirmed the lack of strong correlations. The first principal component explained 37% of the total variation and was determined by HGS and facial masculinity. The second and third each explained about 25% of the total variation and each reflected one other variable, 2D:4D and EME angle respectively (Fig.6). Thus, in order to capture a large amount of the variation, three components were required, one of which only combined variation across two variables.

#### **3.2 Asymmetry measurements**

300 Sex Hormones

Next to the facial masculinity studied here (which was significantly dimorphic: t97=5.69, p<0.0001), only one other measure of masculinity/femininity also showed a significant sexual dimorphism in our sample. Males showed significantly higher HGS (t97=10.1, p<0.0001), but no differences were observed for 2D:4D (t94=1.54, p=0.12) and the EME angle (t97=0.92, p=0.36) (see also Fig. 4). Across males and females, only HGS and facial masculinity showed a significant positive correlation (Table 3), a pattern that appeared consistent among both sexes (Fig.5), albeit not significantly so within sexes (Table 3). In woman, the EME angle and facial masculinity were negatively correlated, yet, unexpectedly,

slightly positively in males (Table 3, Fig. 5). A principal component analysis of the

Fig. 4. Boxplots of the differences in measures of potentially sexually dimorphic traits: from top left to bottom right: facial masculinity based on the 19 landmarks, eye-mouth-eye angle,

Fig. 5. Associations between measures of masculinity that were statistically significant

handgrip strength and digit ratio)

(Table 3).

The degree of measurement error (ME) of FA (i.e., the percentage of variance due to ME relative to the total variance (FA+ME)) due to scanning and placing landmarks, were the following: D2: 16%; D3: 20%; D4: 18%; D5: 13%. None of the hand measurements showed significant directional asymmetry (all p > 0.05). After standardization, asymmetries of handtraits were averaged into a single measure of asymmetry per individual (FAhand). Both handedness and asymmetry in power between right and left hand were significantly correlated with the signed asymmetry of the hand (handedness: r = 0.33, p = 0.001; power: r = 0.30, p = 0.001) (see also Van Dongen et al., 2009). For the three facial characteristics measurements were less accurate (EW: 57%; EN: 14%; CM: 73%). Facial FA showed significant directional asymmetry (all p > 0.05), two facial traits (EW: t99 = 3.2, p = 0.002 and PN: t99 = 1.98, p = 0.05) showed larger values on the right side, on average.

Fig. 6. 3D biplot of associations among the 4 measures of masculinity/Femininity (see text for details).

Because the three measurements of facial FA did not show high accuracy (though did show associations with sexual behavior, see Van Dongen et al., 2009 and below) we decided to take additional measurement on the face in the form of 19 landmarks. Procrustes ANOVA showed significant directional asymmetry (F31,1798=2.42, p<0.0001) and significant FA (F1798,1426=2.28, p<0.0001).

Hand Grip Strength in Relation to Morphological Measures of

**4. Discussion** 

**sexual behaviour** 

Masculinity, Fluctuating Asymmetry and Sexual Behaviour in Males And Females 303

0.12 (0.08)) and self rated dominance, aggression and popularity (average effect size: 0.09 (0.05)). These differences appeared comparable between males and females as there was no significant interaction (F5,68=2.00, p=0.09), but the power to detect an interaction was

probably small. Therefore, we also present average effect sizes by sex (Table 4).

Category sex effect size (SE) p-value 2D:4D males **0.15 (0.06)** 0.02 females **0.11 (0.06)** 0.10 Attractiveness\* males **0.24 (0.09)** 0.01 females -0.07 (0.12) 0.57 Masculinity males **0.24 (0.06)** <0.001 females **0.24 (0.06)** <0.001 Ratings\* males **0.29 (0.05)** <0.001 females 0.10 (0.08) 0.24 self ratings males **0.12 (0.05)** 0.04 females 0.06 (0.05) 0.30 sexual behavior\* males **0.24 (0.06)** <0.001 females 0.04 (0.06) 0.52 Table 4. Average weighted effect sizes of the associations between hand grip strength and other measures of masculinity (masculinity: objective measurements; ratings: ratings of masculinity and dominance by opposite sex raters; self ratings: own evaluations of

masculinity, dominance, popularity; digit ratios (2D:4D), attractiveness and sexual behavior) for males and females. Categories where males have a significantly higher effect size are indicated by a \* (although the interaction was not statistically significant, see text for details).

**4.1 Associations between masculinity, attractiveness, fluctuating asymmetry and** 

This study, albeit small in terms of new data added to the existing literature, did not provide strong evidence that measures of masculinity would be related to sexual behaviour, attractiveness or fluctuating asymmetry. Clearly, sample sizes were relatively small, yet, it did allow to detect robust associations between FA and measures of sexual behaviour (see Van Dongen et al., 2009 for further discussion), but not attractiveness (this study). Thus, this suggests that sample sizes were sufficiently large for some aspects (i.e., associations with FA), and that asymmetry may be more closely related to sexual behaviour and promiscuity that masculinity. Nevertheless, many others have shown associations between masculinity and both attractiveness and sexual behaviour, such that this small study clearly cannot cast any doubt on the relevance of masculinity and hormone levels in human sexual behaviour and attractiveness. However, there is some doubt about the associations among different measures of masculinity and their association with sex-hormone levels (e.g., Koehler et al., 2004; Campbell et al., 2010). In this study, associations among the four objective measures were weak, with the exception of the association between facial masculinity and hand grip

Fig. 7. Associations between eye-mouth-eye angle and facial asymmetry in males (dashed line) and females (solid line).

#### **3.3 Correlations among FA, masculinity and sexual behaviour**

All correlations, across both sexes and for males and females separately, are provided in Table 3. It is important to realize that many tests are being performed and some of them are significant at the 5% level just by chance. It is, therefore, only relevant to interpret correlations significant at the 5%/135=0.04% level (after Bonferonni correction). The only correlations which are significant at this level are situated between FA and sexual behaviour, between the number of partners and age of first sexual contact, between the two measures of facial FA, and between facial masculinity and HGS (Table 3). One correlation that is worth mentioning (albeit not significant after Bonferonni correction) is the negative association between EME angle and facial FA in woman (Table 3) indicating a wider (more feminine) EME angle to be associated with higher facial FA (Fig.7).

#### **3.4 Meta-analysis**

A funnel graph of all available effect sizes is provided in figure 8. There does not appear to be a problem of publication bias (correlation between sample size and effect size = -0.11, d.f.=81, p=0.29). 16 out of 83 estimates were statistically significant (20%) and 64 out of 83 estimates were in the expected direction (i.e., a positive effect size) (77%), a proportion that is significantly higher than 50% (p<0.0001). The average weighted effect size across all estimates equalled 0.19 (0.05), which was significantly different from zero (t7=4.00, p=0.007). Thus, on average there appears to be a robust correlation. However, average effect sizes were only half as high in females (difference=-0.10 (0.03), F1,74=11.3, p=0.001), and differed significantly among the broad categories of masculinity and sexual behaviour (F1,66=3.06, p=0.015). Although most two-by-two comparisons were not statistically significant, averaged across males and females, the highest effect sizes that were significant at the 0.01 level were found for objective measurements of bodily and facial masculinity (average effect size: 0.24 (0.05)) and ratings of dominance and attractiveness of opposite sex raters (average effect size: 0.22 (0.05)). Lower effect sizes, albeit still significant at the 0.05 level, were observed for 2D:4D (average effect size: 0.13 (0.05)) and measures of sexual behaviour (average effect size: 0.14 (0.05)). The remaining two were even somewhat lower, no longer statistically significant but still in the expected direction: attractiveness (average effect size: 0.12 (0.08)) and self rated dominance, aggression and popularity (average effect size: 0.09 (0.05)). These differences appeared comparable between males and females as there was no significant interaction (F5,68=2.00, p=0.09), but the power to detect an interaction was probably small. Therefore, we also present average effect sizes by sex (Table 4).


Table 4. Average weighted effect sizes of the associations between hand grip strength and other measures of masculinity (masculinity: objective measurements; ratings: ratings of masculinity and dominance by opposite sex raters; self ratings: own evaluations of masculinity, dominance, popularity; digit ratios (2D:4D), attractiveness and sexual behavior) for males and females. Categories where males have a significantly higher effect size are indicated by a \* (although the interaction was not statistically significant, see text for details).

#### **4. Discussion**

302 Sex Hormones

Fig. 7. Associations between eye-mouth-eye angle and facial asymmetry in males (dashed

40 45 50 55

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All correlations, across both sexes and for males and females separately, are provided in Table 3. It is important to realize that many tests are being performed and some of them are significant at the 5% level just by chance. It is, therefore, only relevant to interpret correlations significant at the 5%/135=0.04% level (after Bonferonni correction). The only correlations which are significant at this level are situated between FA and sexual behaviour, between the number of partners and age of first sexual contact, between the two measures of facial FA, and between facial masculinity and HGS (Table 3). One correlation that is worth mentioning (albeit not significant after Bonferonni correction) is the negative association between EME angle and facial FA in woman (Table 3) indicating a wider (more

A funnel graph of all available effect sizes is provided in figure 8. There does not appear to be a problem of publication bias (correlation between sample size and effect size = -0.11, d.f.=81, p=0.29). 16 out of 83 estimates were statistically significant (20%) and 64 out of 83 estimates were in the expected direction (i.e., a positive effect size) (77%), a proportion that is significantly higher than 50% (p<0.0001). The average weighted effect size across all estimates equalled 0.19 (0.05), which was significantly different from zero (t7=4.00, p=0.007). Thus, on average there appears to be a robust correlation. However, average effect sizes were only half as high in females (difference=-0.10 (0.03), F1,74=11.3, p=0.001), and differed significantly among the broad categories of masculinity and sexual behaviour (F1,66=3.06, p=0.015). Although most two-by-two comparisons were not statistically significant, averaged across males and females, the highest effect sizes that were significant at the 0.01 level were found for objective measurements of bodily and facial masculinity (average effect size: 0.24 (0.05)) and ratings of dominance and attractiveness of opposite sex raters (average effect size: 0.22 (0.05)). Lower effect sizes, albeit still significant at the 0.05 level, were observed for 2D:4D (average effect size: 0.13 (0.05)) and measures of sexual behaviour (average effect size: 0.14 (0.05)). The remaining two were even somewhat lower, no longer statistically significant but still in the expected direction: attractiveness (average effect size:

**3.3 Correlations among FA, masculinity and sexual behaviour** 

feminine) EME angle to be associated with higher facial FA (Fig.7).

line) and females (solid line).


 0 1

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**3.4 Meta-analysis** 

#### **4.1 Associations between masculinity, attractiveness, fluctuating asymmetry and sexual behaviour**

This study, albeit small in terms of new data added to the existing literature, did not provide strong evidence that measures of masculinity would be related to sexual behaviour, attractiveness or fluctuating asymmetry. Clearly, sample sizes were relatively small, yet, it did allow to detect robust associations between FA and measures of sexual behaviour (see Van Dongen et al., 2009 for further discussion), but not attractiveness (this study). Thus, this suggests that sample sizes were sufficiently large for some aspects (i.e., associations with FA), and that asymmetry may be more closely related to sexual behaviour and promiscuity that masculinity. Nevertheless, many others have shown associations between masculinity and both attractiveness and sexual behaviour, such that this small study clearly cannot cast any doubt on the relevance of masculinity and hormone levels in human sexual behaviour and attractiveness. However, there is some doubt about the associations among different measures of masculinity and their association with sex-hormone levels (e.g., Koehler et al., 2004; Campbell et al., 2010). In this study, associations among the four objective measures were weak, with the exception of the association between facial masculinity and hand grip

Hand Grip Strength in Relation to Morphological Measures of

**4.2 Handgrip strength as a measure of masculinity** 

**5. Conclusion** 

**6. References** 

Masculinity, Fluctuating Asymmetry and Sexual Behaviour in Males And Females 305

Our results show that HGS relates to facial masculinity (but not 2D:4D) in both males and females. HGS has only recently been put forward as a useful measure of masculinity (Table 1), and we here present an overview of the current literature. There appears to be a highly significant and robust average weighted effect size of about 0.2, of correlations between HGS and different correlates of masculinity/femininity. There also appears to be some variation in the effect sizes. On the average, effect sizes were smaller in females and lowest for sexual behaviour and self rated dominance, aggression and popularity. Although there was no significant interaction between sex and type of masculinity measure, the p value was only 0.09, suggesting that the difference between may not have been similar for the different categories. Although we should interpret these test with caution (and await further study), the differences in effect sizes between males and females were strongest for attractiveness, sexual behaviour and rated masculinity. In each of these, relatively strong average effect sizes were observed for males, and nearly zero for females (Table 4). Thus, HGS appears to be related to objectively measured masculinity in both males and females (Table 4 and data from this study), and to a lesser extent with 2D:4D (Table 4). For all other categories, no significant associations were found for females (Table 4). Although it may be to preliminary at this point to make any firm conclusions, our results and the combined analysis of the data from the literature suggests that HGS relates to morphological measures of masculinity alone in females, but also to attractiveness, rated and self-rated masculinity and dominance and sexual behaviour in males.

In this paper we study associations between objective morphological measures of masculinity/femininity and physical strength (handgrip strength) in relation to developmental instability (as measured by fluctuating asymmetry, FA), attractiveness and sexual behaviour. In spite of the relatively small sample sizes, we were able to detect associations between FA and sexual behaviour (further discussed in Van Dongen et al., 2009), yet not with our measures of masculinity. We next focussed on a relatively recently studied measure of masculinity/femininity, namely physical strength expressed as handgrip strength (HGS). We reviewed results from the recent literature and demonstrated a robust association between HGS and other measures of masculinity/femininity. In addition, we were able to detect some sources of variation. On the one hand, HGS related to morphological features of bodily masculinity (and to a lesser extent but still significantly so to 2D:4D ratios) equally strong in both males and females. However, associations between HGS and either attractiveness, (self-)ratings of dominance, masculinity and popularity and sexual behaviour were weaker or absent in females compared to males. Thus, based on the available literature we conclude that physical strength is determined by circulating hormones affecting morphologically dimorphic structures, yet affects behaviour and the physical expression of it in males only. Physical strength and masculinity is thus likely to play a role in male-male competition and as a signal of mate value in sexual selection.

Campbell, B.C., Dreber, A., Apicella, C.L., Eisenberg, D.T.A, Gray, P.B., Little, A.C., Garcia,

and sensation seeking in young men. Physiology and behavior 99: 451-456.

