**Meet the editor**

Dr Raghvendra K. Dubey is currently a professor at the Clinic for Reproductive Endocrinology, University Hospital, Zurich. Born in Lucknow, India, he completed his education there, and later obtained his Masters degree in Biochemistry (MSc) from Lucknow University, and went on to earn his PhD from Kanpur University in India. Subsequently, he completed a three year postdoc-

toral fellowship in Clinical Pharmacology at Vanderbilt University, USA, and later joined the Department of Medicine at West Virginia University as Assistant Professor. After three years, he joined University of Basel as a visiting Professor, and later became Associate Professor at the Department of Medicine in the University of Pittsburgh Medical Center. His current research topic is to study the role of sex hormones in various pathophysiologies, with specific relevance to women's health. He has published more than 100 original peer reviewed papers, several reviews, and book chapters. He is a fellow of the American Heart Association, a member of the American Endocrine Society, and the American Physiological Society. He held a joint associate professorship from 1996 -2010 at the Center for Clinical Pharmacology, Department of Medicine, University of Pittsburgh Medical Center, USA. He has several patents on investigative therapeutic molecules, and he has had competitive research funding for the last two decades. Dr. Dubey's work on estradiol metabolism and the development of 2-methoxyestradiol as a non-carcinogenic therapeutic molecule is ongoing. He has mentored several PhD and Masters students both at ETH Zurich and University of Zurich, Switzerland, and at the University of Pittsburgh, USA.

Contents

**Preface IX** 

Gorazd Drevenšek

Chapter 4 **Sex Hormones and Infertility 81** 

Ylva Vladic Stjernholm

Andrzej Gomuła

Sae Chul Kim

Üner Tan

Chapter 1 **Sex Hormones and Vascular Function 1** 

Robert Hilgers and Dongqi Xing

Chapter 3 **Serum Free Testosterone and Estradiol Levels** 

Iptisam Ipek Muderris and Gokalp Oner

Meaghan Bowling, Suzanne Oparil, Fadi Hage,

**in Perceptual-Verbal and Spatial Abilities; Differences in Sex and Hand Preference 65** 

Chapter 5 **Progesterone in Human Pregnancy and Parturition 99** 

Chapter 6 **Late - Onset Hypogonadism - New Point of View 115** 

Chapter 7 **Hypogonadism After Childhood Cancer Treatment 161** 

Michał Rabijewski and Lucyna Papierska

Chapter 9 **Testosterone Deficiency Linked to Lower Urinary Tract Symptoms 215** 

Chapter 10 **Sex Hormones and Bacterial Infections 237**  Marc Leone, Julien Textoris,

Christian Capo and Jean-Louis Mege

Lorna Zadravec Zaletel, Ljupčo Todorovski and Berta Jereb

Chapter 8 **Osteoporosis in Men - A Crucial Role of Sex Hormones 197** 

Chapter 2 **The Role of Sex Hormones in the Cardiovascular System 31** 

### Contents

#### **Preface XI**


Christian Capo and Jean-Louis Mege

X Contents


### Preface

Sex hormones not only regulate reproductive function, but they also play a prominent role in the biology and physiology of several organs/tissues, as well as in the pathophysiology of several diseases. Indeed, accumulating evidence suggests that sex hormones influence different organs and diseases, thereby highlighting the need to assess their roles individually.

During the last two decades, the information on the mechanisms of action of female sex hormones like estradiol have evolved from the conventional nuclear estrogen receptor (ER-alpha) dependent mechanisms to include additional non-nuclear (membrane), non-genomic, and ER-independent (estradiol metabolism driven) mechanisms. This highlights the need to update the current knowledge on sex hormones. Similar to estrogens, the impact of male sex hormones, androgens, and their mechanisms of actions, and association with various disorders, has evolved and needs to be addressed in greater detail. Increasing evidence that exogenous/epigenetic factors can influence sex hormone production and action emphasises the need to understand and update the mechanisms involved.

The current book provides an in-depth and systematic overview of the sex hormones (male and female hormones) and their impact in the biology and pathophysiology of various diseases. Bowling and colleagues provide an overview of the effects of sex hormones on vascular function, Gorazd Drevensek reviews the association with cardiovascular disease, and Leone et al. discuss their role in regulating bacterial infections. Testosterone's impact upon and association with various diseases, and the use of testosterone measurement to diagnose and treat patients is discussed in two chapters on hypogonadism by Andrzej Gomula and Lorna Z. Zaletel, respectively. Additional discussion on this topic is also found in the chapter on urinary tract symptoms, by Sae Chul Kim, and the chapter on osteoporosis by M. Rabijewski and L. Papierska. A unique perspective on sex differences in developmental programming of adult diseases is provided in the chapter by J.S. Gilbert and C.T. Banek. M. Norlin and K. Wikvall go on to lead the readers through the intricate pathways of sex hormone biosynthesis and metabolism. The importance of sex hormones in reproduction is examined in three chapters by S.E. Seeger and U. Kemmerer, by Y.V. Stjernholm, and by I.I. Muderris and Gokalp Oner, respectively. The role of sex hormones in neuromuscular control and physical training is discussed by E.L. Cadore, L.F.M.

#### X Preface

Kruel, and Rose Fouladi. Continuing, Stefan Van Dongen and S. Ellen research sex hormones' role in masculinity and sex behavior, and Uner Tan discusses the association of sex hormones with perceptual-verbal and spatial abilities. The impact and importance of sex hormones in sex differentiation is provided in the chapter by I. Negri and M. Pellecchia, and the role of WW domain containing oxidoreductase in regulating hormone action and cancer metastasis is overviewed by W.P. Su and colleagues. Because development of therapeutic estrogens is essential for hormone replacement, the pharmacological aspects of drug development are also reviewed in great detail by C. Wiranidchapong.

The overall aim of this book is to introduce readers to the ever-growing importance of sex hormones in regulating human biology and physiology. The book also discusses the association of sex hormones in various clinical pathologies, as well as their therapeutic importance.

> **Dr. Raghvendra K. Dubey**  Department of Obstetrics & Gynecology, Clinic for Endocrinology, University of Zurich Hospital, Zurich, Switzerland

X Preface

great detail by C. Wiranidchapong.

therapeutic importance.

Kruel, and Rose Fouladi. Continuing, Stefan Van Dongen and S. Ellen research sex hormones' role in masculinity and sex behavior, and Uner Tan discusses the association of sex hormones with perceptual-verbal and spatial abilities. The impact and importance of sex hormones in sex differentiation is provided in the chapter by I. Negri and M. Pellecchia, and the role of WW domain containing oxidoreductase in regulating hormone action and cancer metastasis is overviewed by W.P. Su and colleagues. Because development of therapeutic estrogens is essential for hormone replacement, the pharmacological aspects of drug development are also reviewed in

The overall aim of this book is to introduce readers to the ever-growing importance of sex hormones in regulating human biology and physiology. The book also discusses the association of sex hormones in various clinical pathologies, as well as their

**Dr. Raghvendra K. Dubey** 

Clinic for Endocrinology,

Switzerland

Department of Obstetrics & Gynecology,

University of Zurich Hospital, Zurich,

**1** 

*USA* 

**Sex Hormones and Vascular Function** 

The relationship between sex hormones and cardiovascular function and disease has long been recognized. As early as the 1950s, researchers concluded that although levels of cholesterol played a major role in the development of cardiovascular disease (CVD), other factors, including gender and hormones, played a role as well. (Anonymous, 1958). Since that time, despite extensive research focusing on the effects of estrogen on vascular function, the relationship remains poorly understood. Furthermore, clinical treatment of postmenopausal women with hormone replacement therapy (HRT) continues to be

Until the 1990s, extensive observational data suggested that HRT was cardioprotective. However, results from the Heart and Estrogen/Progestin Replacement Study (HERS-I and – II) did not confirm a protective effect of HRT on the heart. (Hulley et al 1996; Grady et al 2002). Later, data from the Women's Health Initiative (WHI) reported an increase in coronary heart disease (CHD) risk in women treated with combined estrogen-progestin compared to placebo, while the WHI unopposed-estrogen arm showed no increase in CHD events (Roussow et al, 2002; Anderson et al, 2004). Since release of the WHI, follow-up analyses have shown that the timing of initiation of HRT makes a difference in outcomes. These analyses showed that younger postmenopausal women who initiate therapy at the time of menopause are not at increased risk of CHD events compared to women who

In this chapter, we will discuss the pathophysiologic effects of sex hormones on the vasculature, describe both clinical and basic research that has led us to our current understanding, and conclude with future perspectives on avenues of investigation that may

Estrogen is a steroid hormone that is produced by aromatization of androgen precursors, specifically androstenedione. (Speroff and Fritz, 2005) Estrogens are synthesized primarily in the ovary, with minor contribution from adipose, skin, muscle, and endometrial tissue. In premenopausal women, the primary form of estrogen is 17β-estradiol, often simply referred to as estradiol or E2 (for the two hydroxyl groups located on the basic estrogen ring

controversial due to conflicting findings in clinical trials.

lead to innovative treatments for postmenopausal women.