J.R., Zamore, R.S. & Lum, J.K. 2010. Testosterone exposure, dopaminergic reward,

strength. HGS also showed a clear sexual dimorphism, as did facial masculinity. However, eye-mouth-eye angle and 2D:4D did not show correlations with facial masculinity or HGS and were not sexually dimorphic. Results for 2D:4D are discussed elsewhere (Van Dongen 2009). For EME angle, one study of similar size as this one did show a sexual dimorphism and associations with attractiveness (Danel & Pawlowski, 2007). The results presented here thus question the generality of the usefulness of EME angle as a measure of masculinity and calls for further research. In spite of the fact that EME angle did not show a sexual dimorphism in this study and did not relate to masculinity (except perhaps weakly in woman), sexual behaviour or attractiveness, there was some suggestion that it correlated with facial FA. This certainly warrants further study since associations between FA and measures of masculinity are at best very weak and results vary among studies (Van Dongen submitted manuscript).

Fig. 8. Funnel graph (effect sizes vs. sample size) of the associations between hand grip strength and other measures of masculinity (masc: objective measurements; ratings: ratings of masculinity and dominance by opposite sex raters), self ratings (own evaluations of masculinity, dominance, popularity, …), digit ratios (DR: 2D:4D), attractiveness (attr) and sexual behavior (sex\_beh). Dash-dotted lines represent critical values for the effect sizes, where more extreme values are statistically significant at the 0.05 level. The solid line the lowess curve of the association (or rather the lack of it in this case). effect sizes for males are given in black, those of females in grey, and estimates from this study are provided in a larger bold font.

#### **4.2 Handgrip strength as a measure of masculinity**

Our results show that HGS relates to facial masculinity (but not 2D:4D) in both males and females. HGS has only recently been put forward as a useful measure of masculinity (Table 1), and we here present an overview of the current literature. There appears to be a highly significant and robust average weighted effect size of about 0.2, of correlations between HGS and different correlates of masculinity/femininity. There also appears to be some variation in the effect sizes. On the average, effect sizes were smaller in females and lowest for sexual behaviour and self rated dominance, aggression and popularity. Although there was no significant interaction between sex and type of masculinity measure, the p value was only 0.09, suggesting that the difference between may not have been similar for the different categories. Although we should interpret these test with caution (and await further study), the differences in effect sizes between males and females were strongest for attractiveness, sexual behaviour and rated masculinity. In each of these, relatively strong average effect sizes were observed for males, and nearly zero for females (Table 4). Thus, HGS appears to be related to objectively measured masculinity in both males and females (Table 4 and data from this study), and to a lesser extent with 2D:4D (Table 4). For all other categories, no significant associations were found for females (Table 4). Although it may be to preliminary at this point to make any firm conclusions, our results and the combined analysis of the data from the literature suggests that HGS relates to morphological measures of masculinity alone in females, but also to attractiveness, rated and self-rated masculinity and dominance and sexual behaviour in males.

#### **5. Conclusion**

304 Sex Hormones

strength. HGS also showed a clear sexual dimorphism, as did facial masculinity. However, eye-mouth-eye angle and 2D:4D did not show correlations with facial masculinity or HGS and were not sexually dimorphic. Results for 2D:4D are discussed elsewhere (Van Dongen 2009). For EME angle, one study of similar size as this one did show a sexual dimorphism and associations with attractiveness (Danel & Pawlowski, 2007). The results presented here thus question the generality of the usefulness of EME angle as a measure of masculinity and calls for further research. In spite of the fact that EME angle did not show a sexual dimorphism in this study and did not relate to masculinity (except perhaps weakly in woman), sexual behaviour or attractiveness, there was some suggestion that it correlated with facial FA. This certainly warrants further study since associations between FA and measures of masculinity are at best very weak and results vary among studies (Van Dongen submitted manuscript).

Fig. 8. Funnel graph (effect sizes vs. sample size) of the associations between hand grip strength and other measures of masculinity (masc: objective measurements; ratings: ratings of masculinity and dominance by opposite sex raters), self ratings (own evaluations of masculinity, dominance, popularity, …), digit ratios (DR: 2D:4D), attractiveness (attr) and sexual behavior (sex\_beh). Dash-dotted lines represent critical values for the effect sizes, where more extreme values are statistically significant at the 0.05 level. The solid line the lowess curve of the association (or rather the lack of it in this case). effect sizes for males are given in black, those of females in grey, and estimates from this study are provided in a

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In this paper we study associations between objective morphological measures of masculinity/femininity and physical strength (handgrip strength) in relation to developmental instability (as measured by fluctuating asymmetry, FA), attractiveness and sexual behaviour. In spite of the relatively small sample sizes, we were able to detect associations between FA and sexual behaviour (further discussed in Van Dongen et al., 2009), yet not with our measures of masculinity. We next focussed on a relatively recently studied measure of masculinity/femininity, namely physical strength expressed as handgrip strength (HGS). We reviewed results from the recent literature and demonstrated a robust association between HGS and other measures of masculinity/femininity. In addition, we were able to detect some sources of variation. On the one hand, HGS related to morphological features of bodily masculinity (and to a lesser extent but still significantly so to 2D:4D ratios) equally strong in both males and females. However, associations between HGS and either attractiveness, (self-)ratings of dominance, masculinity and popularity and sexual behaviour were weaker or absent in females compared to males. Thus, based on the available literature we conclude that physical strength is determined by circulating hormones affecting morphologically dimorphic structures, yet affects behaviour and the physical expression of it in males only. Physical strength and masculinity is thus likely to play a role in male-male competition and as a signal of mate value in sexual selection.

#### **6. References**

Campbell, B.C., Dreber, A., Apicella, C.L., Eisenberg, D.T.A, Gray, P.B., Little, A.C., Garcia, J.R., Zamore, R.S. & Lum, J.K. 2010. Testosterone exposure, dopaminergic reward, and sensation seeking in young men. Physiology and behavior 99: 451-456.

**1. Introduction** 

**15** 

*USA* 

**Sex Differences in the Developmental** 

A significant body of knowledge has established that stressors in early life have long-term health consequences on the adult organism. This has given rise to the Developmental Origins of Health and Adult Disease (DOHAD) hypothesis. Among the several broad themes that have emerged from the clinical and experimental investigations into the DOHAD hypothesis; perhaps none is as intriguing as the role of biological sex and sex hormones in the progression and development of adult diseases. Despite the significant progress in recent years, many uncertainties remain with respect to the roles of biological sex and gonadal steroids in the progression of human diseases in general, and in the mechanisms underlying sex differences in developmental programming in particular. While sexual dimorphism is widely recognized in the progression of many diseases (e.g. cardiovascular), it appears that the primary pathways leading to these differences exert distinct influences during fetal and adult life. The mechanisms by which biological sex contributes to these processes is a rapidly expanding area of investigation drawing upon studies interrogating systems at the molecular, cellular and whole organism physiological levels. From these investigations several intriguing hypotheses have been proposed. These include developmental programming due to: 1) endocrine disruption resulting from exogenous sex steroids and/or analogs or nutritional stress during development; 2) chromosomal regulation of sex dimorphism in the transcriptome of mammalian tissues; and 3) sex specific responses to stressors during fetal life. The goal of this chapter is to place into perspective the current body of knowledge in the rapidly growing area of sex differences in

developmental programming with a primary focus on cardiovascular diseases.

Developmental programming can be defined as the response by the developing organism to specific stimuli during critical periods of organogenesis that results in persistent effects on the adult phenotype. It is now recognized as an important determinant of adult health. Acceptance and understanding of this concept derives from human epidemiological studies suggesting that many metabolic diseases such as cardiovascular disease (Barker, 1993), chronic kidney disease (Li *et al.*, 2007), type II diabetes mellitus (Hovi *et al.*, 2007;Hofman *et al.*, 1997), and hypertension (Roseboom *et al.*, 2001) are associated with low birth weight. Since developing organisms pass more physiological benchmarks prior to birth than during

**2. Periods of susceptibility to developmental programming** 

**Programming of Adult Disease** 

Jeffrey S. Gilbert and Christopher T. Banek *Department of Human Physiology, University of Oregon* 


### **Sex Differences in the Developmental Programming of Adult Disease**

Jeffrey S. Gilbert and Christopher T. Banek *Department of Human Physiology, University of Oregon USA* 

#### **1. Introduction**

306 Sex Hormones

Danel, D. & Pawlowski, B. 2007. Eye-mouth-eye angle as a good indicator of face

Fink, B., Neave, N. & Seydel, H. 2007. Male facial appearance signals physical strength to

Gallup, A.C., White, D.D. & Gallup G.G. 2007. Handgrip strength predicts sexual behavior,

Gallup, A.C., O'Brien, D.T., White, D.D. & Wilson, D.S. 2010. Handgrip strength and socially dominant behavior in male adolescents. Evolutionary Psychology 8: 229-243. Garver-Apgar, C.E., Eaton, M.A., Tybur, J.M. & Thompson, M.E. 2011. Evidence of

Klingenberg, C. P. 2011. MorphoJ: an integrated software package for geometric

Koehler, N., Simmons, L.W., & Rhodes, G. 2004. How well does second-to-fourth-digit ratio

Kokko, H., Brooks, R., McNamara, J.M. & Houston, A.I. (2002). The sexual selection continuum. *Proceedings of the Royal Society of London* B, 269,1331-1340. Little, A. C., Jones, B. C., Waitt, C., Tiddeman, B. P., Feinberg, D. R., Perrett, D. I. Apicella, C.

Puts, D. A. 2010. Beauty and the beast: Mechanisms of sexual selection in humans. *Evolution* 

Shoup, M.L. & Gallup, G.G. 2008. Men's faces convey information about their bodies and their behavior: what you see is what you get. Evolutionary psychology 6: 469-479. Thornhill, R. & Gangestad, S.W. 1999. Facial attractiveness. trends in Cognitive Sciences. 3:

Van Dongen Stefan. (2009). A critical re-evaluation of the association between 2D:4D ratios and fluctuating asymmetry in humans. *Annals of human biology, 36,* 186-198. Van Dongen, S., Cornille, R. & Lens, L. 2009. Sex and asymmetry in humans: What is the role of developmental instability. *Journal of Evolutionary Biology*, 22, 612-622

Getty, T. (2002). Signaling health versus parasites. *American Naturalist, 159,* 363-371.

morphometrics. *Molecular Ecology Resources* 11: 353-357

Data across culture and species. *PLoS One*, *3*, e2106.

comparative Psychology 121: 221-225.

behavior. 28: 423-429.

B (suppl.) 271: S296-S298.

*and Human Behavior, 31,*157-175.

452-460

women. American Journal of Human Biology 19: 82-87.

masculinization, asymmetry, and attractiveness (Homo sapiens). Journal of

body morphology, and aggression in male college students. Evolution and human

intralocus sexual conflict: physically and hormonally masculine individuals have more attractive brothers relative to sisters. Evolution and human behavior in press.

in hands correlate with other indications of masculinity in males? Proc R Soc. Lond

L. & Marlowe, F. W. (2008). Symmetry is related to sexual dimorphism in faces:

A significant body of knowledge has established that stressors in early life have long-term health consequences on the adult organism. This has given rise to the Developmental Origins of Health and Adult Disease (DOHAD) hypothesis. Among the several broad themes that have emerged from the clinical and experimental investigations into the DOHAD hypothesis; perhaps none is as intriguing as the role of biological sex and sex hormones in the progression and development of adult diseases. Despite the significant progress in recent years, many uncertainties remain with respect to the roles of biological sex and gonadal steroids in the progression of human diseases in general, and in the mechanisms underlying sex differences in developmental programming in particular.

While sexual dimorphism is widely recognized in the progression of many diseases (e.g. cardiovascular), it appears that the primary pathways leading to these differences exert distinct influences during fetal and adult life. The mechanisms by which biological sex contributes to these processes is a rapidly expanding area of investigation drawing upon studies interrogating systems at the molecular, cellular and whole organism physiological levels. From these investigations several intriguing hypotheses have been proposed. These include developmental programming due to: 1) endocrine disruption resulting from exogenous sex steroids and/or analogs or nutritional stress during development; 2) chromosomal regulation of sex dimorphism in the transcriptome of mammalian tissues; and 3) sex specific responses to stressors during fetal life. The goal of this chapter is to place into perspective the current body of knowledge in the rapidly growing area of sex differences in developmental programming with a primary focus on cardiovascular diseases.

#### **2. Periods of susceptibility to developmental programming**

Developmental programming can be defined as the response by the developing organism to specific stimuli during critical periods of organogenesis that results in persistent effects on the adult phenotype. It is now recognized as an important determinant of adult health. Acceptance and understanding of this concept derives from human epidemiological studies suggesting that many metabolic diseases such as cardiovascular disease (Barker, 1993), chronic kidney disease (Li *et al.*, 2007), type II diabetes mellitus (Hovi *et al.*, 2007;Hofman *et al.*, 1997), and hypertension (Roseboom *et al.*, 2001) are associated with low birth weight. Since developing organisms pass more physiological benchmarks prior to birth than during

Sex Differences in the Developmental Programming of Adult Disease 309

as male fetuses become larger than age-matched females (Hindmarsh *et al.*, 2002;Crawford *et al.*, 1987;Parker *et al.*, 1984). From clinical and experimental studies we know that this size difference persists to term (Hindmarsh *et al.*, 2002;Parker *et al.*, 1984;Gilbert *et al.*, 2007a;Gilbert *et al.*, 2006a). Underlying these morphological differences are specific sex related differences in endocrinology and metabolism. While androgens are recognized for their role in male maturation, they are also essential to development of the female fetus. Production of androgens in both the ovaries and the adrenal cortex in females is essential to folliculogenesis and mammary development. Levels below the required amount for normal development have been shown to diminish the development of the tissues aforementioned. Exposure of female fetuses to androgens may not necessarily disrupt normal development of the ovaries, but may result in altered expression of steroidogenic proteins (Hogg *et al.*, 2011). Increased androgens are also associated with female fetuses developing male-like sexual behavior, increased aggression, delayed vaginal opening (Meisel & Ward, 1981). Sex differences at the molecular level also persist from embryonic into fetal life. Baserga *et al.* have reported that gestation in the rat cyclooxygenase-2 (COX-2) levels were higher in the female than the male kidney at day of gestation (DG) 8, although not significantly increased at DG 21. In contrast, 11β-Hydroxysteroid Dehydrogenase 2 (11β-HSD2) levels were higher in the male control kidney at DG 21. Both of these gene products play important roles in renal function and alterations in either could have developmental and/or functional effects in the kidney (Baserga *et al.*, 2007b). Similarly, sex differences have also been reported in the

Similar to the kidney, the mammary gland undergoes discrete phases of development. However, in contrast to the kidney, the mammary has important developmental phases that extend into adulthood. In early pregnancy, the processes of fetal mammary development are thought to occur independently of influences from systemic hormones (Hennighausen & Robinson, 2001). After mid-gestation, placental hormones enter fetal circulation and initiate canalization of the early ductal system. It is during this period that exposure to endocrine mimetics may exert an influence on subsequent risk of breast cancer in the offspring (Xue & Michels, 2007). While the origin and the purpose of these sex differences in fetal development remain unclear, it may simply reflect different trajectories of fetal development between the sexes; however, this may also underlie sex differences in developmental

It has long been observed that growth restricted fetuses which develop metabolic syndrome often experience "catch-up growth" and surpass normal birth weight controls (Hales & Ozanne, 2003). While it is clear that catch-up growth plays a role in the manifestations of developmental programming it has been difficult to identify the specific peri-natal *vs*. postnatal influences involved. Disordered vascular function is thought to contribute to programming of cardiovascular health but the cause and effect relationships remain uncertain (Martin *et al.*, 2000;Leeson *et al.*, 2001;Goodfellow *et al.*, 1998). There are recognized sex differences in arterial pressure and the progression of renal disease, both of which are thought to involve interactions of the renin angiotensin system and sex steroids (Sandberg & Ji, 2003;Silva-Antonialli *et al.*, 2004). Most current evidence points to sex steroids as the most

important factor influencing sex differences in post-natal cardiovascular function.

ontogeny of gene expression in the renal RAS (Gilbert *et al.*, 2007a).

programming.

**2.3 Post-natal** 

any other time in life, it is not surprising that deviations in the timing or nature of these developmental steps have functional consequences in later life. Hence, it is vitally important to understand early life gene-environment interactions that can increase predisposition to adult disease. Figure 1 provides an overview of the timing of the susceptibilities for various organs systems and processes in developmental programming.

Fig. 1. *Organogenesis and Maturation Disruption.* Timeline depicts separate stages of mammalian organ development and maturation and the relative sensitivity to various programming stimuli. Listed above are necessary processes during this period for normal growth and the activity (light blue). At the top are the perturbations that can interfere with proper development.

#### **2.1 Embryonic development**

Susceptibility to programming events begins very early in life and surprisingly sex differences are already present. The transcriptome of male and female pre-implantation embryos differ such that several genes located on the X chromosome are more expressed in bovine and human female versus male embryos (Gutierrez-Adan *et al.*, 2000;Taylor *et al.*, 2001;Wrenzycki *et al.*, 2002;Peippo *et al.*, 2002) while autosomal genes expressed in trophoblast cells, such as those for interferon-γ (Larson *et al.*, 2001), human choriogonadotropic hormone (Haning, Jr. *et al.*, 1989), and numerous other imprinted genes (Paldi *et al.*, 1995;Kovtun *et al.*, 2000;Durcova-Hills *et al.*, 2004) are also not expressed or methylated the same across the sexes. Morphological differences exist as well; male and female embryos differ in rates of development as early as the first few days post-fertilization. Bovine (Avery *et al.*, 1992;Yadav *et al.*, 1993), murine (Valdivia *et al.*, 1993) and ovine embryos (Bernardi & Delouis, 1996) produced *in vitro* often fall into fast-cleaving and slow-cleaving groups that are predominantly male and female, respectively. Interestingly, Sood *et al.* reported a sex dichotomy in the genes expressed in male and female placentas (Sood *et al.*, 2006) using microarray analysis and identified genes in villous samples such as JAK1, IL2RB, Clusterin, LTBP, CXCL1, and IL1RL1 that were expressed at higher levels in female placentas.

#### **2.2 Fetal development**

The fetal period is a critical time for organogenesis. As gestation progresses and the embryo becomes a fetus, the sex dimorphism of early development re-appears around mid-gestation

as male fetuses become larger than age-matched females (Hindmarsh *et al.*, 2002;Crawford *et al.*, 1987;Parker *et al.*, 1984). From clinical and experimental studies we know that this size difference persists to term (Hindmarsh *et al.*, 2002;Parker *et al.*, 1984;Gilbert *et al.*, 2007a;Gilbert *et al.*, 2006a). Underlying these morphological differences are specific sex related differences in endocrinology and metabolism. While androgens are recognized for their role in male maturation, they are also essential to development of the female fetus. Production of androgens in both the ovaries and the adrenal cortex in females is essential to folliculogenesis and mammary development. Levels below the required amount for normal development have been shown to diminish the development of the tissues aforementioned. Exposure of female fetuses to androgens may not necessarily disrupt normal development of the ovaries, but may result in altered expression of steroidogenic proteins (Hogg *et al.*, 2011). Increased androgens are also associated with female fetuses developing male-like sexual behavior, increased aggression, delayed vaginal opening (Meisel & Ward, 1981).

Sex differences at the molecular level also persist from embryonic into fetal life. Baserga *et al.* have reported that gestation in the rat cyclooxygenase-2 (COX-2) levels were higher in the female than the male kidney at day of gestation (DG) 8, although not significantly increased at DG 21. In contrast, 11β-Hydroxysteroid Dehydrogenase 2 (11β-HSD2) levels were higher in the male control kidney at DG 21. Both of these gene products play important roles in renal function and alterations in either could have developmental and/or functional effects in the kidney (Baserga *et al.*, 2007b). Similarly, sex differences have also been reported in the ontogeny of gene expression in the renal RAS (Gilbert *et al.*, 2007a).

Similar to the kidney, the mammary gland undergoes discrete phases of development. However, in contrast to the kidney, the mammary has important developmental phases that extend into adulthood. In early pregnancy, the processes of fetal mammary development are thought to occur independently of influences from systemic hormones (Hennighausen & Robinson, 2001). After mid-gestation, placental hormones enter fetal circulation and initiate canalization of the early ductal system. It is during this period that exposure to endocrine mimetics may exert an influence on subsequent risk of breast cancer in the offspring (Xue & Michels, 2007). While the origin and the purpose of these sex differences in fetal development remain unclear, it may simply reflect different trajectories of fetal development between the sexes; however, this may also underlie sex differences in developmental programming.

#### **2.3 Post-natal**

308 Sex Hormones

any other time in life, it is not surprising that deviations in the timing or nature of these developmental steps have functional consequences in later life. Hence, it is vitally important to understand early life gene-environment interactions that can increase predisposition to adult disease. Figure 1 provides an overview of the timing of the susceptibilities for various

> **Endogenous and exogenous interference in sex hormone levels result in disrupted reproductive organ development, as well as cardiovascular, renal, sexual, and behavior health perturbation**

Embryonic Prenatal Perinatal Pubescent Adult

Susceptibility to programming events begins very early in life and surprisingly sex differences are already present. The transcriptome of male and female pre-implantation embryos differ such that several genes located on the X chromosome are more expressed in bovine and human female versus male embryos (Gutierrez-Adan *et al.*, 2000;Taylor *et al.*, 2001;Wrenzycki *et al.*, 2002;Peippo *et al.*, 2002) while autosomal genes expressed in trophoblast cells, such as those for interferon-γ (Larson *et al.*, 2001), human choriogonadotropic hormone (Haning, Jr. *et al.*, 1989), and numerous other imprinted genes (Paldi *et al.*, 1995;Kovtun *et al.*, 2000;Durcova-Hills *et al.*, 2004) are also not expressed or methylated the same across the sexes. Morphological differences exist as well; male and female embryos differ in rates of development as early as the first few days post-fertilization. Bovine (Avery *et al.*, 1992;Yadav *et al.*, 1993), murine (Valdivia *et al.*, 1993) and ovine embryos (Bernardi & Delouis, 1996) produced *in vitro* often fall into fast-cleaving and slow-cleaving groups that are predominantly male and female, respectively. Interestingly, Sood *et al.* reported a sex dichotomy in the genes expressed in male and female placentas (Sood *et al.*, 2006) using microarray analysis and identified genes in villous samples such as JAK1, IL2RB, Clusterin, LTBP, CXCL1, and IL1RL1 that were

The fetal period is a critical time for organogenesis. As gestation progresses and the embryo becomes a fetus, the sex dimorphism of early development re-appears around mid-gestation

Fig. 1. *Organogenesis and Maturation Disruption.* Timeline depicts separate stages of mammalian organ development and maturation and the relative sensitivity to various programming stimuli. Listed above are necessary processes during this period for normal growth and the activity (light blue). At the top are the perturbations that can interfere with

**Reproductive Organ Development and Maturation**

**Cardiovascular and Renal Function Decrease**

**Decreases in circulating sex hormones and steroids**

organs systems and processes in developmental programming.

**Cell Signaling, Organization, and Migration**

proper development.

**2.1 Embryonic development** 

expressed at higher levels in female placentas.

**2.2 Fetal development** 

**Epigenetic and genetic insults can prevent necessary genes from normal expression activity and have a lifetime impact**

**Organ Programming, Development, and Maturation**

It has long been observed that growth restricted fetuses which develop metabolic syndrome often experience "catch-up growth" and surpass normal birth weight controls (Hales & Ozanne, 2003). While it is clear that catch-up growth plays a role in the manifestations of developmental programming it has been difficult to identify the specific peri-natal *vs*. postnatal influences involved. Disordered vascular function is thought to contribute to programming of cardiovascular health but the cause and effect relationships remain uncertain (Martin *et al.*, 2000;Leeson *et al.*, 2001;Goodfellow *et al.*, 1998). There are recognized sex differences in arterial pressure and the progression of renal disease, both of which are thought to involve interactions of the renin angiotensin system and sex steroids (Sandberg & Ji, 2003;Silva-Antonialli *et al.*, 2004). Most current evidence points to sex steroids as the most important factor influencing sex differences in post-natal cardiovascular function.

Sex Differences in the Developmental Programming of Adult Disease 311

**Disruption Reported Mimetics Reported Conduits of** 

Polychlorinated (PCB) and polybrominated (PBB) biphenyls, dichlorodiphenyltrichloroethane (DDT), Dichlordiphenyldichloroethylene (DDE), methoxychlor, diethylstilbestrol (DES),

Kepone , procymidone,

compounds occur through various types of materials in a variety of settings.

reproductive development (Manikkam *et al.*, 2004;Bormann *et al.*, 2011).

castration of normal growth, hypertensive male rats (Ojeda *et al.*, 2007a).

A (BPA).

Prostaglandins Phthalates, COX inhibiting

cardioprotective properties as well (Manolakou *et al.*, 2009).

alkylphenols, cadmium, bisphenyl-

dichlorodiphenyldichloroethylene (DDE), vinclosolin, 2,3,7,8 tetrachlorodibenzodioxin (TCDD)

Table 1. *Overview of exogenous steroid mimetics.* Exogenous endocrine mimetics have been reported to have agonistic and/or antagonistic behavior in mammals. Exposures to these

and renal protective component that is attenuated during post-menopausal state (Rubinow & Girdler, 2011;Ojeda *et al.*, 2007a). But, estrogen differences between the sexes cannot alone explain the disease development differences because androgens have been observed to have

Androgens are important in fetal development regardless of sex. During the first trimester of development, the male fetus maturation is dictated by the presence of androgens, which if disrupted can lead to several conditions such as testicular cancer, lower sperm count and motility, and cryptorchidism (Manikkam *et al.*, 2004;Bormann *et al.*, 2011;Recabarren *et al.*, 2008). With below-normal levels of testosterone, the male fetus fails to properly develop the testes, known as testicular dysgenesis. In contrast, excess testosterone is linked to altered development of the seminiferous tubules and lower sperm count and motility. Taken together these studies show proper control of androgen levels in males is essential to normal

In growth restricted males, increased testosterone levels have been shown to lead to an increase in angiotensin II sensitivity and this in turn may lead to increased susceptibility to hypertension (Ojeda *et al.*, 2010). Recently, Ojeda et al. showed high incidence of hypertension coinciding with growth restriction is dependent on circulating testosterone levels. The castration of adult growth restricted males resulted in mitigation of the hypertension, which contrasted the observations of no blood pressure change after

The concept of endocrine disruption leading to developmental programming can be extended to the fetal renin-angiotensin system (RAS) as well. Interestingly, this system is responsive not only to pharmacological manipulation but also to nutritional stress as well. Indeed, work from several laboratories has provided insights regarding the role of the RAS in cardiac development (Beinlich *et al.*, 1991;Beinlich & Morgan, 1993;Beinlich *et al.*, 1995;Samyn *et al.*, 1998;Segar *et al.*, 1997;Segar *et al.*, 2001;Sundgren *et al.*, 2003). In particular, Sundgren *et al*. demonstrated that Ang II promotes hyperplastic growth during early gestation, whereas Beinlich *et al*. have reported neonatal hypertrophic growth in the pig

pharmaceuticals Soft plastics, paints, inks;

**Exposure**

Dielectric fluids and electronics; hard plastics; pesticides; insecticides; synthetic estrogens; sewage degradation, batteries and television screens; plastics

and dental sealants

Insecticides; pesticides; fungicides; herbicides

prescription pharmaceuticals

**Sex Hormone** 

Estrogens

Androgens

In contrast to organs such as the kidney that complete development *in utero*, several reproductive organs such as the mammary undergo significant developmental changes during post-natal life and sometimes well into adulthood. A significant portion of mammary development begins at puberty and continues throughout an individual's reproductive years (Hinck & Silberstein, 2005) and renders this particular organ to more critical periods susceptible to programming influences. Aberrant signaling during these phases may initiate abnormal growth of the ductal epithelium, possibly resulting in alterations in risk for subsequently developing mammary cancer.

Interestingly, Wlodek, *et al.* found that uteroplacental insufficiency, via uterine restriction in rats, resulted in reduced alveolar proliferation (Wlodek *et al.*, 2009). We have reported that growth restricted female rats from hypertensive mothers have a much higher incidence of mammary tumors when exposed to N-nitroso-N-methylurea than normal birthweight control rats (Gingery *et al.*, 2011). Thus, differentiation events of the mammary epithelium occurring mid- to late-gestation may provide a substrate sensitive to sub-optimal intrauterine conditions or environmental exposures that could set the stage for subsequent development of cancer.