**1. Introduction** 

initiate therapy at a later age.

**2.1 Estrogen metabolism** 

**2. Physiology of estrogen actions** 

Meaghan Bowling, Suzanne Oparil,

*University of Alabama at Birmingham* 

Fadi Hage, Robert Hilgers and Dongqi Xing

### **Sex Hormones and Vascular Function**

Meaghan Bowling, Suzanne Oparil, Fadi Hage, Robert Hilgers and Dongqi Xing *University of Alabama at Birmingham USA* 

#### **1. Introduction**

The relationship between sex hormones and cardiovascular function and disease has long been recognized. As early as the 1950s, researchers concluded that although levels of cholesterol played a major role in the development of cardiovascular disease (CVD), other factors, including gender and hormones, played a role as well. (Anonymous, 1958). Since that time, despite extensive research focusing on the effects of estrogen on vascular function, the relationship remains poorly understood. Furthermore, clinical treatment of postmenopausal women with hormone replacement therapy (HRT) continues to be controversial due to conflicting findings in clinical trials.

Until the 1990s, extensive observational data suggested that HRT was cardioprotective. However, results from the Heart and Estrogen/Progestin Replacement Study (HERS-I and – II) did not confirm a protective effect of HRT on the heart. (Hulley et al 1996; Grady et al 2002). Later, data from the Women's Health Initiative (WHI) reported an increase in coronary heart disease (CHD) risk in women treated with combined estrogen-progestin compared to placebo, while the WHI unopposed-estrogen arm showed no increase in CHD events (Roussow et al, 2002; Anderson et al, 2004). Since release of the WHI, follow-up analyses have shown that the timing of initiation of HRT makes a difference in outcomes. These analyses showed that younger postmenopausal women who initiate therapy at the time of menopause are not at increased risk of CHD events compared to women who initiate therapy at a later age.

In this chapter, we will discuss the pathophysiologic effects of sex hormones on the vasculature, describe both clinical and basic research that has led us to our current understanding, and conclude with future perspectives on avenues of investigation that may lead to innovative treatments for postmenopausal women.

#### **2. Physiology of estrogen actions**

#### **2.1 Estrogen metabolism**

Estrogen is a steroid hormone that is produced by aromatization of androgen precursors, specifically androstenedione. (Speroff and Fritz, 2005) Estrogens are synthesized primarily in the ovary, with minor contribution from adipose, skin, muscle, and endometrial tissue. In premenopausal women, the primary form of estrogen is 17β-estradiol, often simply referred to as estradiol or E2 (for the two hydroxyl groups located on the basic estrogen ring

Sex Hormones and Vascular Function 3

GPER (GPR30) is an intracellular transmembrane ER that initiates many rapid non-genomic signaling events, including intracellular calcium mobilization and synthesis of phosphatidyl-inositol 3,4,5-triphosphate in the nucleus of multiple cell types. (Revankar et al, 2005). GPER has been indentified in human internal mammary arteries, saphenous veins, and contributes to vasorelaxation in arteries, although this mechanism remains to be fully

Estrogen acts on various cell types through both genomic and non–genomic mechanisms. Genomic effects occur when estrogen binds to ERs in target tissue cell nuclei, resulting in changes in gene expression. Multiple genes in both the nuclear and mitochondrial genomes are regulated by ERα and ER-β. (O'Lone et al, 2007) In aortic smooth muscle cells and endothelial cells of wild-type ovariectomized mice, E2 treatment resulted in both up- and down-regulation of multiple genes involved in mitochondrial fuction. ERα upregulated four clusters of genes, while ERβ downregulated a different set of mitochondrial genes. E2 also stimulates oxidative phosphorylation and inhibits production of superoxide and other reactive oxygen species in mitochondria. (O'Lone et al, 2007) It is hypothesized that this mechanism decreases the rate of accumulation of mitochondrial DNA mutations over a lifespan, and therefore protects against age-related disease. This notion is relevant to the

Estrogen can also trigger non-genomic events by binding to targets other than nuclear receptors, eg., cell membrane ERs. (Kelly and Levin, 2001) Non-genomic effects, such as direct activation of intracellular signaling pathways, can be rapid and do not require changes in gene expression, although the long-term consequences include altered

Progesterone is a steroid hormone that is synthesized from the precursor pregnenolone (a cholesterol metabolite) by 3β-hydroxysteroid dehydrogenase in the ovaries and adrenal

Progesterone acts on two major progesterone receptors (PRs), PR-A and PR-B. (Speroff and Fritz, 2005) While the role of ERs in vascular physiology and pathophysiology is well studied, the literature on PRs is limited and most of what is known about their biological function is derived from studies of reproductive tissues. PR-A and PR-B can form homodimers (AA and BB) or heterodimers (AB) upon binding to a progestin. Downstream effects include protein phosphorylation and modulation of gene transcription. (Speroff and Fritz, 2005) Based on in-vitro studies of endometrium and breast tissue, PR-B is a positive

Fig. 2. Progesterone Metabolism ((modified from Science Slides Suite © 2010))

development of CVD and timing of HRT initiation. (O'Lone et al, 2007)

understood (Haas et al, 2009)

transcription of targeted genes.

glands. (Figure 2)

**3. Physiology of progesterone actions** 

**3.1 Progesterone metabolism and progesterone receptors** 

structure). (Speroff and Fritz, 2005) Estradiol is the form of estrogen used in most preclinical studies, and will be abbreviated as 'E2' in this chapter. Clinical studies, particularly studies of HRT, have employed a variety of naturally occurring or synthetic estrogens, which will be identified specifically in the text. Other forms of estrogen include estrone and estriol. Estrone, like estradiol, is produced by aromatization of androstenedione, and is the primary estrogen in postmenopausal women. Estriol is a peripheral metabolite of estrone and estradiol and is not secreted by the ovary. Estriol is the dominant form of estrogen in pregnant women. (Speroff and Fritz, 2005)

Fig. 1. Estrogen Metabolism (modified from Science Slides Suite © 2010)

The majority of circulating estrogens are bound to carrier proteins, including albumin and sex hormone-binding globulin (SHBG). Albumin binds 30 percent of circulating estrogen and SHBG binds another 69 percent. (Speroff and Fritz, 2005) Only the remaining 1 percent of estrogen that is not protein bound is physiologically active.

#### **2.2 Estrogen receptors**

Estrogens act on specific estrogen receptors (ERs) that are differentially expressed in various tissues. There are at least three, and possibly four distinct estrogen receptors. Two of these are the classic ERs: ERα and ER-β. Other ERs include the more recently discovered G protein-coupled receptor (GPER, GPR30, and a putative receptor (ER-X), that has been studied mainly in brain. (Miller et al, 2008)

ERα and ERβ are members of the nuclear steroid hormone receptor superfamily and function as ligand-activated transcription factors. (Speroff and Fritz, 2005) They are expressed in the vasculature and play a role in mediating/modulating responses to vascular injury. Once an estrogen ligand binds to its receptor, the receptor undergoes a conformational change that leads to downstream events in the nucleus, activating or inactivating transcription factors that lead to alterations in gene expression. The conformational plasticity of the ERs is a major reason that estrogen is able to have a variety of agonist/antagonist effects in a given cell or tissue. (Speroff and Fritz, 2005) ERα and ERβ play a pivotal role in vascular remodeling in response to vascular injury.

structure). (Speroff and Fritz, 2005) Estradiol is the form of estrogen used in most preclinical studies, and will be abbreviated as 'E2' in this chapter. Clinical studies, particularly studies of HRT, have employed a variety of naturally occurring or synthetic estrogens, which will be identified specifically in the text. Other forms of estrogen include estrone and estriol. Estrone, like estradiol, is produced by aromatization of androstenedione, and is the primary estrogen in postmenopausal women. Estriol is a peripheral metabolite of estrone and estradiol and is not secreted by the ovary. Estriol is the dominant form of estrogen in

Fig. 1. Estrogen Metabolism (modified from Science Slides Suite © 2010)

play a pivotal role in vascular remodeling in response to vascular injury.

of estrogen that is not protein bound is physiologically active.