The transcriptome continues to display sex differences in adulthood, such as in differences in expression of mRNA for osmoregulatory, drug and steroid metabolizing proteins in the murine kidney and liver (Rinn *et al.*, 2004). It is therefore not unreasonable to hypothesize sex differences exist within a molecular framework and that there are many potential avenues, from embryonic life on into adulthood, through which sex differences may interact with developmental programming stimuli to result in sex specific alterations.

#### **3. Endocrine disruption**

It has become nearly axiomatic that endocrine signaling by gonadal steroids like estradiol and testosterone are important contributors to the development and maintenance of longterm health and/or disease. Endocrine disruption generally occurs as a consequence of one or more of four main characteristics of the compound under study: agonist, antagonist, modification, and/or altering synthesis (Derfoul *et al.*, 2003). Some compounds can have a pleiotropic effect in which at least two signaling pathways known to be independent from each other are impacted. Moreover, endogenous sexual hormones and mimetics include: estrone, estriol, estradiol, human chorionic gonadotropin, testosterone, progesterone, prostaglandins, and several other estrogens and androgens. While endocrine disruptors are traditionally considered to be environmental pollutants, there are numerous physiological stressors that may generate disturbances of endocrine signaling pathways. Further, in Table 1 we highlight the main classes of steroidogenic endocrine disruptors and the manners in which they become accessible to organisms. To this end, we have considered a variety of physiological models under the general theme of endocrine disruption.

#### **3.1 Endogenous hormones/mimetics**

Females on average are at lesser of a risk for cardiovascular disease during the premenopausal state, but significantly are at increased risk for cardiovascular and renal disease after menopause, nearing comparable rates to male disease development (Gilbert & Nijland, 2008;Sakemi *et al.*, 1995). In adult growth-restricted females, an ovariectomy can lead to an increase in renal-induced hypertension, compared to subjects with the ovaries still intact(Ojeda *et al.*, 2007a). These observations allude to the idea that estrogens have a cardiac

In contrast to organs such as the kidney that complete development *in utero*, several reproductive organs such as the mammary undergo significant developmental changes during post-natal life and sometimes well into adulthood. A significant portion of mammary development begins at puberty and continues throughout an individual's reproductive years (Hinck & Silberstein, 2005) and renders this particular organ to more critical periods susceptible to programming influences. Aberrant signaling during these phases may initiate abnormal growth of the ductal epithelium, possibly resulting in

Interestingly, Wlodek, *et al.* found that uteroplacental insufficiency, via uterine restriction in rats, resulted in reduced alveolar proliferation (Wlodek *et al.*, 2009). We have reported that growth restricted female rats from hypertensive mothers have a much higher incidence of mammary tumors when exposed to N-nitroso-N-methylurea than normal birthweight control rats (Gingery *et al.*, 2011). Thus, differentiation events of the mammary epithelium occurring mid- to late-gestation may provide a substrate sensitive to sub-optimal intrauterine conditions or environmental exposures that could set the stage for subsequent

The transcriptome continues to display sex differences in adulthood, such as in differences in expression of mRNA for osmoregulatory, drug and steroid metabolizing proteins in the murine kidney and liver (Rinn *et al.*, 2004). It is therefore not unreasonable to hypothesize sex differences exist within a molecular framework and that there are many potential avenues, from embryonic life on into adulthood, through which sex differences may interact

It has become nearly axiomatic that endocrine signaling by gonadal steroids like estradiol and testosterone are important contributors to the development and maintenance of longterm health and/or disease. Endocrine disruption generally occurs as a consequence of one or more of four main characteristics of the compound under study: agonist, antagonist, modification, and/or altering synthesis (Derfoul *et al.*, 2003). Some compounds can have a pleiotropic effect in which at least two signaling pathways known to be independent from each other are impacted. Moreover, endogenous sexual hormones and mimetics include: estrone, estriol, estradiol, human chorionic gonadotropin, testosterone, progesterone, prostaglandins, and several other estrogens and androgens. While endocrine disruptors are traditionally considered to be environmental pollutants, there are numerous physiological stressors that may generate disturbances of endocrine signaling pathways. Further, in Table 1 we highlight the main classes of steroidogenic endocrine disruptors and the manners in which they become accessible to organisms. To this end, we have considered a variety of

Females on average are at lesser of a risk for cardiovascular disease during the premenopausal state, but significantly are at increased risk for cardiovascular and renal disease after menopause, nearing comparable rates to male disease development (Gilbert & Nijland, 2008;Sakemi *et al.*, 1995). In adult growth-restricted females, an ovariectomy can lead to an increase in renal-induced hypertension, compared to subjects with the ovaries still intact(Ojeda *et al.*, 2007a). These observations allude to the idea that estrogens have a cardiac

with developmental programming stimuli to result in sex specific alterations.

physiological models under the general theme of endocrine disruption.

alterations in risk for subsequently developing mammary cancer.

development of cancer.

**3. Endocrine disruption** 

**3.1 Endogenous hormones/mimetics** 


Table 1. *Overview of exogenous steroid mimetics.* Exogenous endocrine mimetics have been reported to have agonistic and/or antagonistic behavior in mammals. Exposures to these compounds occur through various types of materials in a variety of settings.

and renal protective component that is attenuated during post-menopausal state (Rubinow & Girdler, 2011;Ojeda *et al.*, 2007a). But, estrogen differences between the sexes cannot alone explain the disease development differences because androgens have been observed to have cardioprotective properties as well (Manolakou *et al.*, 2009).

Androgens are important in fetal development regardless of sex. During the first trimester of development, the male fetus maturation is dictated by the presence of androgens, which if disrupted can lead to several conditions such as testicular cancer, lower sperm count and motility, and cryptorchidism (Manikkam *et al.*, 2004;Bormann *et al.*, 2011;Recabarren *et al.*, 2008). With below-normal levels of testosterone, the male fetus fails to properly develop the testes, known as testicular dysgenesis. In contrast, excess testosterone is linked to altered development of the seminiferous tubules and lower sperm count and motility. Taken together these studies show proper control of androgen levels in males is essential to normal reproductive development (Manikkam *et al.*, 2004;Bormann *et al.*, 2011).

In growth restricted males, increased testosterone levels have been shown to lead to an increase in angiotensin II sensitivity and this in turn may lead to increased susceptibility to hypertension (Ojeda *et al.*, 2010). Recently, Ojeda et al. showed high incidence of hypertension coinciding with growth restriction is dependent on circulating testosterone levels. The castration of adult growth restricted males resulted in mitigation of the hypertension, which contrasted the observations of no blood pressure change after castration of normal growth, hypertensive male rats (Ojeda *et al.*, 2007a).

The concept of endocrine disruption leading to developmental programming can be extended to the fetal renin-angiotensin system (RAS) as well. Interestingly, this system is responsive not only to pharmacological manipulation but also to nutritional stress as well. Indeed, work from several laboratories has provided insights regarding the role of the RAS in cardiac development (Beinlich *et al.*, 1991;Beinlich & Morgan, 1993;Beinlich *et al.*, 1995;Samyn *et al.*, 1998;Segar *et al.*, 1997;Segar *et al.*, 2001;Sundgren *et al.*, 2003). In particular, Sundgren *et al*. demonstrated that Ang II promotes hyperplastic growth during early gestation, whereas Beinlich *et al*. have reported neonatal hypertrophic growth in the pig

Sex Differences in the Developmental Programming of Adult Disease 313

and sudden cardiac death (Gilbert *et al.*, 2006b;Ojeda *et al.*, 2008;Grigore *et al.*, 2008). These differences in the expression of cardiovascular disease may be related in part to intrinsic sexdifferences in myocardial function. Many recent studies have provided evidence that indicates a sex dichotomy also exists in the physiological responses to developmental challenges as they relate to the programming of subsequent cardio-renal function. These studies have largely been interpreted in one of two ways: 1) that male and female fetuses adapt differently to developmental stressors; or 2) that male and female sex steroids have a profound influence on the development and progression of developmentally programmed disease states. Moreover, since sex differences are apparent quite early in embryonic development and are independent of sex hormones; developing a third line of reasoning to suggest innate differences between the sexes play a role in the response of the developing organism to stressors may yield useful insights. Viewed in concert several primary remaining questions emerge: Do innate sex differences originating in fetal life predispose organisms to adult diseases in general and developmentally programmed outcomes in particular? Do post-natal sex differences drive specific fetal adaptations to *in utero* stressors that generate differential outcomes? Or perhaps it is a combination of these scenarios?

A small number of clinical studies have investigated sex differences in renal function as it relates to developmentally programmed hypertension. The larger body of work in this area has detailed differences in cardiovascular parameters and stress responses. Nevertheless, several interesting findings have been reported that confirm the idea that women are "renoprotected" during early adulthood. A recent report from the Nord Trøndelag Health Study (1995-1997) in Norway found intrauterine growth restriction (IUGR), high blood pressure and low normal renal function were associated in 20-30 year olds (Hallan *et al.*, 2008). Although the degree of impaired renal function was small in these young adults, it was significant and more consistent in men than women (Hallan *et al.*, 2008). Similarly, Kistner *et al.* reported women born pre-term had increased blood pressure but no signs of adverse

Other studies have evaluated cardiovascular responses between male and female subjects that were growth restricted *in utero*. In one such study, Ward and colleagues reported women born small were far more susceptible to stress-induced increases in systolic blood pressure (Ward *et al.*, 2004). A recent study by Jones *et al.* has shown that there are marked sex differences in the way size at birth is associated with alterations in cardiovascular physiology established in childhood (Jones *et al.*, 2008). Further evidence that markers of impaired fetal growth are related to autonomic cardiovascular control involving modulation of both sympathetic and parasympathetic function but in a sex-specific manner has also been provided in an adult Australian cohort by the same group (Jones *et al.*, 2007). The authors reported women, but not men, who were small at birth demonstrated increased low-frequency blood pressure variability at rest and during stress, reduced levels of high-

Studies utilizing animal models have employed a range of stressors in a variety of species to induce fetal growth restriction and test hypotheses regarding the developmental origins of disease (summarized in Table 2). Perhaps the most common model to date has focused on maternal nutrient restriction (MNR), either as a decrease in total caloric intake or an

frequency heart period variability and a reduction in baroreflex sensitivity.

**4.1 Human studies** 

**4.2 Animal studies** 

renal function as young adults (Kistner *et al.*, 2000).

(Beinlich *et al.*, 1995;Sundgren *et al.*, 2003). The intra-cardiac RAS also appears to be sensitive to nutritional stress as demonstrated recently by Gilbert et al. in a study that shows decreased immunoreactive AT1 and AT2 in the mid-gestation left ventricle of fetal sheep gestated in ewes that were subjected to 50% global nutrient restriction (Gilbert *et al.*, 2005b).

#### **3.2 Exogenous hormones/mimetics/antagonists**

Exposure to a variety of environmental factors may generate exogenous interference with endocrine systems through mimetic or antagonistic activity. This is a rapidly growing area of research with implications for both environmental and public health. Several known exogenous estrogen antagonists include polychlorinated (PCB) and polybrominated (PBB) biphenyls, dichlorodiphenyltrichloroethane (DDT), methoxychlor, diethylstilbestrol, 17βestradiol, alkylphenols sewage degradation, cadmium, and the infamous bisphenyl-A (BPA) (Sonnenschein & Soto, 1998;Derfoul *et al.*, 2003). Xenoestrogens may exist in the system at levels that do not elicit strong or detectable estrogenic effects individually, but it has been shown that these have additive effects and several xenoestrogens in the system can act together to induce estrogenic activity. (Sonnenschein & Soto, 1998) This additive effect brings rise to the idea that some materials we are exposed to may pass inspection individually for tolerable levels of these endocrine disrupting compounds, but in concert with low levels of the xenoestrogens may have phenotypic effects *in utero*. Since estrogen is mainly required in maturation and growth, it is common to not see congenital endocrine complications until adolescence or even as late as adulthood (Derfoul *et al.*, 2003). Overexposure of estrogens to the fetus has been suggested to stunt growth and alter bone development (Derfoul *et al.*, 2003;Sonnenschein & Soto, 1998). In contrast to the large number of estrogen disruptors, only several androgen antagonists have been identified and studied. These are insecticide ingredients such as kepone and procymidone, dichlorodiphenyldichloroethyle (DDE), vinclosolin, and 2,3,7,8-tetrachlorodibenzodioxin (TCDD) (Sonnenschein & Soto, 1998). While very little to no androgen agonists have been discovered (Sonnenschein & Soto, 1998) it has been reported that exposure to androgen antagonists like DDE is linked to development of recurrent respiratory tract infection (Carey *et al.*, 2007).

Prostaglandins are important to many sexual processes in both men and women, but little has been done on that research to describe endocrine disruption targeting prostaglandins. Interference in prostaglandin pathways has been associated with the development of several types of cancer and cardiovascular disorders. The alteration of synthesis of prostaglandins from arachadonic acid through the COX enzyme has been shown disrupt endocrine processes. Several phthalates that are similar to pharmaceutical COX inhibitors, were found to disrupt the levels of prostaglandin synthesis (Kristensen *et al.*, 2011). Chronic inhibition of COX activity is known to have deleterious effects on renal and cardiovascular function, resulting in mild to moderate hypertension and even renal failure. Developmental sex differences in this system have been reported and show that renal COX-2 expression is higher in female fetuses at gestational day 21 than age matched male fetuses (Baserga *et al.*, 2007a). Prostaglandin synthesis pathways are relatively understudied but may provide important insights into the development of sex differences in adult disease.

#### **4. Sex differences in developmental programming**

Numerous studies have documented sex-differences in the incidence and severity of cardiovascular diseases such as coronary artery disease, heart failure, cardiac hypertrophy, and sudden cardiac death (Gilbert *et al.*, 2006b;Ojeda *et al.*, 2008;Grigore *et al.*, 2008). These differences in the expression of cardiovascular disease may be related in part to intrinsic sexdifferences in myocardial function. Many recent studies have provided evidence that indicates a sex dichotomy also exists in the physiological responses to developmental challenges as they relate to the programming of subsequent cardio-renal function. These studies have largely been interpreted in one of two ways: 1) that male and female fetuses adapt differently to developmental stressors; or 2) that male and female sex steroids have a profound influence on the development and progression of developmentally programmed disease states. Moreover, since sex differences are apparent quite early in embryonic development and are independent of sex hormones; developing a third line of reasoning to suggest innate differences between the sexes play a role in the response of the developing organism to stressors may yield useful insights. Viewed in concert several primary remaining questions emerge: Do innate sex differences originating in fetal life predispose organisms to adult diseases in general and developmentally programmed outcomes in particular? Do post-natal sex differences drive specific fetal adaptations to *in utero* stressors that generate differential outcomes? Or perhaps it is a combination of these scenarios?