**2.2 Estrogen receptors** 

studied mainly in brain. (Miller et al, 2008)

The majority of circulating estrogens are bound to carrier proteins, including albumin and sex hormone-binding globulin (SHBG). Albumin binds 30 percent of circulating estrogen and SHBG binds another 69 percent. (Speroff and Fritz, 2005) Only the remaining 1 percent

Estrogens act on specific estrogen receptors (ERs) that are differentially expressed in various tissues. There are at least three, and possibly four distinct estrogen receptors. Two of these are the classic ERs: ERα and ER-β. Other ERs include the more recently discovered G protein-coupled receptor (GPER, GPR30, and a putative receptor (ER-X), that has been

ERα and ERβ are members of the nuclear steroid hormone receptor superfamily and function as ligand-activated transcription factors. (Speroff and Fritz, 2005) They are expressed in the vasculature and play a role in mediating/modulating responses to vascular injury. Once an estrogen ligand binds to its receptor, the receptor undergoes a conformational change that leads to downstream events in the nucleus, activating or inactivating transcription factors that lead to alterations in gene expression. The conformational plasticity of the ERs is a major reason that estrogen is able to have a variety of agonist/antagonist effects in a given cell or tissue. (Speroff and Fritz, 2005) ERα and ERβ

pregnant women. (Speroff and Fritz, 2005)

GPER (GPR30) is an intracellular transmembrane ER that initiates many rapid non-genomic signaling events, including intracellular calcium mobilization and synthesis of phosphatidyl-inositol 3,4,5-triphosphate in the nucleus of multiple cell types. (Revankar et al, 2005). GPER has been indentified in human internal mammary arteries, saphenous veins, and contributes to vasorelaxation in arteries, although this mechanism remains to be fully understood (Haas et al, 2009)

Estrogen acts on various cell types through both genomic and non–genomic mechanisms. Genomic effects occur when estrogen binds to ERs in target tissue cell nuclei, resulting in changes in gene expression. Multiple genes in both the nuclear and mitochondrial genomes are regulated by ERα and ER-β. (O'Lone et al, 2007) In aortic smooth muscle cells and endothelial cells of wild-type ovariectomized mice, E2 treatment resulted in both up- and down-regulation of multiple genes involved in mitochondrial fuction. ERα upregulated four clusters of genes, while ERβ downregulated a different set of mitochondrial genes. E2 also stimulates oxidative phosphorylation and inhibits production of superoxide and other reactive oxygen species in mitochondria. (O'Lone et al, 2007) It is hypothesized that this mechanism decreases the rate of accumulation of mitochondrial DNA mutations over a lifespan, and therefore protects against age-related disease. This notion is relevant to the development of CVD and timing of HRT initiation. (O'Lone et al, 2007)

Estrogen can also trigger non-genomic events by binding to targets other than nuclear receptors, eg., cell membrane ERs. (Kelly and Levin, 2001) Non-genomic effects, such as direct activation of intracellular signaling pathways, can be rapid and do not require changes in gene expression, although the long-term consequences include altered transcription of targeted genes.

#### **3. Physiology of progesterone actions**

#### **3.1 Progesterone metabolism and progesterone receptors**

Progesterone is a steroid hormone that is synthesized from the precursor pregnenolone (a cholesterol metabolite) by 3β-hydroxysteroid dehydrogenase in the ovaries and adrenal glands. (Figure 2)

Fig. 2. Progesterone Metabolism ((modified from Science Slides Suite © 2010))

Progesterone acts on two major progesterone receptors (PRs), PR-A and PR-B. (Speroff and Fritz, 2005) While the role of ERs in vascular physiology and pathophysiology is well studied, the literature on PRs is limited and most of what is known about their biological function is derived from studies of reproductive tissues. PR-A and PR-B can form homodimers (AA and BB) or heterodimers (AB) upon binding to a progestin. Downstream effects include protein phosphorylation and modulation of gene transcription. (Speroff and Fritz, 2005) Based on in-vitro studies of endometrium and breast tissue, PR-B is a positive

Sex Hormones and Vascular Function 5

channels by stimulating neuronal NOS via a signal pathway involving PI3-kinase and Akt

In summary, E2 at pharmacological concentrations causes vasorelaxation via a combination of endothelium-dependent, ER-mediated actions and contraction-modulating effects at the level of the smooth muscle cell. The E2-induced relaxing profile in a specific vascular bed depends on the species, gender, expression patterns and degree of dimerization and

Endogenous E2 lowers BP. Observational studies have demonstrated that BP is lower when E2 levels peak during the luteal phase than when they are at their nadir during the follicular phase of the menstrual cycle (Dunne et al, 1991; Karpanou et al, 1993; Chapman et al, 1997). Menopause is associated with a significant increase in BP in cross-sectional studies (Staessen et al, 1998). In a prospective study of BP in premenopausal, perimenopausal, and postmenopausal women, an age-independent 4-5 mmHg increase in systolic BP was found in postmenopausal women (Staessen et al, 1997). Further, BP is reduced when endogenous E2 levels are elevated during pregnancy (Siamopoulos et al, 1996). Data on the BP effects of estrogen replacement therapy (ERT) in menopausal women have been inconsistent, with reports of BP neutral (PEPI Trial Writing Group, 1995), BP lowering (Mercuro et al, 1997; Mercuro et al, 1998; Cagnacci et al, 1999; Seely et al, 1999; Butkevich et al, 2000) and BP elevating effects (Anderson et al, 2004; Wassertheil-Smoller et al, 2000). In the Postmenopausal Estrogen/Progestin interventions (PEPI) trial, which enrolled 875 healthy normotensive early postmenopausal women, assignment to conjugated equine estrogens (CEE), 0.625 mg/d ± a progestin did not impact systolic or diastolic BP when compared with placebo controls (PEPI Trial Writing Group, 1995). In contrast, when transdermal E2 was administered at physiologic doses to healthy postmenopausal women in two studies that evaluated ambulatory BP, active treatment significantly lowered nocturnal systolic, diastolic and mean BP by 3-7 mmHg compared with placebo (Cagnacci et al, 1999; Seely et al, 1999). The observational study component of the WHI (WHI-OS) collected data on risk factors for CVD, including BP, from 98,705 women aged 50-79 yr, the largest multiethnic, best characterized cohort of postmenopausal women ever studied (Wassertheil-Smoller et al, 2000). WHI-OS found that current HRT use was associated with a 25% greater likelihood of having hypertension compared with past use or no prior use. Further, among 5310 postmenopausal women randomized to CEE (0.625 mg/d) alone compared to a placebo group as part of the randomized controlled trial component of WHI, there was a 1.1-mmHg increase from baseline in systolic BP that persisted throughout the 6.8 yr of follow up (Anderson et al,

2004). There was no difference in diastolic BP between treatment groups.

Similar to estrogens, the effects of progestins on BP are dependent on the type of progestin. Natural progesterone has been associated with BP lowering or neutral effects. Higher levels of progesterone correlate with lower systolic but not diastolic BP during the second and third trimesters of pregnancy (Kristiansson et al, 2001). In a crossover study of 15 postmenopausal women assigned to placebo or transdermal E2 ± intravaginal progesterone,

**4.2.2 Progestin effects on blood pressure** 

(Han et al, 2007).

crosstalk between the ER subtypes.

**4.2 Sex hormone effects on blood pressure 4.2.1 Estrogen effects on blood pressure** 

regulator of progesterone-responsive genes, whereas PR-A activation inhibits PR-B activity. Like estrogen, progesterone has both genomic and non-genomic effects, binding both nuclear and cell membrane receptors.

#### **4. Physiologic effects of sex hormones on the vasculature**

#### **4.1 Estrogen effects on vascular reactivity**

E2 has rapid non-genomic actions on the arterial wall, resulting in vasodilation. Administration of E2 to ovariectomized ewes results in rapid uterine vasodilation, leading to a rise in uterine blood flow within 30 to 45 min (Killam et al*,* 1973). This rise in uterine blood flow is partially mediated by ER activation and release of nitric oxide (NO), as shown by local infusion of the nonselective ER blocker ICI 182,780 or the NO synthase blocker L-NAME, respectively, into the main uterine artery of nonpregnant ewes (Van Buren et al, 1992) A better understanding of the mechanism(s) by which E2 increases NO production in the vasculature comes from in vitro experiments with cultured endothelial cells. E2 stimulates eNOS activity via an ERα-mediated process in endothelial cells (Chen et al, 1999). ERα and ERβ are present on the endothelial cell membrane and are expressed in a wide range of blood vessels from different vascular beds and species (Andersson et al, 2001). The ERα and eNOS proteins are organized into a functional signaling module in caveolae located on endothelial cell membranes (Chambliss et al, 2000). The role of the ERβ in vasodilation is less clear, but studies from ERβ knockout mice have shown an inhibitory role of ERβ in ER-mediated NO relaxation (Petterson et al, 2000).

GPER is a seven-transmembrane G protein-coupled ER that has only recently been shown to play a role in the vasculature (Haas et al, 2007). Isoflavones, natural estrogenic compounds (phytoestrogens) found in soy products, e.g. genistein and dadein, and selective ER modulators (SERMs), e.g. tamoxifen and raloxifene, bind to GPER (Filardo et al, 2000). Selective stimulation of GPER by intravenous infusion of the GPER agonist G-1 results in an acute reduction in blood pressure in rats (Haas et al, 2009). G-1 relaxes ex vivo rat and human arteries via an endothelium-dependent and L-NAME-sensitive mechanism (Haas et al, 2009). It is uncertain whether E2-induced relaxing responses are mediated via GPER or whether crosstalk between ER/ERβ and GPER exists. Selective GPER antagonists like G-15 (Dennis et al, 2009) might unravel a role for GPER-dependent vasorelaxation upon E2 signaling in the vasculature.

E2 results in vasorelaxation even in the absence of a functional endothelium (Jiang et al. 1991), due primarily to Ca2+-antagonistic effects in smooth muscle cells. E2 inhibits voltage-dependent calcium inward currents on smooth muscle cells, but not on endothelial cells (Shan et al, 1994; Kitazawa et al, 1997). This leads to a reduction in intracellular Ca2+ concentration and lower Ca2+-calmodulin-dependent myosin light chain phosphorylation and contraction (Somlyo and Somlyo, 1994). In addition to these Ca2+ antagonistic effects on smooth muscle cells, a variety of endothelium-independent mechanisms have been proposed to account for E2-induced vasodilation. E2 has been reported to increase cAMP and cGMP levels in the vasculature, thus suggesting a cyclic nucleotide-dependent mechanism of relaxation (Kuehl et al, 1974). For instance, in the porcine coronary artery, E2 causes relaxation via protein kinase G activation and cAMPdependent opening of large-conductance Ca2+-activated K+ channels (BKCa) (Rosenfeld et al, 2000). In human coronary artery smooth muscle cells, E2 has been shown to open BKCa

regulator of progesterone-responsive genes, whereas PR-A activation inhibits PR-B activity. Like estrogen, progesterone has both genomic and non-genomic effects, binding both

E2 has rapid non-genomic actions on the arterial wall, resulting in vasodilation. Administration of E2 to ovariectomized ewes results in rapid uterine vasodilation, leading to a rise in uterine blood flow within 30 to 45 min (Killam et al*,* 1973). This rise in uterine blood flow is partially mediated by ER activation and release of nitric oxide (NO), as shown by local infusion of the nonselective ER blocker ICI 182,780 or the NO synthase blocker L-NAME, respectively, into the main uterine artery of nonpregnant ewes (Van Buren et al, 1992) A better understanding of the mechanism(s) by which E2 increases NO production in the vasculature comes from in vitro experiments with cultured endothelial cells. E2 stimulates eNOS activity via an ERα-mediated process in endothelial cells (Chen et al, 1999). ERα and ERβ are present on the endothelial cell membrane and are expressed in a wide range of blood vessels from different vascular beds and species (Andersson et al, 2001). The ERα and eNOS proteins are organized into a functional signaling module in caveolae located on endothelial cell membranes (Chambliss et al, 2000). The role of the ERβ in vasodilation is less clear, but studies from ERβ knockout mice have shown an inhibitory role

GPER is a seven-transmembrane G protein-coupled ER that has only recently been shown to play a role in the vasculature (Haas et al, 2007). Isoflavones, natural estrogenic compounds (phytoestrogens) found in soy products, e.g. genistein and dadein, and selective ER modulators (SERMs), e.g. tamoxifen and raloxifene, bind to GPER (Filardo et al, 2000). Selective stimulation of GPER by intravenous infusion of the GPER agonist G-1 results in an acute reduction in blood pressure in rats (Haas et al, 2009). G-1 relaxes ex vivo rat and human arteries via an endothelium-dependent and L-NAME-sensitive mechanism (Haas et al, 2009). It is uncertain whether E2-induced relaxing responses are mediated via GPER or whether crosstalk between ER/ERβ and GPER exists. Selective GPER antagonists like G-15 (Dennis et al, 2009) might unravel a role for GPER-dependent vasorelaxation upon E2

E2 results in vasorelaxation even in the absence of a functional endothelium (Jiang et al. 1991), due primarily to Ca2+-antagonistic effects in smooth muscle cells. E2 inhibits voltage-dependent calcium inward currents on smooth muscle cells, but not on endothelial cells (Shan et al, 1994; Kitazawa et al, 1997). This leads to a reduction in intracellular Ca2+ concentration and lower Ca2+-calmodulin-dependent myosin light chain phosphorylation and contraction (Somlyo and Somlyo, 1994). In addition to these Ca2+ antagonistic effects on smooth muscle cells, a variety of endothelium-independent mechanisms have been proposed to account for E2-induced vasodilation. E2 has been reported to increase cAMP and cGMP levels in the vasculature, thus suggesting a cyclic nucleotide-dependent mechanism of relaxation (Kuehl et al, 1974). For instance, in the porcine coronary artery, E2 causes relaxation via protein kinase G activation and cAMPdependent opening of large-conductance Ca2+-activated K+ channels (BKCa) (Rosenfeld et al, 2000). In human coronary artery smooth muscle cells, E2 has been shown to open BKCa

**4. Physiologic effects of sex hormones on the vasculature** 

of ERβ in ER-mediated NO relaxation (Petterson et al, 2000).

nuclear and cell membrane receptors.

signaling in the vasculature.

**4.1 Estrogen effects on vascular reactivity** 

channels by stimulating neuronal NOS via a signal pathway involving PI3-kinase and Akt (Han et al, 2007).

In summary, E2 at pharmacological concentrations causes vasorelaxation via a combination of endothelium-dependent, ER-mediated actions and contraction-modulating effects at the level of the smooth muscle cell. The E2-induced relaxing profile in a specific vascular bed depends on the species, gender, expression patterns and degree of dimerization and crosstalk between the ER subtypes.

#### **4.2 Sex hormone effects on blood pressure 4.2.1 Estrogen effects on blood pressure**

Endogenous E2 lowers BP. Observational studies have demonstrated that BP is lower when E2 levels peak during the luteal phase than when they are at their nadir during the follicular phase of the menstrual cycle (Dunne et al, 1991; Karpanou et al, 1993; Chapman et al, 1997). Menopause is associated with a significant increase in BP in cross-sectional studies (Staessen et al, 1998). In a prospective study of BP in premenopausal, perimenopausal, and postmenopausal women, an age-independent 4-5 mmHg increase in systolic BP was found in postmenopausal women (Staessen et al, 1997). Further, BP is reduced when endogenous E2 levels are elevated during pregnancy (Siamopoulos et al, 1996). Data on the BP effects of estrogen replacement therapy (ERT) in menopausal women have been inconsistent, with reports of BP neutral (PEPI Trial Writing Group, 1995), BP lowering (Mercuro et al, 1997; Mercuro et al, 1998; Cagnacci et al, 1999; Seely et al, 1999; Butkevich et al, 2000) and BP elevating effects (Anderson et al, 2004; Wassertheil-Smoller et al, 2000). In the Postmenopausal Estrogen/Progestin interventions (PEPI) trial, which enrolled 875 healthy normotensive early postmenopausal women, assignment to conjugated equine estrogens (CEE), 0.625 mg/d ± a progestin did not impact systolic or diastolic BP when compared with placebo controls (PEPI Trial Writing Group, 1995). In contrast, when transdermal E2 was administered at physiologic doses to healthy postmenopausal women in two studies that evaluated ambulatory BP, active treatment significantly lowered nocturnal systolic, diastolic and mean BP by 3-7 mmHg compared with placebo (Cagnacci et al, 1999; Seely et al, 1999). The observational study component of the WHI (WHI-OS) collected data on risk factors for CVD, including BP, from 98,705 women aged 50-79 yr, the largest multiethnic, best characterized cohort of postmenopausal women ever studied (Wassertheil-Smoller et al, 2000). WHI-OS found that current HRT use was associated with a 25% greater likelihood of having hypertension compared with past use or no prior use. Further, among 5310 postmenopausal women randomized to CEE (0.625 mg/d) alone compared to a placebo group as part of the randomized controlled trial component of WHI, there was a 1.1-mmHg increase from baseline in systolic BP that persisted throughout the 6.8 yr of follow up (Anderson et al, 2004). There was no difference in diastolic BP between treatment groups.