#### **4.1 Human studies**

312 Sex Hormones

(Beinlich *et al.*, 1995;Sundgren *et al.*, 2003). The intra-cardiac RAS also appears to be sensitive to nutritional stress as demonstrated recently by Gilbert et al. in a study that shows decreased immunoreactive AT1 and AT2 in the mid-gestation left ventricle of fetal sheep gestated in ewes that were subjected to 50% global nutrient restriction (Gilbert *et al.*, 2005b).

Exposure to a variety of environmental factors may generate exogenous interference with endocrine systems through mimetic or antagonistic activity. This is a rapidly growing area of research with implications for both environmental and public health. Several known exogenous estrogen antagonists include polychlorinated (PCB) and polybrominated (PBB) biphenyls, dichlorodiphenyltrichloroethane (DDT), methoxychlor, diethylstilbestrol, 17βestradiol, alkylphenols sewage degradation, cadmium, and the infamous bisphenyl-A (BPA) (Sonnenschein & Soto, 1998;Derfoul *et al.*, 2003). Xenoestrogens may exist in the system at levels that do not elicit strong or detectable estrogenic effects individually, but it has been shown that these have additive effects and several xenoestrogens in the system can act together to induce estrogenic activity. (Sonnenschein & Soto, 1998) This additive effect brings rise to the idea that some materials we are exposed to may pass inspection individually for tolerable levels of these endocrine disrupting compounds, but in concert with low levels of the xenoestrogens may have phenotypic effects *in utero*. Since estrogen is mainly required in maturation and growth, it is common to not see congenital endocrine complications until adolescence or even as late as adulthood (Derfoul *et al.*, 2003). Overexposure of estrogens to the fetus has been suggested to stunt growth and alter bone development (Derfoul *et al.*, 2003;Sonnenschein & Soto, 1998). In contrast to the large number of estrogen disruptors, only several androgen antagonists have been identified and studied. These are insecticide ingredients such as kepone and procymidone, dichlorodiphenyldichloroethyle (DDE), vinclosolin, and 2,3,7,8-tetrachlorodibenzodioxin (TCDD) (Sonnenschein & Soto, 1998). While very little to no androgen agonists have been discovered (Sonnenschein & Soto, 1998) it has been reported that exposure to androgen antagonists like DDE is linked to development of

Prostaglandins are important to many sexual processes in both men and women, but little has been done on that research to describe endocrine disruption targeting prostaglandins. Interference in prostaglandin pathways has been associated with the development of several types of cancer and cardiovascular disorders. The alteration of synthesis of prostaglandins from arachadonic acid through the COX enzyme has been shown disrupt endocrine processes. Several phthalates that are similar to pharmaceutical COX inhibitors, were found to disrupt the levels of prostaglandin synthesis (Kristensen *et al.*, 2011). Chronic inhibition of COX activity is known to have deleterious effects on renal and cardiovascular function, resulting in mild to moderate hypertension and even renal failure. Developmental sex differences in this system have been reported and show that renal COX-2 expression is higher in female fetuses at gestational day 21 than age matched male fetuses (Baserga *et al.*, 2007a). Prostaglandin synthesis pathways are relatively understudied but may provide

Numerous studies have documented sex-differences in the incidence and severity of cardiovascular diseases such as coronary artery disease, heart failure, cardiac hypertrophy,

important insights into the development of sex differences in adult disease.

**4. Sex differences in developmental programming** 

**3.2 Exogenous hormones/mimetics/antagonists** 

recurrent respiratory tract infection (Carey *et al.*, 2007).

A small number of clinical studies have investigated sex differences in renal function as it relates to developmentally programmed hypertension. The larger body of work in this area has detailed differences in cardiovascular parameters and stress responses. Nevertheless, several interesting findings have been reported that confirm the idea that women are "renoprotected" during early adulthood. A recent report from the Nord Trøndelag Health Study (1995-1997) in Norway found intrauterine growth restriction (IUGR), high blood pressure and low normal renal function were associated in 20-30 year olds (Hallan *et al.*, 2008). Although the degree of impaired renal function was small in these young adults, it was significant and more consistent in men than women (Hallan *et al.*, 2008). Similarly, Kistner *et al.* reported women born pre-term had increased blood pressure but no signs of adverse renal function as young adults (Kistner *et al.*, 2000).

Other studies have evaluated cardiovascular responses between male and female subjects that were growth restricted *in utero*. In one such study, Ward and colleagues reported women born small were far more susceptible to stress-induced increases in systolic blood pressure (Ward *et al.*, 2004). A recent study by Jones *et al.* has shown that there are marked sex differences in the way size at birth is associated with alterations in cardiovascular physiology established in childhood (Jones *et al.*, 2008). Further evidence that markers of impaired fetal growth are related to autonomic cardiovascular control involving modulation of both sympathetic and parasympathetic function but in a sex-specific manner has also been provided in an adult Australian cohort by the same group (Jones *et al.*, 2007). The authors reported women, but not men, who were small at birth demonstrated increased low-frequency blood pressure variability at rest and during stress, reduced levels of highfrequency heart period variability and a reduction in baroreflex sensitivity.

#### **4.2 Animal studies**

Studies utilizing animal models have employed a range of stressors in a variety of species to induce fetal growth restriction and test hypotheses regarding the developmental origins of disease (summarized in Table 2). Perhaps the most common model to date has focused on maternal nutrient restriction (MNR), either as a decrease in total caloric intake or an

Sex Differences in the Developmental Programming of Adult Disease 315

response in the mesenteric vascular bed of pregnant adult females exposed to MNR during development (Hemmings *et al.*, 2005). MNR during the pre-implantation period in the rat resulted in elevated BP in male offspring only (Kwong *et al.*, 2000). Restriction of specific nutrients other than protein has also been evaluated. A maternal low-sodium diet in rats has recently been associated with increased maternal plasma renin activity and correlated with IUGR, increased blood pressure, and reduced creatinine clearance in female offspring but

Similar to the results observed in many small animal models, not all large animal models show clear effects of MNR on the offspring. In addition, only a subset of these studies has been evaluated for sex differences. Previous work has shown male sheep and baboon fetuses are more susceptible to the effects of poor maternal nutrition (Gilbert *et al.*, 2005a;Gilbert *et al.*, 2006a;Gilbert *et al.*, 2007a). Studies in sheep have shown global caloric restriction impairs nephrogenesis and alters intrarenal immunoreactive AT1, AT2 and renin expression in gestational age and gender specific ways (Gilbert *et al.*, 2007a). Further, only male offspring of these NR ewes are hypertensive (Gilbert *et al.*, 2005a). While the mechanisms by which NR alters gene expression remains unclear in our model, data from Lillycrop *et al.* and Burdge *et al.,* both employing protein restriction in the rat suggests deficiency of methyl donors may alter gene methylation patterns and in turn effect changes in gene expression (Lillycrop *et al.*, 2005;Burdge *et al.*, 2007;Lillycrop *et al.*, 2007;Lillycrop *et al.*, 2008). It remains unclear whether the increased risk to the males is a result of geneenvironment interactions originating during or after gestation. Further studies are needed to

Models of utero-placental insufficiency are quite intriguing as they are relevant to multiple maternal health issues as well as to the developmental programming of hypertension. Alexander *et al.* have shown that reduced uterine perfusion pressure during the last trimester of pregnancy in the rat programs hypertension in the offspring and in a sex specific manner (Grigore *et al.*, 2007;Alexander, 2003). Further, in this model both the RAS and sex steroids have been implicated in the observed sex differences in hypertension (Ojeda *et al.*, 2007b;Ojeda *et al.*, 2007a;Grigore *et al.*, 2007). In contrast, the two kidney-one wrapped kidney (2K,1W) model of hypertension resulted in hypertension in 30 week old female offspring only (Denton *et al.*, 2003). Interestingly, plasma renin activity was significantly lower in the female offspring of hypertensive mothers at 10 weeks of age (*P*<0.05), suggesting that development of the renin-angiotensin system was altered. The differences in the factors elaborated by the ischemic placenta and poorly perfused kidney illustrate the complexity of the interactions between the maternal endocrine milieu and fetal development. Whereas reduced renal perfusion primarily activates the RAS, the ischemic placenta produces a variety of humoral and locally acting factors such as sFlt-1 (soluble fmslike tyrosine kinase-1) and tumor necrosis factor (TNF)-α that have far reaching effects. Recent studies in the rat and baboon have shown chronic reductions of utero-placental blood flow elevates levels of sFlt-1 in the placenta, amniotic fluid and maternal plasma (Gilbert *et al.*, 2007b;Makris *et al.*, 2007). In the rat, this has been associated with decreased fetal growth and subsequent hypertension that is sex dependent (Alexander, 2003;Ojeda *et al.*, 2007a). Recent studies in rodents have shown elevated sFlt-1 levels alone results in fetal growth restriction (Lu *et al.*, 2007b;Bridges *et al.*, 2008). Furthermore, Lu *et al*. have followed

not in males (Battista *et al.*, 2002).

thoroughly investigate these possibilities.

**4.2.2 Models of utero-placental and renal insufficiency** 

**Programming Insult Species Adult outcomes reported in the literature** Maternal overnutrition Rat, mouse, sheep Hypertension (females), reduced vascular compliance, Endothelial dysfunction, aortic hypoplasia, decreased renal Na+, K+ ATPase activity, decreased locomotor activity (female>male) Maternal undernutrition Rat, sheep, baboon Hypertension (male>female), growth restriction, altered expression of reninangiotensin system Placental insufficiency Rat Hypertension (male>female), growth restriction, glucose intolerance Maternal renal insufficiency Rat, rabbit Hypertension (female>male) Ang II receptor inhibition Rat Hypertension, decreased nephron number, glomerulosclerosis (male>female), interstitial fibrosis (male>female) Glucocorticoid excess Rat, sheep Hypertension (sex and age dependent), glomerulosclerosis Androgen excess Rat, mouse, sheep, human Hypertension (male>female), decreased endothelial function, ovarian dysgenesis, increased ANG-2 sensitivity (GR males), increased male-like sexual behavior (female), growth retardation, delayed vaginal opening, increased aggression Estrogen deficiency Rat, mouse, sheep, human Hypertension (postmenopausal women), infertility, abnormal mammary growth

isocaloric decrease in protein content; studies to understand the consequences of maternal obesity from the DOHAD perspective are gaining (Grigore *et al.*, 2008;Mcmillen & Robinson, 2005;Gallou-Kabani *et al.*, 2007;Khan *et al.*, 2005;Khan *et al.*, 2003;Taylor *et al.*, 2004).

Table 2. *Summary of developmental programming studies and outcomes.* A variety of developmental insults lead to long-term health consequences for the offspring. (References found in Sections 2 and 3.)

#### **4.2.1 Models of nutrient restriction**

Others have shown that considerable sex differences are observed in the response to MNR between male and female baboon fetuses near term (Cox *et al.*, 2008). Evidence from MNR studies suggest female progeny are less affected than their male siblings (Ozaki *et al.*, 2001;Woods *et al.*, 2005;McMullen & Langley-Evans, 2005b;McMullen & Langley-Evans, 2005a) although these observations may depend on the extent of the nutrient restriction (Hoppe *et al.*, 2007). These studies generally show decreased nephron endowment and altered expression of components of the intra-renal renin-angiotensin system (Ozaki *et al.*, 2001;Woods *et al.*, 2005;McMullen & Langley-Evans, 2005b;McMullen & Langley-Evans, 2005a;Hoppe *et al.*, 2007). Hemmings *et al.* have reported impairment of the myogenic

isocaloric decrease in protein content; studies to understand the consequences of maternal obesity from the DOHAD perspective are gaining (Grigore *et al.*, 2008;Mcmillen & Robinson,

**Programming Insult Species Adult outcomes reported in the literature**

Placental insufficiency Rat Hypertension (male>female), growth

Glucocorticoid excess Rat, sheep Hypertension (sex and age dependent),

insufficiency Rat, rabbit Hypertension (female>male)

human

Rat, mouse, sheep,

Table 2. *Summary of developmental programming studies and outcomes.* A variety of

developmental insults lead to long-term health consequences for the offspring. (References

Others have shown that considerable sex differences are observed in the response to MNR between male and female baboon fetuses near term (Cox *et al.*, 2008). Evidence from MNR studies suggest female progeny are less affected than their male siblings (Ozaki *et al.*, 2001;Woods *et al.*, 2005;McMullen & Langley-Evans, 2005b;McMullen & Langley-Evans, 2005a) although these observations may depend on the extent of the nutrient restriction (Hoppe *et al.*, 2007). These studies generally show decreased nephron endowment and altered expression of components of the intra-renal renin-angiotensin system (Ozaki *et al.*, 2001;Woods *et al.*, 2005;McMullen & Langley-Evans, 2005b;McMullen & Langley-Evans, 2005a;Hoppe *et al.*, 2007). Hemmings *et al.* have reported impairment of the myogenic

Hypertension (females), reduced vascular compliance, Endothelial dysfunction, aortic hypoplasia, decreased renal Na+, K+ ATPase activity, decreased locomotor

Hypertension (male>female), growth restriction, altered expression of renin-

Hypertension, decreased nephron number, glomerulosclerosis (male>female), interstitial fibrosis (male>female)

Hypertension (male>female), decreased endothelial function, ovarian dysgenesis, increased ANG-2 sensitivity (GR males), increased male-like sexual behavior (female), growth retardation, delayed vaginal opening, increased aggression

infertility, abnormal mammary growth

restriction, glucose intolerance

activity (female>male)

angiotensin system

glomerulosclerosis

human Hypertension (postmenopausal women),

2005;Gallou-Kabani *et al.*, 2007;Khan *et al.*, 2005;Khan *et al.*, 2003;Taylor *et al.*, 2004).

Maternal overnutrition Rat, mouse, sheep

Maternal undernutrition Rat, sheep, baboon

inhibition Rat

Androgen excess Rat, mouse, sheep,

Maternal renal

Ang II receptor

Estrogen deficiency

found in Sections 2 and 3.)