#### **4.2.2 Progestin effects on blood pressure**

Similar to estrogens, the effects of progestins on BP are dependent on the type of progestin. Natural progesterone has been associated with BP lowering or neutral effects. Higher levels of progesterone correlate with lower systolic but not diastolic BP during the second and third trimesters of pregnancy (Kristiansson et al, 2001). In a crossover study of 15 postmenopausal women assigned to placebo or transdermal E2 ± intravaginal progesterone,

Sex Hormones and Vascular Function 7

C-reactive protein (CRP) is an acute phase reactant that has been shown to be both a marker and a mediator of vascular disease. There is an E2-dependent sexual dimorphism in expression of human CRP in experimental models, i.e., the transgenic mouse expressing human CRP (CRPtg) (Szalai et al 1997, 1998, 2002) and in some human populations (Yamada et al, 2001). E2 treatment of male CRPtg can lower baseline CRP levels and removal of E2 can restore its high baseline expression. (Szalai et al, 1998) In postmenopausal women, oral CEE increases baseline CRP levels, but low dose oral or transdermal E2 supplementation does not affect CRP. (Cushman et al, 1999; Vongpatanasin et al, 2003; Lakoski et al, 2005; Mosca et al, 2004) This HRT-induced CRP increase occurs without a significant change in IL-6 or TNF-α, major regulators of CRP under inflammatory conditions, suggesting that the effects of menopausal hormones on CRP do not reflect a generalized inflammatory state. (Vongpatanasin et al, 2003; Mosca et al, 2004) Data from the WHI and the Women's Health Study have demonstrated that CRP predicts CVD risk in post-menopausal women independent of HRT. (Kurtz et al, 2011) HRT use had less predictive value than CRP levels in these studies. Thus, the clinical significance of hormone-related changes in circulating

**5. Estrogen effects on inflammation and vascular pathology** 

**5.1 Estrogen modulates pro-inflammatory mediator expression after vascular injury**  Inflammation plays a critical role in the pathogenesis of atherosclerosis and subsequent CVD. (Hansson et al, 2005) The process is initiated by activation of endothelial cells due to deposition of lipoproteins, pressure overload, and/or hyperglycemia, leading to increased expression of adhesion molecules (including selectin, vascular cell adhesion molecule 1 [ VCAM-1], and intercellular adhesion molecule 1 [ICAM-1]). These molecules cause circulating leukocytes to bind to vascular endothelial cells and release pro-inflammatory cytokines and growth factors. The bound leukocytes then infiltrate the vascular smooth muscle cells layer, leading to a cascade of cytokine secretion, further contributing to the local

Based on extensive studies using the rat carotid injury model, E2 has been shown to be a negative modulator of injury-induced vascular inflammation and neointima formation. (Bakir et al, 2000; Miller et al, 2004; Xing et al, 2004) There is a sexual dimorphism in the response to vascular injury, with males demonstrating increased neointima formation compared to females. (Chen et al 1996, 1998; Levine et al 1996; Miller et al, 2004) This sexual dimorphism is E2-dependent, based on evidence that physiologic levels of circulating E2 (40-60 pg/ml) decrease neointima formation in both male and female gonadectomized animals. Furthermore, addition of MPA, the progestin used in the Women's Health Initiative, opposes the effects of E2 on injury-induced vascular inflammation and neointima

E2 modulates the vascular response to injury by reducing local expression of inflammatory mediators and influx of leukocytes into balloon-injured carotid arteries of ovariectomized rats. (Miller et al, 2004; Xing et al, 2004) In particular, E2 decreases expression of cytokineinduced neutrophil chemoattractant (CINC-2β), a chemoattractant for neutrophils and monocyte chemoattractant protein (MCP-1) in injured arteries. (Xing et al, 2004) This results in significant reductions in influx of these inflammatory leukocyte subtypes, limiting the

**4.4 Estrogen effects on C-reactive protein** 

CRP levels remains uncertain.

formation. (Levine et al, 1996)

injury response. (Figure 3)

inflammatory environment within the vessel.

addition of progesterone did not affect the nocturnal BP lowering seen with E2 compared with placebo (Seely et al, 1999). Similarly, medroxyprogesterone acetate (MPA) appears to have BP neutral or lowering effects. In a double-blind, crossover study of postmenopausal women assigned to CEE and placebo or increasing doses of MPA, there was a dosedependent decrease in ambulatory daytime diastolic and mean BPs for women assigned to the progestin compared with placebo (Harvey et al, 2001). In contrast, most studies of synthetic progestins for contraception or hormone therapy have revealed a BP-elevating effect. Oral contraceptives in particular appear to precipitate or accelerate hypertension (Rosenthal et al, 2000).

#### **4.3 Estrogen effects on lipoproteins**

E2 also affects serum lipoprotein levels and the interaction of lipoproteins with cellular elements in the vessel wall. E2 has been shown to protect against atherosclerotic lesion formation in multiple animal models. In primate models, E2 results in up to a 66 percent decrease in aortic atherosclerotic plaque size. (Bjarnson et al, 1997) Mouse models have been widely used to study atherosclerosis because of the ability to easily inactivate targeted genes coding for apolipoprotein E (*Apoe*) and the LDL receptor (*Ldlr*) which lead to spontaneous development of atherosclerosis. E2 prevents both initiation and progression of atherosclerotic plaque development in these models. Using subcutaneous implanted E2 releasing pellets to achieve physiologic serum levels, atherosclerotic plaques did not progress beyond the fatty streak stage in apolipoprotein E-deficient mice. (Elhage et al, 1997) Similarly, E2 has been shown to reduce atherosclerotic lesion size in male *Apoe-/-* mice. (Tse et al, 1999) Treatment of minimally-oxidized LDL with E2 leads to decreased cytotoxicity in cultured endothelial cells. (Negre-Salvayre et al, 1993) E2 also inhibits LDL oxidation and decreases formation of cholesterol esters. (Huber et al, 1990)

E2 effects on lipoproteins and atherosclerosis are mediated by both ER and ER. When E2 treated mice that were deficient in ApoE alone were compared to mice that were deficient in both ApoE and ER, E2 reduced atherosclerotic plaque size in *Apoe-/-* mice by 80%. This effect was not seen in the *Apoe-/- , ER-/-* mice, indicating that ER plays a critical role in prevention of aterhosclerosis in this model. (Hodgin et al, 2001)

Clinical studies have shown reductions in serum lipoproteins following oral estrogen replacement therapy. One study randomized women to treatment with CEE (Premarin 0.625 mg) daily versus placebo. (Walsh et al, 1991) The estrogen treatment group had a 15% reduction in serum concentrations of LDL cholesterol and a 16% increase in high density lipoprotein (HDL) cholesterol. Triglyceride levels increased by 24%. These results were consistent across the age spectrum; even women in their 8th decade of life showed similar changes in serum cholesterol levels. Oral estrogens facilitate postprandial clearance of atherogenic lipoproteins and increase serum HDL levels, specifically HDL2, which may play a major role in reduction of atherogenesis. Oral CEEs appear to increase HDL levels to a greater degree than oral E2. Triglyceride levels are increased with administration of both oral CEE and oral E2, though to a lesser extent by oral E2.

Transdermal E2 formulations also decrease LDL levels, but to a lesser extent than oral preparations. (Stevenson et al 2009) Transdermal estrogens have not been shown to alter postprandial lipoprotein clearance or circulating HDL levels, but may lower triglyceride levels. (Godsland et al, 2001)

addition of progesterone did not affect the nocturnal BP lowering seen with E2 compared with placebo (Seely et al, 1999). Similarly, medroxyprogesterone acetate (MPA) appears to have BP neutral or lowering effects. In a double-blind, crossover study of postmenopausal women assigned to CEE and placebo or increasing doses of MPA, there was a dosedependent decrease in ambulatory daytime diastolic and mean BPs for women assigned to the progestin compared with placebo (Harvey et al, 2001). In contrast, most studies of synthetic progestins for contraception or hormone therapy have revealed a BP-elevating effect. Oral contraceptives in particular appear to precipitate or accelerate hypertension

E2 also affects serum lipoprotein levels and the interaction of lipoproteins with cellular elements in the vessel wall. E2 has been shown to protect against atherosclerotic lesion formation in multiple animal models. In primate models, E2 results in up to a 66 percent decrease in aortic atherosclerotic plaque size. (Bjarnson et al, 1997) Mouse models have been widely used to study atherosclerosis because of the ability to easily inactivate targeted genes coding for apolipoprotein E (*Apoe*) and the LDL receptor (*Ldlr*) which lead to spontaneous development of atherosclerosis. E2 prevents both initiation and progression of atherosclerotic plaque development in these models. Using subcutaneous implanted E2 releasing pellets to achieve physiologic serum levels, atherosclerotic plaques did not progress beyond the fatty streak stage in apolipoprotein E-deficient mice. (Elhage et al, 1997) Similarly, E2 has been shown to reduce atherosclerotic lesion size in male *Apoe-/-* mice. (Tse et al, 1999) Treatment of minimally-oxidized LDL with E2 leads to decreased cytotoxicity in cultured endothelial cells. (Negre-Salvayre et al, 1993) E2 also inhibits LDL oxidation and

E2 effects on lipoproteins and atherosclerosis are mediated by both ER and ER. When E2 treated mice that were deficient in ApoE alone were compared to mice that were deficient in both ApoE and ER, E2 reduced atherosclerotic plaque size in *Apoe-/-* mice by 80%. This

Clinical studies have shown reductions in serum lipoproteins following oral estrogen replacement therapy. One study randomized women to treatment with CEE (Premarin 0.625 mg) daily versus placebo. (Walsh et al, 1991) The estrogen treatment group had a 15% reduction in serum concentrations of LDL cholesterol and a 16% increase in high density lipoprotein (HDL) cholesterol. Triglyceride levels increased by 24%. These results were consistent across the age spectrum; even women in their 8th decade of life showed similar changes in serum cholesterol levels. Oral estrogens facilitate postprandial clearance of atherogenic lipoproteins and increase serum HDL levels, specifically HDL2, which may play a major role in reduction of atherogenesis. Oral CEEs appear to increase HDL levels to a greater degree than oral E2. Triglyceride levels are increased with administration of both

Transdermal E2 formulations also decrease LDL levels, but to a lesser extent than oral preparations. (Stevenson et al 2009) Transdermal estrogens have not been shown to alter postprandial lipoprotein clearance or circulating HDL levels, but may lower triglyceride

*-/-* mice, indicating that ER plays a critical role in

(Rosenthal et al, 2000).

**4.3 Estrogen effects on lipoproteins** 

effect was not seen in the *Apoe-/- , ER*

levels. (Godsland et al, 2001)

decreases formation of cholesterol esters. (Huber et al, 1990)

prevention of aterhosclerosis in this model. (Hodgin et al, 2001)

oral CEE and oral E2, though to a lesser extent by oral E2.

#### **4.4 Estrogen effects on C-reactive protein**

C-reactive protein (CRP) is an acute phase reactant that has been shown to be both a marker and a mediator of vascular disease. There is an E2-dependent sexual dimorphism in expression of human CRP in experimental models, i.e., the transgenic mouse expressing human CRP (CRPtg) (Szalai et al 1997, 1998, 2002) and in some human populations (Yamada et al, 2001). E2 treatment of male CRPtg can lower baseline CRP levels and removal of E2 can restore its high baseline expression. (Szalai et al, 1998) In postmenopausal women, oral CEE increases baseline CRP levels, but low dose oral or transdermal E2 supplementation does not affect CRP. (Cushman et al, 1999; Vongpatanasin et al, 2003; Lakoski et al, 2005; Mosca et al, 2004) This HRT-induced CRP increase occurs without a significant change in IL-6 or TNF-α, major regulators of CRP under inflammatory conditions, suggesting that the effects of menopausal hormones on CRP do not reflect a generalized inflammatory state. (Vongpatanasin et al, 2003; Mosca et al, 2004) Data from the WHI and the Women's Health Study have demonstrated that CRP predicts CVD risk in post-menopausal women independent of HRT. (Kurtz et al, 2011) HRT use had less predictive value than CRP levels in these studies. Thus, the clinical significance of hormone-related changes in circulating CRP levels remains uncertain.

#### **5. Estrogen effects on inflammation and vascular pathology**

**5.1 Estrogen modulates pro-inflammatory mediator expression after vascular injury** 

Inflammation plays a critical role in the pathogenesis of atherosclerosis and subsequent CVD. (Hansson et al, 2005) The process is initiated by activation of endothelial cells due to deposition of lipoproteins, pressure overload, and/or hyperglycemia, leading to increased expression of adhesion molecules (including selectin, vascular cell adhesion molecule 1 [ VCAM-1], and intercellular adhesion molecule 1 [ICAM-1]). These molecules cause circulating leukocytes to bind to vascular endothelial cells and release pro-inflammatory cytokines and growth factors. The bound leukocytes then infiltrate the vascular smooth muscle cells layer, leading to a cascade of cytokine secretion, further contributing to the local inflammatory environment within the vessel.

Based on extensive studies using the rat carotid injury model, E2 has been shown to be a negative modulator of injury-induced vascular inflammation and neointima formation. (Bakir et al, 2000; Miller et al, 2004; Xing et al, 2004) There is a sexual dimorphism in the response to vascular injury, with males demonstrating increased neointima formation compared to females. (Chen et al 1996, 1998; Levine et al 1996; Miller et al, 2004) This sexual dimorphism is E2-dependent, based on evidence that physiologic levels of circulating E2 (40-60 pg/ml) decrease neointima formation in both male and female gonadectomized animals. Furthermore, addition of MPA, the progestin used in the Women's Health Initiative, opposes the effects of E2 on injury-induced vascular inflammation and neointima formation. (Levine et al, 1996)

E2 modulates the vascular response to injury by reducing local expression of inflammatory mediators and influx of leukocytes into balloon-injured carotid arteries of ovariectomized rats. (Miller et al, 2004; Xing et al, 2004) In particular, E2 decreases expression of cytokineinduced neutrophil chemoattractant (CINC-2β), a chemoattractant for neutrophils and monocyte chemoattractant protein (MCP-1) in injured arteries. (Xing et al, 2004) This results in significant reductions in influx of these inflammatory leukocyte subtypes, limiting the injury response. (Figure 3)

Sex Hormones and Vascular Function 9

of the excitatory FcRs on these cells, (Kramer et al, 2004, 2007) it is plausible that the vasoprotective effects of E2 against CRP-mediated vascular injury response in female mice are regulated by its modulation of macrophage phenotype in order to express less activating

Studies in ERα- and ERβ-deficient mice and in rats treated with pharmacologic antagonists of ERs have provided evidence that both ER subtypes contribute to the vasoprotective effects of E2 in the setting of acute injury. (Mori et al, 2000; Brouchet et al, 2001; Karas et al, 1999; Geraldes et al, 2003; Xing et al, 2007). The ER subtypes contribute to vasoprotection in a cell-specific manner. In porcine endothelial and vascular smooth muscle cells, E2 acts through inhibition of PDGF-BB-induced p38 and p42/44 mitogen-activated protein kinase (MAPK) phosphorylation to stimulate migration and proliferation. (Geraldes et al, 2003) Down-regulation of ERβ, but not ERα, prevented the effects of E2 on smooth muscle cell migration and proliferation. In contrast, in porcine endothelial cells, down-regulation of ERα prevented E2-induced p38 and p42/44 MAPK activation, while down-regulation of