**4.2.1 Models of nutrient restriction** 

response in the mesenteric vascular bed of pregnant adult females exposed to MNR during development (Hemmings *et al.*, 2005). MNR during the pre-implantation period in the rat resulted in elevated BP in male offspring only (Kwong *et al.*, 2000). Restriction of specific nutrients other than protein has also been evaluated. A maternal low-sodium diet in rats has recently been associated with increased maternal plasma renin activity and correlated with IUGR, increased blood pressure, and reduced creatinine clearance in female offspring but not in males (Battista *et al.*, 2002).

Similar to the results observed in many small animal models, not all large animal models show clear effects of MNR on the offspring. In addition, only a subset of these studies has been evaluated for sex differences. Previous work has shown male sheep and baboon fetuses are more susceptible to the effects of poor maternal nutrition (Gilbert *et al.*, 2005a;Gilbert *et al.*, 2006a;Gilbert *et al.*, 2007a). Studies in sheep have shown global caloric restriction impairs nephrogenesis and alters intrarenal immunoreactive AT1, AT2 and renin expression in gestational age and gender specific ways (Gilbert *et al.*, 2007a). Further, only male offspring of these NR ewes are hypertensive (Gilbert *et al.*, 2005a). While the mechanisms by which NR alters gene expression remains unclear in our model, data from Lillycrop *et al.* and Burdge *et al.,* both employing protein restriction in the rat suggests deficiency of methyl donors may alter gene methylation patterns and in turn effect changes in gene expression (Lillycrop *et al.*, 2005;Burdge *et al.*, 2007;Lillycrop *et al.*, 2007;Lillycrop *et al.*, 2008). It remains unclear whether the increased risk to the males is a result of geneenvironment interactions originating during or after gestation. Further studies are needed to thoroughly investigate these possibilities.

#### **4.2.2 Models of utero-placental and renal insufficiency**

Models of utero-placental insufficiency are quite intriguing as they are relevant to multiple maternal health issues as well as to the developmental programming of hypertension. Alexander *et al.* have shown that reduced uterine perfusion pressure during the last trimester of pregnancy in the rat programs hypertension in the offspring and in a sex specific manner (Grigore *et al.*, 2007;Alexander, 2003). Further, in this model both the RAS and sex steroids have been implicated in the observed sex differences in hypertension (Ojeda *et al.*, 2007b;Ojeda *et al.*, 2007a;Grigore *et al.*, 2007). In contrast, the two kidney-one wrapped kidney (2K,1W) model of hypertension resulted in hypertension in 30 week old female offspring only (Denton *et al.*, 2003). Interestingly, plasma renin activity was significantly lower in the female offspring of hypertensive mothers at 10 weeks of age (*P*<0.05), suggesting that development of the renin-angiotensin system was altered. The differences in the factors elaborated by the ischemic placenta and poorly perfused kidney illustrate the complexity of the interactions between the maternal endocrine milieu and fetal development. Whereas reduced renal perfusion primarily activates the RAS, the ischemic placenta produces a variety of humoral and locally acting factors such as sFlt-1 (soluble fmslike tyrosine kinase-1) and tumor necrosis factor (TNF)-α that have far reaching effects.

Recent studies in the rat and baboon have shown chronic reductions of utero-placental blood flow elevates levels of sFlt-1 in the placenta, amniotic fluid and maternal plasma (Gilbert *et al.*, 2007b;Makris *et al.*, 2007). In the rat, this has been associated with decreased fetal growth and subsequent hypertension that is sex dependent (Alexander, 2003;Ojeda *et al.*, 2007a). Recent studies in rodents have shown elevated sFlt-1 levels alone results in fetal growth restriction (Lu *et al.*, 2007b;Bridges *et al.*, 2008). Furthermore, Lu *et al*. have followed

Sex Differences in the Developmental Programming of Adult Disease 317

The importance of environmental exposures to endocrine disruptors during pregnancy has long been noted. Factors derived from *Pinus ponderosa* needles (e.g. isocupressic acid) and leaves from *Veratrum californicum* have long been observed to have profound impacts on the pregnancies of livestock (Short *et al.*, 1995;Panter *et al.*, 1992;Wu *et al.*, 2002). Moreover, the observations that ingestion of *Veratrum californicum* by sheep at specific times of gestation resulted in fetal malformations and prolonged gestation laid the foundation for experimental evidence that supports a crucial role for glucocorticoids and the fetal hypothalamic pituitary axis in the onset of parturition (Liggins, 1994;Challis *et al.*, 2000). Similarly, carbenoxolone, an active ingredient of licorice may also inhibit production of cortisol and disrupt normal HPA signaling between the mother and fetus. These findings point to a role for maternal and fetal stressors that alter glucocorticoid levels during pregnancy as important mediators of developmental programming. One such physiological stressor is exercise during pregnancy which has been reported to have a variety of effects on the offspring in hypertensive rats (Gilbert *et al.*, 2008). Whereas moderate exercise lowered blood pressure in female offspring and increased body density in both male and female progeny, a high volume of exercise resulted in post-natal growth failure followed by catchup growth but only females suffered exacerbated hypertension (Gilbert *et al.*, 2002). Using a dexamethazone injection model, O'Reagan *et al.* showed similar effects on BP in males and females but the magnitude of hypertension and a greater stress-induced hypertension was observed in males. In another study, prenatal dexamethasone (DEX) treatment significantly enhanced the arterial pressure response to acute stress only in female Wistar rats, while DEX augmented the elevation in heart rate during stress only in male rats (Bechtold *et al.*, 2008). Ortiz *et al.* have shown antenatal DEX elevates blood pressure in female offspring at three weeks of age while only male offspring had increased blood pressure at six months of age (Ortiz *et al.*, 2003). Interestingly, despite the observation only male DEX-treated rats were hypertensive at six months of age, both male and female offspring showed signs of glomerulosclerosis when compared to control rats (Ortiz *et al.*, 2003). Similar work has shown that a postnatal diet rich in ω-3 (n-3) fatty acids attenuates the effects of DEX on blood pressure in the offspring (Wyrwoll *et al.*, 2006) in a sex independent manner. With the wide ranging effects reported in the glucocorticoid models, continued studies are required

to tease out the mechanisms of sex-specific responsivity in this programming model.

following acute volume expansion (Loria *et al.*, 2007a).

Another intriguing area of investigation garnering attention involves the role of the maternal RAS during pregnancy and/or lactation in pregnancy outcome and offspring health. These approaches may be in the form of administration of RAS inhibitors (Salazar *et al.*, 2008) or altered sodium diet as described above (Battista *et al.*, 2002). RAS inhibition at the level of the AT1 receptor is reported to have several sex specific effects that manifest post-partum (Loria *et al.*, 2007b;Saez *et al.*, 2007;Salazar *et al.*, 2008). Saez *et al.* found that AT1 inhibition reduces nephron number similarly in male and female rats, but the subsequent glomerulosclerosis and interstitial fibrosis are greater in males than in females. Further, the male rats are also reported to have a significant papillary atrophy (Saez *et al.*, 2007). Functional differences include impaired urinary-concentrating ability during a prolonged dehydration in the male offspring (Loria *et al.*, 2007b) and impaired excretory capacity

Although the present data clearly indicate inhibition of the RAS during pregnancy has well defined and deleterious effects on renal development and function in the offspring, current studies are less clear on the effects of more subtle perturbations of the RAS (e.g. via dietary

**4.2.4 Endocrine disruption** 

the mouse offspring of these pregnancies and reported sex specific effects regarding the development of hypertension as only male mice have higher blood pressure in this model (Lu *et al.*, 2007a). Viewed together, these studies strongly suggest that in addition to the immediate well being of the mother, a long term outlook with regards to the well being of the fetus must also be considered during complicated and/or high risk pregnancies.

#### **4.2.3 Maternal obesity**

Maternal obesity is associated with a variety of conditions including maternal hypertension, hypertriglyceridemia, hyperglycemia and insulin resistance (Wilson & Grundy, 2003), that are independently correlated with a suboptimal *in utero* environment and consequently linked to DOHAD. Several human studies have described a positive correlation between maternal weight and/or adiposity and blood pressure of teenage children (Lawlor *et al.*, 2004;Cho *et al.*, 2000;Laor *et al.*, 1997), leading Boney *et al* to conclude from their examination of large for gestational age babies and the incidence of childhood metabolic syndrome, that "given the increased obesity prevalence in children exposed to either maternal diabetes or maternal obesity, there are implications for perpetuating the cycle of obesity, insulin resistance, and their consequences in subsequent generations." Few, if any, of the studies in humans include offspring sex as a co-variable (Boney *et al.*, 2005).

Important information with regard to maternal nutrient excess and sex-associated difference comes largely from animal models. Studies show hypertension in male rat offspring after exposure to a maternal diet high in saturated fat (or low in linoleic acid) that is not present in females (Langley-Evans, 1996). In contrast, Elahi *et al.* reported mice fed high fat diets long before the onset of gestation are hypercholesterolemic, hypertensive and produce hypertensive, hypercholesterolemic female offspring (Elahi *et al.*, 2008). Moreover, treatment of the dams with pravastatin lowered blood pressure and cholesterol levels in those offspring (Elahi *et al.*, 2008). Because the numerous pleiotropic effects of statins the mechanisms for these effects remain unclear, nevertheless these observations provide insights for further studies.

In a model more resembling high fat food consumption in humans, Armitage *et al.* demonstrated that a diet rich in fat fed to pregnant rats results in male offspring gaining more body weight and presenting with decreased renal renin activity when compared to females (Armitage *et al.*, 2005). Offspring from this model are reportedly hypertensive, exhibit increased aortic stiffness, decreased aortic smooth muscle cell number, endothelial dysfunction and decreased renal Na+, K+-ATPase activity. The bulk of these changes were independent of sex except for increased blood pressure where female offspring were hypertensive while the males were not (Khan *et al.*, 2003;Samuelsson *et al.*, 2008). Further, Khan et al. reported female offspring have reduced locomotor activity at 180 days of age compared to male offspring of pregnant rats fed a high fat diet during pregnancy (Khan *et al.*, 2003). In addition, this research group used cross-fostering techniques after birth to show that the hypertension in females is attained whether exposure to maternal high fat diet occurs before and during pregnancy or during the suckling period (Khan *et al.*, 2005). While the mechanisms responsible for programming due to high fat diets remain unclear, the report that statin treatment has beneficial effects on the offspring highlights at least one potential mechanism, alterations in lipid metabolism (Elahi *et al.*, 2008). In addition, it has been suggested that high levels of butyric acid that may result from a high fat diet could lead to changes in chromatin structure and result in epigenetic alterations (Junien, 2006). Taken together these observations highlight an important role for nutrition and intermediate metabolites in developmental programming.

#### **4.2.4 Endocrine disruption**

316 Sex Hormones

the mouse offspring of these pregnancies and reported sex specific effects regarding the development of hypertension as only male mice have higher blood pressure in this model (Lu *et al.*, 2007a). Viewed together, these studies strongly suggest that in addition to the immediate well being of the mother, a long term outlook with regards to the well being of

Maternal obesity is associated with a variety of conditions including maternal hypertension, hypertriglyceridemia, hyperglycemia and insulin resistance (Wilson & Grundy, 2003), that are independently correlated with a suboptimal *in utero* environment and consequently linked to DOHAD. Several human studies have described a positive correlation between maternal weight and/or adiposity and blood pressure of teenage children (Lawlor *et al.*, 2004;Cho *et al.*, 2000;Laor *et al.*, 1997), leading Boney *et al* to conclude from their examination of large for gestational age babies and the incidence of childhood metabolic syndrome, that "given the increased obesity prevalence in children exposed to either maternal diabetes or maternal obesity, there are implications for perpetuating the cycle of obesity, insulin resistance, and their consequences in subsequent generations." Few, if any,

the fetus must also be considered during complicated and/or high risk pregnancies.

of the studies in humans include offspring sex as a co-variable (Boney *et al.*, 2005).

Important information with regard to maternal nutrient excess and sex-associated difference comes largely from animal models. Studies show hypertension in male rat offspring after exposure to a maternal diet high in saturated fat (or low in linoleic acid) that is not present in females (Langley-Evans, 1996). In contrast, Elahi *et al.* reported mice fed high fat diets long before the onset of gestation are hypercholesterolemic, hypertensive and produce hypertensive, hypercholesterolemic female offspring (Elahi *et al.*, 2008). Moreover, treatment of the dams with pravastatin lowered blood pressure and cholesterol levels in those offspring (Elahi *et al.*, 2008). Because the numerous pleiotropic effects of statins the mechanisms for these effects remain unclear, nevertheless these observations provide insights for further studies. In a model more resembling high fat food consumption in humans, Armitage *et al.* demonstrated that a diet rich in fat fed to pregnant rats results in male offspring gaining more body weight and presenting with decreased renal renin activity when compared to females (Armitage *et al.*, 2005). Offspring from this model are reportedly hypertensive, exhibit increased aortic stiffness, decreased aortic smooth muscle cell number, endothelial dysfunction and decreased renal Na+, K+-ATPase activity. The bulk of these changes were independent of sex except for increased blood pressure where female offspring were hypertensive while the males were not (Khan *et al.*, 2003;Samuelsson *et al.*, 2008). Further, Khan et al. reported female offspring have reduced locomotor activity at 180 days of age compared to male offspring of pregnant rats fed a high fat diet during pregnancy (Khan *et al.*, 2003). In addition, this research group used cross-fostering techniques after birth to show that the hypertension in females is attained whether exposure to maternal high fat diet occurs before and during pregnancy or during the suckling period (Khan *et al.*, 2005). While the mechanisms responsible for programming due to high fat diets remain unclear, the report that statin treatment has beneficial effects on the offspring highlights at least one potential mechanism, alterations in lipid metabolism (Elahi *et al.*, 2008). In addition, it has been suggested that high levels of butyric acid that may result from a high fat diet could lead to changes in chromatin structure and result in epigenetic alterations (Junien, 2006). Taken together these observations highlight an important role for nutrition and intermediate

**4.2.3 Maternal obesity** 

metabolites in developmental programming.