Administration of the ERβ selective agonist DPN has been shown to result in dosedependednt attenuation of neointima formation induced by injury of the mouse femoral artery. (Krom et al, 2007) The ERα selective agonist PPT prevented neointima formation at low but not high concentrations in this study. In a subsequent study, MPP, an ERα selective antagonist, blocked the inhibitory effect of PPT on neointima formation, but did not block the effects of E2 or DPN. (Harrington et al, 2003) This suggests that E2 acts through a selective ERβ

The TNF-α-stimulated vascular smooth muscle cell has been used as an in-vitro model of the vascular injury response in order to examine cellular/molecular mechanisms of E2 induced vasoprotection. (Xing et al, 2007) E2 has been shown to attenuate TNF-α induced expression of pro-inflammatory mediators in rat aortic smooth muscle cells through ERβ. (Xing et al, 2007) In this model, DPN reduced TNF-α-induced expression of the neutrophil cytokine CINC-2β in a dose-dependent fashion, while PPT had no effect. The non-selective ER antagonist ICI-182,780 blocked the anti-inflammatory effects of both DPN and E2. Furthermore, both DPN and E2 reduced neutrophil chemotactic activity in TNF-α-treated

In order to reconcile laboratory findings that E2 provides vascular protection with clinical trial results indicating harmful effects of E2 on the cardiovascular system, studies have been done in models comparing young versus aged animals. Results from one study showed opposing effects of E2 based on age: E2 increased neointima formation in balloon-injured carotid arteries of aged (+75%) versus young (10-12 weeks; -40%) ovariectomized rats. (Miller et al, 2007) The attenuating effect of E2 on inflammatory mediator expression and neutrophil and monocyte infiltration was lost in the injured arteries of aged rats. ERα and ERβ expression was similar in both the young and aged animals. This laboratory evidence was the first of its kind to show that E2 exacerbates the vascular response to injury in aged animals. This seminal finding indicates that the protective effect of E2 is impaired following long periods of hormone deprivation, supporting the timing hypothesis. (Pinna et al, 2008)

pathway to attenuate neointima formation following restenosis in this mouse model.

**5.4 The role of aging in loss of estrogen-induced vasoprotection** 

receptors (FcRI and FcRIII) and more inhibitory receptors (FcRIIb).

**5.3 Estrogen receptors and vascular inflammation** 

ERβ had no effect.

rat aortic SMCs.

Fig. 3. E2 effects on the early vascular injury response. (*Adapted from Xing et al, 2009*)

#### **5.2 The role of C-reactive protein in E2 modulation of vascular inflammation**

E2 also exerts an anti-inflammatory and vasoprotective effect in injured arteries of CRP transgenic (CRPtg) mice. CRPtg mice carry a transgene containing the entire human *CRP* gene and its promoters while the mouse supplies all the required *trans*-acting factors. (Hage et al, 2008) Since in CRPtg mice, human CRP increases several hundred-fold during an acute phase response, analogous to the human condition, this model is convenient for the *in vivo* study of the biologic activities, including vascular effects, of human CRP. Using CRPtg mice, we and others have established that human CRP is a pathogenic mediator of cardiovascular disease. (Danenberg et al, 2003; Paul et al, 2004; Zhang et al, 2010; Nagai et al, 2011; Takahashi et al, 2010)

Using the carotid ligation model of acute vascular injury, we showed that young ovariectomized CRPtg mice develop twofold greater neointima formation than control non transgenic (NTG) mice and that there are extensive deposits of human CRP in the neointima of injured vessels of these animals in the absence of an increase in blood levels of the protein. (Kumar et al, 1997; Hage et al, 2010; Wang et al, 2005; Xing et al, 2008) These findings suggest that local expression of human CRP may exacerbate the adverse remodeling seen after acute arterial injury in the CRPtg model. To test the hypothesis that E2 can inhibit the vascular injury response attributed to human CRP, we treated ovariectomized CRPtg and control NTG mice with E2 prior to carotid ligation and observed that E2-treated CRPtg mice had a significant, ~85%, reduction in neointima formation compared to vehicle-treated CRPtg mice. The E2 effect was directionally similar but somewhat smaller in magnitude in control NTG mice. (Wang et al, 2005) Since the exaggerated vascular injury response in CRPtg mice is mediated by immunoglobulin G Fc receptors (FcRs) on macrophages, (Xing et al, 2008) and since E2, via its interaction with ERs, can reduce inflammatory cytokine release from activated human macrophages by decreasing expression

Fig. 3. E2 effects on the early vascular injury response. (*Adapted from Xing et al, 2009*)

**5.2 The role of C-reactive protein in E2 modulation of vascular inflammation** 

Takahashi et al, 2010)

E2 also exerts an anti-inflammatory and vasoprotective effect in injured arteries of CRP transgenic (CRPtg) mice. CRPtg mice carry a transgene containing the entire human *CRP* gene and its promoters while the mouse supplies all the required *trans*-acting factors. (Hage et al, 2008) Since in CRPtg mice, human CRP increases several hundred-fold during an acute phase response, analogous to the human condition, this model is convenient for the *in vivo* study of the biologic activities, including vascular effects, of human CRP. Using CRPtg mice, we and others have established that human CRP is a pathogenic mediator of cardiovascular disease. (Danenberg et al, 2003; Paul et al, 2004; Zhang et al, 2010; Nagai et al, 2011;

Using the carotid ligation model of acute vascular injury, we showed that young ovariectomized CRPtg mice develop twofold greater neointima formation than control non transgenic (NTG) mice and that there are extensive deposits of human CRP in the neointima of injured vessels of these animals in the absence of an increase in blood levels of the protein. (Kumar et al, 1997; Hage et al, 2010; Wang et al, 2005; Xing et al, 2008) These findings suggest that local expression of human CRP may exacerbate the adverse remodeling seen after acute arterial injury in the CRPtg model. To test the hypothesis that E2 can inhibit the vascular injury response attributed to human CRP, we treated ovariectomized CRPtg and control NTG mice with E2 prior to carotid ligation and observed that E2-treated CRPtg mice had a significant, ~85%, reduction in neointima formation compared to vehicle-treated CRPtg mice. The E2 effect was directionally similar but somewhat smaller in magnitude in control NTG mice. (Wang et al, 2005) Since the exaggerated vascular injury response in CRPtg mice is mediated by immunoglobulin G Fc receptors (FcRs) on macrophages, (Xing et al, 2008) and since E2, via its interaction with ERs, can reduce inflammatory cytokine release from activated human macrophages by decreasing expression of the excitatory FcRs on these cells, (Kramer et al, 2004, 2007) it is plausible that the vasoprotective effects of E2 against CRP-mediated vascular injury response in female mice are regulated by its modulation of macrophage phenotype in order to express less activating receptors (FcRI and FcRIII) and more inhibitory receptors (FcRIIb).

#### **5.3 Estrogen receptors and vascular inflammation**

Studies in ERα- and ERβ-deficient mice and in rats treated with pharmacologic antagonists of ERs have provided evidence that both ER subtypes contribute to the vasoprotective effects of E2 in the setting of acute injury. (Mori et al, 2000; Brouchet et al, 2001; Karas et al, 1999; Geraldes et al, 2003; Xing et al, 2007). The ER subtypes contribute to vasoprotection in a cell-specific manner. In porcine endothelial and vascular smooth muscle cells, E2 acts through inhibition of PDGF-BB-induced p38 and p42/44 mitogen-activated protein kinase (MAPK) phosphorylation to stimulate migration and proliferation. (Geraldes et al, 2003) Down-regulation of ERβ, but not ERα, prevented the effects of E2 on smooth muscle cell migration and proliferation. In contrast, in porcine endothelial cells, down-regulation of ERα prevented E2-induced p38 and p42/44 MAPK activation, while down-regulation of ERβ had no effect.

Administration of the ERβ selective agonist DPN has been shown to result in dosedependednt attenuation of neointima formation induced by injury of the mouse femoral artery. (Krom et al, 2007) The ERα selective agonist PPT prevented neointima formation at low but not high concentrations in this study. In a subsequent study, MPP, an ERα selective antagonist, blocked the inhibitory effect of PPT on neointima formation, but did not block the effects of E2 or DPN. (Harrington et al, 2003) This suggests that E2 acts through a selective ERβ pathway to attenuate neointima formation following restenosis in this mouse model.