The importance of environmental exposures to endocrine disruptors during pregnancy has long been noted. Factors derived from *Pinus ponderosa* needles (e.g. isocupressic acid) and leaves from *Veratrum californicum* have long been observed to have profound impacts on the pregnancies of livestock (Short *et al.*, 1995;Panter *et al.*, 1992;Wu *et al.*, 2002). Moreover, the observations that ingestion of *Veratrum californicum* by sheep at specific times of gestation resulted in fetal malformations and prolonged gestation laid the foundation for experimental evidence that supports a crucial role for glucocorticoids and the fetal hypothalamic pituitary axis in the onset of parturition (Liggins, 1994;Challis *et al.*, 2000). Similarly, carbenoxolone, an active ingredient of licorice may also inhibit production of cortisol and disrupt normal HPA signaling between the mother and fetus. These findings point to a role for maternal and fetal stressors that alter glucocorticoid levels during pregnancy as important mediators of developmental programming. One such physiological stressor is exercise during pregnancy which has been reported to have a variety of effects on the offspring in hypertensive rats (Gilbert *et al.*, 2008). Whereas moderate exercise lowered blood pressure in female offspring and increased body density in both male and female progeny, a high volume of exercise resulted in post-natal growth failure followed by catchup growth but only females suffered exacerbated hypertension (Gilbert *et al.*, 2002). Using a dexamethazone injection model, O'Reagan *et al.* showed similar effects on BP in males and females but the magnitude of hypertension and a greater stress-induced hypertension was observed in males. In another study, prenatal dexamethasone (DEX) treatment significantly enhanced the arterial pressure response to acute stress only in female Wistar rats, while DEX augmented the elevation in heart rate during stress only in male rats (Bechtold *et al.*, 2008).

Ortiz *et al.* have shown antenatal DEX elevates blood pressure in female offspring at three weeks of age while only male offspring had increased blood pressure at six months of age (Ortiz *et al.*, 2003). Interestingly, despite the observation only male DEX-treated rats were hypertensive at six months of age, both male and female offspring showed signs of glomerulosclerosis when compared to control rats (Ortiz *et al.*, 2003). Similar work has shown that a postnatal diet rich in ω-3 (n-3) fatty acids attenuates the effects of DEX on blood pressure in the offspring (Wyrwoll *et al.*, 2006) in a sex independent manner. With the wide ranging effects reported in the glucocorticoid models, continued studies are required to tease out the mechanisms of sex-specific responsivity in this programming model.

Another intriguing area of investigation garnering attention involves the role of the maternal RAS during pregnancy and/or lactation in pregnancy outcome and offspring health. These approaches may be in the form of administration of RAS inhibitors (Salazar *et al.*, 2008) or altered sodium diet as described above (Battista *et al.*, 2002). RAS inhibition at the level of the AT1 receptor is reported to have several sex specific effects that manifest post-partum (Loria *et al.*, 2007b;Saez *et al.*, 2007;Salazar *et al.*, 2008). Saez *et al.* found that AT1 inhibition reduces nephron number similarly in male and female rats, but the subsequent glomerulosclerosis and interstitial fibrosis are greater in males than in females. Further, the male rats are also reported to have a significant papillary atrophy (Saez *et al.*, 2007). Functional differences include impaired urinary-concentrating ability during a prolonged dehydration in the male offspring (Loria *et al.*, 2007b) and impaired excretory capacity following acute volume expansion (Loria *et al.*, 2007a).

Although the present data clearly indicate inhibition of the RAS during pregnancy has well defined and deleterious effects on renal development and function in the offspring, current studies are less clear on the effects of more subtle perturbations of the RAS (e.g. via dietary

Sex Differences in the Developmental Programming of Adult Disease 319

chromosomal complement of the fetus may affect maternal metabolism and as the mother carrying a male fetus endures NR, the male fetus will face greater hardship than a female fetus in an equivalent pregnancy. In contrast, the female fetus in a pregnancy with an over-

While the existence of sexually dimorphic phenotypes is rather obvious, the mechanisms that underlie this process have remained a matter of interest. Using a theoretical model to examine the evolutionary association between X-linkage and sexually dimorphic phenotypes, Rice concluded that "sex chromosomes facilitate the evolution of sexual dimorphism and that X-linked genes have a predominant role in coding for sexually dimorphic traits" (Rice, 1984). In the ensuing twenty-five years support for this thesis has grown to include functional grouping of X chromosome gene content. Genes expressed in brain (Zechner *et al.*, 2001), for example, are particularly abundant on the X chromosome. In contrast, and perhaps of importance to potential paternal contributions to the interactions between fetus and the maternal environment, placentally expressed genes are relatively rare

It has been recognized in humans that blood pressure is higher in men than in women (Burt *et al.*, 1995) and this difference originates during adolescence and persists into adulthood (Yong *et al.*, 1993). Further, males show an enhanced propensity to progress towards renal injury and decreased renal function than do females in several species (Neugarten *et al.*, 2002;Reckelhoff *et al.*, 1998;Sandberg & Ji, 2003). Although the roots of this difference have been linked to the RAS (Miller *et al.*, 1999), a role for an alteration in the ratio of sex steroids has also been proposed. Androgens have been linked with the progression of renal injury (Reckelhoff *et al.*, 1998;Sandberg & Ji, 2003) while estrogens have been proposed as being protective of renal function (Sandberg & Ji, 2003). Moreover, it seems that sex may exert distinctly different influences during fetal and adult life. Whereas male fetuses may be more susceptible to *in utero* nutrient privation (Gilbert *et al.*, 2007a), female fetuses appear to have increased susceptibility to gestational over-nutrition (Khan *et al.*, 2003). The reasons for this are not clear; however, one clue may be held in the long observed differences in growth rates exhibited by male and female fetuses *in utero* (Parker *et al.*, 1984). Despite findings that seem to clearly identify sex hormones as a likely culprit, recent efforts have raised many further questions and much remains unclear regarding the role of innate sex *vs.* sex steroids

Epigenetic phenomena appear to be central to the induction of persistent and heritable changes in gene expression that occur without alteration of DNA sequence (Akintola *et al.*, 2008;Bird, 1986;Holliday & Ho, 2002;Wyrwoll *et al.*, 2007). While most cells in an organism contain the same DNA, gene expression varies widely across various tissues. Epigenetic mechanisms underlie this tissue- and cell-type-specific gene expression (Waterland & Michels, 2007) and include CpG methylation, histone modification (acetylation) and the activity of autoregulatory DNA-binding proteins (Kelly & Trasler, 2004). Moreover, since DNA methylation and histone acetylation are implicated in the silencing of gene expression, X-inactivation and X-linked dosage differences (Chow *et al.*, 2005), one might argue that sexbias in differential gene expression linked to DOHAD also has its roots in methylation.

Indeed, these processes appear to have many sex specific features.

nourished mother could face similar hardship via different pathways.

**5.1 Innate sex differences** 

on the X chromosome (Ko *et al.*, 1998).

in developmental programming.

**5.2 Epigenetic mechanisms** 

alterations, etc.) on the long term health of the offspring. Further work in these areas will help define the importance of these pathways in the developmental programming of health and disease.

#### **5. Potential mechanisms underlying sex differences in developmental programming**

A variety of mechanisms have been postulated with regard to DOHAD (summarized in Figure 2). While the contribution of sex to the developmental origins of disease is widely recognized, it seems sex may exert distinctly different influences during fetal and adult life. For example, while male fetuses may be more susceptible to *in utero* nutrient privation (Gilbert *et al.*, 2007a), female fetuses may have increased susceptibility to gestational overnutrition (Khan *et al.*, 2003). The reasons for this remain nebulous; however, one clue may be held in the long observed differences in growth rates exhibited by male and female fetuses *in utero* (Parker *et al.*, 1984). Hence, a faster growing male fetus may experience greater or lesser degrees of these nutritional insults compared to a female counterpart. Differences in the rate at which the male develops compared to the female likely contribute to gender differences in stress responses during pregnancy (Ozaki *et al.*, 2001). It remains unclear whether male fetuses have increased metabolism compared to female fetuses. Hence, the

Fig. 2. *Proposed mechanisms of sexual dimorphism in developmental programming.* Research has revealed the dependence on the stimuli that result in normal development, but several connections are yet to be defined. Dark blue arrows with solid outline indicate observed pathways, like blue arrows with dotted outline represent putative connections.

chromosomal complement of the fetus may affect maternal metabolism and as the mother carrying a male fetus endures NR, the male fetus will face greater hardship than a female fetus in an equivalent pregnancy. In contrast, the female fetus in a pregnancy with an overnourished mother could face similar hardship via different pathways.

#### **5.1 Innate sex differences**

318 Sex Hormones

alterations, etc.) on the long term health of the offspring. Further work in these areas will help define the importance of these pathways in the developmental programming of health

A variety of mechanisms have been postulated with regard to DOHAD (summarized in Figure 2). While the contribution of sex to the developmental origins of disease is widely recognized, it seems sex may exert distinctly different influences during fetal and adult life. For example, while male fetuses may be more susceptible to *in utero* nutrient privation (Gilbert *et al.*, 2007a), female fetuses may have increased susceptibility to gestational overnutrition (Khan *et al.*, 2003). The reasons for this remain nebulous; however, one clue may be held in the long observed differences in growth rates exhibited by male and female fetuses *in utero* (Parker *et al.*, 1984). Hence, a faster growing male fetus may experience greater or lesser degrees of these nutritional insults compared to a female counterpart. Differences in the rate at which the male develops compared to the female likely contribute to gender differences in stress responses during pregnancy (Ozaki *et al.*, 2001). It remains unclear whether male fetuses have increased metabolism compared to female fetuses. Hence, the

**Maternal environmental factors/stressors (Under/over nutrition, placental ischemia, environmental estrogen/androgens)**

> **Sexual dimorphism in: receptor expression, nephron endowment, vascular vasoactivity, energy metabolism, & locomotor activity**

**?**

**Hypertension, cardiovascular disease, metabolic syndrome, reproductive function cancer incidence**

Fig. 2. *Proposed mechanisms of sexual dimorphism in developmental programming.* Research has revealed the dependence on the stimuli that result in normal development, but several connections are yet to be defined. Dark blue arrows with solid outline indicate observed

pathways, like blue arrows with dotted outline represent putative connections.

**Sex specificity in: paracrine/endocrine pathways**

**?**

**RAS / COX2 / GC**

**Sex hormones: ↑Androgens ↓Estrogens**

**Epigenetic/(nutrigenetic?) sex specific alterations: DNA methylation & histone acetylation** 

**?**

**5. Potential mechanisms underlying sex differences in developmental** 

and disease.

**programming** 

**Sex specificity in: gene expression & phenotype**

**?**

**Morphological & functional deficits ?**

**?**

While the existence of sexually dimorphic phenotypes is rather obvious, the mechanisms that underlie this process have remained a matter of interest. Using a theoretical model to examine the evolutionary association between X-linkage and sexually dimorphic phenotypes, Rice concluded that "sex chromosomes facilitate the evolution of sexual dimorphism and that X-linked genes have a predominant role in coding for sexually dimorphic traits" (Rice, 1984). In the ensuing twenty-five years support for this thesis has grown to include functional grouping of X chromosome gene content. Genes expressed in brain (Zechner *et al.*, 2001), for example, are particularly abundant on the X chromosome. In contrast, and perhaps of importance to potential paternal contributions to the interactions between fetus and the maternal environment, placentally expressed genes are relatively rare on the X chromosome (Ko *et al.*, 1998).

It has been recognized in humans that blood pressure is higher in men than in women (Burt *et al.*, 1995) and this difference originates during adolescence and persists into adulthood (Yong *et al.*, 1993). Further, males show an enhanced propensity to progress towards renal injury and decreased renal function than do females in several species (Neugarten *et al.*, 2002;Reckelhoff *et al.*, 1998;Sandberg & Ji, 2003). Although the roots of this difference have been linked to the RAS (Miller *et al.*, 1999), a role for an alteration in the ratio of sex steroids has also been proposed. Androgens have been linked with the progression of renal injury (Reckelhoff *et al.*, 1998;Sandberg & Ji, 2003) while estrogens have been proposed as being protective of renal function (Sandberg & Ji, 2003). Moreover, it seems that sex may exert distinctly different influences during fetal and adult life. Whereas male fetuses may be more susceptible to *in utero* nutrient privation (Gilbert *et al.*, 2007a), female fetuses appear to have increased susceptibility to gestational over-nutrition (Khan *et al.*, 2003). The reasons for this are not clear; however, one clue may be held in the long observed differences in growth rates exhibited by male and female fetuses *in utero* (Parker *et al.*, 1984). Despite findings that seem to clearly identify sex hormones as a likely culprit, recent efforts have raised many further questions and much remains unclear regarding the role of innate sex *vs.* sex steroids in developmental programming.

#### **5.2 Epigenetic mechanisms**

Epigenetic phenomena appear to be central to the induction of persistent and heritable changes in gene expression that occur without alteration of DNA sequence (Akintola *et al.*, 2008;Bird, 1986;Holliday & Ho, 2002;Wyrwoll *et al.*, 2007). While most cells in an organism contain the same DNA, gene expression varies widely across various tissues. Epigenetic mechanisms underlie this tissue- and cell-type-specific gene expression (Waterland & Michels, 2007) and include CpG methylation, histone modification (acetylation) and the activity of autoregulatory DNA-binding proteins (Kelly & Trasler, 2004). Moreover, since DNA methylation and histone acetylation are implicated in the silencing of gene expression, X-inactivation and X-linked dosage differences (Chow *et al.*, 2005), one might argue that sexbias in differential gene expression linked to DOHAD also has its roots in methylation. Indeed, these processes appear to have many sex specific features.

Sex Differences in the Developmental Programming of Adult Disease 321

studied by Sullivan (Sullivan *et al.*, 2007) who has described a relationship between androgens and the development of albuminuria, and the renal protection afforded by estrogen, in spontaneously hypertensive rats. There is some evidence to suggest that both over activity of the renin angiotensin system and oxidative stress likely contributing to sex differences in the progression to renal injury. Treatment with either an AT1 blocker and/or an ACE inhibitor blunts the occurrence of renal injury in males (Lazaro *et al.*, 2005). Male spontaneously hypertensive rats (SHR), which exhibit some signs of a programming model such as smaller size at birth when compared to Wistar-Kyoto control rats, exhibit androgendependent increases in blood pressure and albuminuria that are independent of renal cortical angiotensin II levels and oxidative stress (Sullivan *et al.*, 2007). In contrast, a female specific form of hypertension during pregnancy, preeclampsia, is reportedly not to be influenced by

Interestingly, the cardio-renal protective effects of estrogens has not been a universal finding (Salazar *et al.*, 2008). Considering the differences between the models employed by different laboratories, one possibility could be the magnitude of the insult to the kidney during development has an influence on the extent of protection that may be afforded by female sex hormones in later life. It is widely recognized that differences in sex hormones contribute to considerable sexual dimorphism in the transcriptome of a variety of mammalian tissues and organs (Rinn & Snyder, 2005); however, it has only recently been recognized that androgen/estrogen independent mechanisms may operate at the transcriptional level to regulate sex differences (Tullis *et al.*, 2003). This possibility represents an alternate pathway that may be at work contributing to the observations that the relationship between sex hormones and blood pressure is far more complex than simply the balance of estrogen *vs.* testosterone (Ojeda *et al.*, 2007a). Taken together, it appears that the influence of sex on the developmental origins of disease may reach far beyond the widely recognized role of sex hormones. Alternatively, recent work implicates growth hormone (GH) in sex dependent differences in renal expression of glomerular AT1 during hypertrophy following uninephrectomy; male rat kidneys show increased glomerular AT1 expression, whereas females do not (Mok *et al.*, 2003). Because there is sexual dimorphism in GH release these observations may hold implications for both normal and pathological growth and

From a clinical perspective it is hoped that increased understanding and awareness of developmental programming will lead to better diagnostic, preventative and therapeutic measures. The persistence of programmed effects is likely due to covalent modifications of the genome resulting from changes in promoter methylation and histone acetylation. The emerging fields of metabolomics and nutrigenetics suggest many of these alterations are likely a result of changes in the metabolic flux during critical periods of development. While epigenetic phenomena are central to the induction of persistent and heritable changes in gene expression that occur without alteration of DNA sequence, their contribution to the intensively studied sex differences in developmental programming remains uncertain. While reversal of these molecular changes may be possible and to improve long-term health outcomes if interventions are timed appropriately, loss of function in existing structures may be difficult to overcome if developmental plasticity is no longer present. For example it is difficult to see how any deficit in nephron endowment can be remedied. Nevertheless,

the levels of circulating testosterone levels during pregnancy (Tuutti *et al.*, 2011).

development of the kidney.

**6. Concluding remarks** 

Because moderate folate depletion can induce genome-wide DNA methylation (Jacob *et al.*, 1998), genomic methylation may be useful as an integrative biomarker of methyl donor nutritional status (Mason, 2003). While considerable work has been initiated in this area with regards to developmental programming, little work has focused specifically on sex differences. Interestingly, sheep exposed to a methyl deficient diet during pregnancy produce hypertensive male offspring compared to females of similar rearing, as well as to male and female controls (Sinclair *et al.*, 2007). The authors then evaluated 1400 CpG sites (primarily gene promoter associated) in fetal liver at 90 days of gestation (term=150d) and reported that more than half of the affected loci were specific to males. These observations suggest male-specific demethylation that could provide a mechanistic basis for the phenotypic sex differences observed in that study (Sinclair *et al.*, 2007). In addition, the emerging fields of nutrigenetics and metabolomics (Mutch *et al.*, 2005;Goodacre, 2007) seem poised to shed further light on these operational characteristics of these mechanisms.

Alternatively, it has also been hypothesized that when genes are expressed in multiple tissues or serve several functions they should show less sex bias than genes that are more specialized (Ellegren & Parsch, 2007). The genes such as those involved in the RAS are certainly expressed in multiple tissues, yet these genes are also closely associated with sex differences in the developmental origins of cardio-renal diseases. Clearly there is a tremendous gap in our understanding of these complex topics and further studies are needed to clarify these matters particularly in the light of the differences reported regarding fetal gender and the developmental response to maternal over- and under-nutrition.

#### **5.3 Sex steroids**

In contrast to the sex-related dichotomy observed in response to nutritional stressors, when faced with a robust stressor such as AT1 antagonism (Loria *et al.*, 2007a;Loria *et al.*, 2007b;Saez *et al.*, 2007;Salazar *et al.*, 2008), severe protein restriction (Woods *et al.*, 2001), or chronic reductions uterine perfusion pressure (Ojeda *et al.*, 2007a;Alexander, 2003) both male and female fetuses are affected similarly *in utero.* Nonetheless a dichotomy emerges later in life with females being less impacted by their suboptimal *in utero* experience (Loria *et al.*, 2007a;Loria *et al.*, 2007b;Saez *et al.*, 2007;Salazar *et al.*, 2008). The apparent benefit of being female in scenarios such as this are supported by recent work that suggested estrogens confer a protective effect on intrauterine growth restricted females that prevents the development of programmed hypertension (Ojeda *et al.*, 2007a). Moreover, the observation that ovariectomy leads to a significant increase in blood pressure in growth-restricted females with no significant effect in controls makes a strong case for the post-developmental involvement of estrogens. Indeed, estrogen replacement reversed the effect of ovariectomy on blood pressure in growth-restricted offspring as did renin angiotensin system blockade (Ojeda *et al.*, 2007a).

Studies on the role of sex hormones in expression of components of renal renin angiotensin in healthy Sprague Dawley rats, have suggested that an estrogen-mediated attenuation of renal AT1 binding is a potential mechanism by which estrogen exerts protection from vascular and renal disease in females (Rogers *et al.*, 2007). When this inhibition is lifted following ovariectomy in their model, or in diabetes or menopause, the resulting increased angiotensin II signaling increases both the degree of susceptibility to vascular and renal disease and the rate of existing disease progression (Rogers *et al.*, 2007).

Testosterone has also been implicated in the progression of hypertension in male growth restricted offspring (Ojeda *et al.*, 2007b). The potential underlying mechanisms have been

Because moderate folate depletion can induce genome-wide DNA methylation (Jacob *et al.*, 1998), genomic methylation may be useful as an integrative biomarker of methyl donor nutritional status (Mason, 2003). While considerable work has been initiated in this area with regards to developmental programming, little work has focused specifically on sex differences. Interestingly, sheep exposed to a methyl deficient diet during pregnancy produce hypertensive male offspring compared to females of similar rearing, as well as to male and female controls (Sinclair *et al.*, 2007). The authors then evaluated 1400 CpG sites (primarily gene promoter associated) in fetal liver at 90 days of gestation (term=150d) and reported that more than half of the affected loci were specific to males. These observations suggest male-specific demethylation that could provide a mechanistic basis for the phenotypic sex differences observed in that study (Sinclair *et al.*, 2007). In addition, the emerging fields of nutrigenetics and metabolomics (Mutch *et al.*, 2005;Goodacre, 2007) seem

poised to shed further light on these operational characteristics of these mechanisms.

fetal gender and the developmental response to maternal over- and under-nutrition.

disease and the rate of existing disease progression (Rogers *et al.*, 2007).

Testosterone has also been implicated in the progression of hypertension in male growth restricted offspring (Ojeda *et al.*, 2007b). The potential underlying mechanisms have been

**5.3 Sex steroids** 

Alternatively, it has also been hypothesized that when genes are expressed in multiple tissues or serve several functions they should show less sex bias than genes that are more specialized (Ellegren & Parsch, 2007). The genes such as those involved in the RAS are certainly expressed in multiple tissues, yet these genes are also closely associated with sex differences in the developmental origins of cardio-renal diseases. Clearly there is a tremendous gap in our understanding of these complex topics and further studies are needed to clarify these matters particularly in the light of the differences reported regarding

In contrast to the sex-related dichotomy observed in response to nutritional stressors, when faced with a robust stressor such as AT1 antagonism (Loria *et al.*, 2007a;Loria *et al.*, 2007b;Saez *et al.*, 2007;Salazar *et al.*, 2008), severe protein restriction (Woods *et al.*, 2001), or chronic reductions uterine perfusion pressure (Ojeda *et al.*, 2007a;Alexander, 2003) both male and female fetuses are affected similarly *in utero.* Nonetheless a dichotomy emerges later in life with females being less impacted by their suboptimal *in utero* experience (Loria *et al.*, 2007a;Loria *et al.*, 2007b;Saez *et al.*, 2007;Salazar *et al.*, 2008). The apparent benefit of being female in scenarios such as this are supported by recent work that suggested estrogens confer a protective effect on intrauterine growth restricted females that prevents the development of programmed hypertension (Ojeda *et al.*, 2007a). Moreover, the observation that ovariectomy leads to a significant increase in blood pressure in growth-restricted females with no significant effect in controls makes a strong case for the post-developmental involvement of estrogens. Indeed, estrogen replacement reversed the effect of ovariectomy on blood pressure in growth-restricted offspring as did renin angiotensin system blockade (Ojeda *et al.*, 2007a). Studies on the role of sex hormones in expression of components of renal renin angiotensin in healthy Sprague Dawley rats, have suggested that an estrogen-mediated attenuation of renal AT1 binding is a potential mechanism by which estrogen exerts protection from vascular and renal disease in females (Rogers *et al.*, 2007). When this inhibition is lifted following ovariectomy in their model, or in diabetes or menopause, the resulting increased angiotensin II signaling increases both the degree of susceptibility to vascular and renal studied by Sullivan (Sullivan *et al.*, 2007) who has described a relationship between androgens and the development of albuminuria, and the renal protection afforded by estrogen, in spontaneously hypertensive rats. There is some evidence to suggest that both over activity of the renin angiotensin system and oxidative stress likely contributing to sex differences in the progression to renal injury. Treatment with either an AT1 blocker and/or an ACE inhibitor blunts the occurrence of renal injury in males (Lazaro *et al.*, 2005). Male spontaneously hypertensive rats (SHR), which exhibit some signs of a programming model such as smaller size at birth when compared to Wistar-Kyoto control rats, exhibit androgendependent increases in blood pressure and albuminuria that are independent of renal cortical angiotensin II levels and oxidative stress (Sullivan *et al.*, 2007). In contrast, a female specific form of hypertension during pregnancy, preeclampsia, is reportedly not to be influenced by the levels of circulating testosterone levels during pregnancy (Tuutti *et al.*, 2011).

Interestingly, the cardio-renal protective effects of estrogens has not been a universal finding (Salazar *et al.*, 2008). Considering the differences between the models employed by different laboratories, one possibility could be the magnitude of the insult to the kidney during development has an influence on the extent of protection that may be afforded by female sex hormones in later life. It is widely recognized that differences in sex hormones contribute to considerable sexual dimorphism in the transcriptome of a variety of mammalian tissues and organs (Rinn & Snyder, 2005); however, it has only recently been recognized that androgen/estrogen independent mechanisms may operate at the transcriptional level to regulate sex differences (Tullis *et al.*, 2003). This possibility represents an alternate pathway that may be at work contributing to the observations that the relationship between sex hormones and blood pressure is far more complex than simply the balance of estrogen *vs.* testosterone (Ojeda *et al.*, 2007a). Taken together, it appears that the influence of sex on the developmental origins of disease may reach far beyond the widely recognized role of sex hormones. Alternatively, recent work implicates growth hormone (GH) in sex dependent differences in renal expression of glomerular AT1 during hypertrophy following uninephrectomy; male rat kidneys show increased glomerular AT1 expression, whereas females do not (Mok *et al.*, 2003). Because there is sexual dimorphism in GH release these observations may hold implications for both normal and pathological growth and development of the kidney.

#### **6. Concluding remarks**

From a clinical perspective it is hoped that increased understanding and awareness of developmental programming will lead to better diagnostic, preventative and therapeutic measures. The persistence of programmed effects is likely due to covalent modifications of the genome resulting from changes in promoter methylation and histone acetylation. The emerging fields of metabolomics and nutrigenetics suggest many of these alterations are likely a result of changes in the metabolic flux during critical periods of development. While epigenetic phenomena are central to the induction of persistent and heritable changes in gene expression that occur without alteration of DNA sequence, their contribution to the intensively studied sex differences in developmental programming remains uncertain. While reversal of these molecular changes may be possible and to improve long-term health outcomes if interventions are timed appropriately, loss of function in existing structures may be difficult to overcome if developmental plasticity is no longer present. For example it is difficult to see how any deficit in nephron endowment can be remedied. Nevertheless,

Sex Differences in the Developmental Programming of Adult Disease 323

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#### **7. Acknowledgements**

The authors regret that, because of space limitations, we have been unable to cite all the primary literature in the field. This work is supported by American Heart Association Grant 10SDG2600040.

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**16** 

*1,2ROC 3,4USA* 

**WW Domain-Containing** 

*1Institute of Molecular Medicine,* 

*2Department of Chemistry,* 

**Oxidoreductase is a Potential** 

Won-Pei Su1, Shu-Hui Chen2, Szu-Jung Chen1,

*National Cheng Kung University, Tainan, Taiwan,* 

*National Cheng Kung University, Tainan, Taiwan, 3Department of Neuroscience and Physiology, SUNY* 

*4Department of Neurochemistry, NYS Institute of Basic Research in Developmental Disabilities, Staten Island, NY,* 

*Upstate Medical University, Syracuse, NY,* 

**Receptor for Sex Steroid Hormones** 

Pei-Yi Chou1, Chun-Cheng Huang1 and Nan-Shan Chang1,3,4

Estrogen is a steroid hormone that comprises a group of compounds, including estrone (E1), estradiol (E2) and estriol (E3). E2 is an ovarian hormone necessary for the development of secondary sexual characteristics and function of the reproductive system in females. It also plays important roles in non-reproductive organs by multiple pathways. Estrogens are produced primarily by developing follicles in the ovaries, the corpus luteum, and the placenta. Some estrogens are also produced in smaller amounts by other tissues such as the liver, adrenal glands, and the breasts. E2 is converted from testosterone and E1 from rostenedione; both conversions are regulated by a dehydrogenase enzyme, aromatase. Estrogens are eliminated from the body by metabolic conversion to hormonally inactive and water-soluble metabolites that are excreted in the urine and/or feces. The metabolic disposition of estrogens includes oxidative metabolism (Martucci et al., 1993) and conjugative metabolism by glucuronidation (Zhu, et al., 1996), sulfonation (Hernandez et al., 1992) and/or *O*-methylation (Ball & Knuppen, 1980). Hydroxylation at the C-2 and C-4 position of E2 (17-Estradiol) yields the catecholestrogens (CEs), 2-hydroxyestrone (2- OHE1) and 2-hydroxyestradiol (2-OHE2), 4-hydroxyestrone (4-OHE1) and 4 hydroxyestradiol (4-OHE2) while hydroxylation at the C-16 position yields 16 hydroxyestrone (16 -OHE1), which is subsequently converted to estriol (E3) (Ball & Knuppen, 1980; Zhu & Conney, 1998). The hydroxylated products exert very different biological properties: the 16 -hydroxy and 4-hydroxy metabolites are active estrogens,

**1. Introduction** 

**1.1 Biosynthesis and metabolism of estrogens** 