The TNF-α-stimulated vascular smooth muscle cell has been used as an in-vitro model of the vascular injury response in order to examine cellular/molecular mechanisms of E2 induced vasoprotection. (Xing et al, 2007) E2 has been shown to attenuate TNF-α induced expression of pro-inflammatory mediators in rat aortic smooth muscle cells through ERβ. (Xing et al, 2007) In this model, DPN reduced TNF-α-induced expression of the neutrophil cytokine CINC-2β in a dose-dependent fashion, while PPT had no effect. The non-selective ER antagonist ICI-182,780 blocked the anti-inflammatory effects of both DPN and E2. Furthermore, both DPN and E2 reduced neutrophil chemotactic activity in TNF-α-treated rat aortic SMCs.

#### **5.4 The role of aging in loss of estrogen-induced vasoprotection**

In order to reconcile laboratory findings that E2 provides vascular protection with clinical trial results indicating harmful effects of E2 on the cardiovascular system, studies have been done in models comparing young versus aged animals. Results from one study showed opposing effects of E2 based on age: E2 increased neointima formation in balloon-injured carotid arteries of aged (+75%) versus young (10-12 weeks; -40%) ovariectomized rats. (Miller et al, 2007) The attenuating effect of E2 on inflammatory mediator expression and neutrophil and monocyte infiltration was lost in the injured arteries of aged rats. ERα and ERβ expression was similar in both the young and aged animals. This laboratory evidence was the first of its kind to show that E2 exacerbates the vascular response to injury in aged animals. This seminal finding indicates that the protective effect of E2 is impaired following long periods of hormone deprivation, supporting the timing hypothesis. (Pinna et al, 2008)

Sex Hormones and Vascular Function 11

are equivalent in ameliorating menopausal symptoms. Among the oral estrogens, E2 is considered to be the most potent estrogen and estrone is reported to be 50-70% less active.

While both oral and transdermal estrogens are absorbed systemically, oral estrogens are unique in that they undergo the "first-pass effect" in the liver. Intestinal absorption of estrogens leads to high concentrations of hormone in the portal vein, stimulating hepatic production of thyroxine-binding globulin, corticosteroid-binding globulin, SHBG, triglycerides, HDL, triglycerides, and clotting factors. Transdermal administration of estrogen does not have this effect and there is no resulting increased hepatic production of

Multiple oral estrogen preparations are available, including CEE, E2, esterified estrogen,

**Estrogen Replacement Therapy Drug Company Dose Company Drug Dose**  *Oral Therapy Transdermal Therapy*  **Estradiol Estradiol** 

0.5, 1, 2 mg *Patches*

0.9, 1.25 mg

2.5 mg

Healthcase 0.5, 1.5, mg *Gel* 

Gynodiol Firlding 0.5, 1, 1.5, 2 mg Alora Watson 0.025, 0.05,

Ogen Pharmacia 0.75, 1.5, 3 mg Menostar Bayer 0.014 mg/d

0.9, 1.25 mg *Emulsions* 

Enjuvia Elan 0.625, 1.25 mg Estrasorb Novavox 0.025

**Conjugated synthetic estrogens** EstroGel Solvay 0.75 mg/pump

*Vaginal Therapy* Divigel Upsher-Smith 0.25, 0.5, 1

**Estradiol** Elestrin Kenwood 0.52 mg/pump

**Esterified Estrogens** Estraderm Novartis 0.05, 0.1 mg/d

0.075, 0.1 mg/d

mg/d

mg/d

mg/d

0.06, 0.075, 0.1

0.05, 0.075, 0.1

0.05, 0.075, 0.1

mg/pouch

mg/pouch

Climara Berlax 0.025, 0.05,

Esclim Women First 0.025, 0.0375,

Vivelle Novartis 0.025, 0.0375,

Estriol is the least potent of the three estrogens, with a potency one-tenth that of E2.

the above proteins.

Estrace Warner

Chilcot

and conjugated synthetic estrogens. (Table 1)

**Conjugated equine estrogens** 

Premarin Wyeth-Ayerst 0.3, 0.45, 0.625,

Menest Monarch 0.3, 0.625, 1.25,

Cenestin Elan 0.3, 0.45, 0.625,

Ortho-Est Women First

The potential role of age-related alterations in ER signaling in these processes remains poorly understood and warrants further study.

#### **6. Reproductive aging, sex hormones, and women's health**

Reproductive aging is a function of decreased production of sex hormones by the ovaries. As a woman enters her fifth decade of life, depletion of remaining ovarian follicles occurs. Estrogen production by the ovaries begins to decrease, and women experience progressive loss of menstrual cyclicity. When total depletion of follicles occurs, menses cease, and the woman enters menopause, defined as the absence of menses for a 12-month period. The average age of menopause in the United States is 51 years. (Speroff and Fritz, 2005) Due to increasing lifespan, women can now expect to spend a significant portion of their lives in the postmenopausal state. This prolonged hypoestrogenism may have important consequences for quality of life, as well as various other health parameters, including cardiovascular function, bone health, and cognitive function.

#### **6.1 Age- and sex-specific trends**

CHD is rare in premenopausal women, but the incidence of myocardial infarction rises dramatically after menopause. Furthermore, women with premature ovarian failure or early natural menopause (≤ 44 years) have an associated increase in the risk of CVD. (Hu et al, 1999) (Mondul et al 2005) These observations led to the belief that menopause itself is a risk factor for CHD. However, the relationship between menopause, age and CHD is complex and it is not clear that menopause per se is a risk factor for CHD. It is important to recognize that women who develop CHD after menopause have more CHD risk factors (dyslipidemia, family history, hypertension, tobacco use, and diabetes mellitus) compared to postmenopausal women who remain free of disease. Currently available data from randomized controlled trials, e.g., WHI and HERS, do not indicate that HRT is useful in the primary or secondary prevention of CHD. However, a growing body of evidence suggests that initiation of treatment with different HRT preparations in the perimenopausal period may have beneficial effects on the vasculature that may delay the progression of CVD and prevent ischemic events.

#### **6.2 Types and routes of administration of postmenopausal estrogen replacement therapy**

ERT is only indicated for the treatment of moderate to severe menopausal symptoms, specifically vasomotor symptoms (eg. hot flushes). Contraindications to ERT include known CHD, breast cancer, a previous venous thromboembolic event or stroke, active liver disease, or high risk for these conditions. ERT should be initiated as close to the time of menopause as possible, typically beginning in the late forties to early fifties. Initiation of therapy beyond age 59 is controversial due to increased risk of CHD events. Most physicians now agree that the benefit of estrogen treatment in healthy, early menopausal women, using the lowest dose and shortest duration of therapy, outweigh the risks of treatment.

Systemic ERT can be given orally or non-orally in the form of transdermal patches and topical creams, gels, and mists. Estrogen can also be given vaginally via tablets, topical formulations and vaginal rings. However, vaginal therapy is only indicated for the treatment of vaginal atrophy and not for systemic/vasomotor symptoms. Assuming that equivalent doses of replacement estrogen are given, the different routes of administration

The potential role of age-related alterations in ER signaling in these processes remains

Reproductive aging is a function of decreased production of sex hormones by the ovaries. As a woman enters her fifth decade of life, depletion of remaining ovarian follicles occurs. Estrogen production by the ovaries begins to decrease, and women experience progressive loss of menstrual cyclicity. When total depletion of follicles occurs, menses cease, and the woman enters menopause, defined as the absence of menses for a 12-month period. The average age of menopause in the United States is 51 years. (Speroff and Fritz, 2005) Due to increasing lifespan, women can now expect to spend a significant portion of their lives in the postmenopausal state. This prolonged hypoestrogenism may have important consequences for quality of life, as well as various other health parameters, including cardiovascular

CHD is rare in premenopausal women, but the incidence of myocardial infarction rises dramatically after menopause. Furthermore, women with premature ovarian failure or early natural menopause (≤ 44 years) have an associated increase in the risk of CVD. (Hu et al, 1999) (Mondul et al 2005) These observations led to the belief that menopause itself is a risk factor for CHD. However, the relationship between menopause, age and CHD is complex and it is not clear that menopause per se is a risk factor for CHD. It is important to recognize that women who develop CHD after menopause have more CHD risk factors (dyslipidemia, family history, hypertension, tobacco use, and diabetes mellitus) compared to postmenopausal women who remain free of disease. Currently available data from randomized controlled trials, e.g., WHI and HERS, do not indicate that HRT is useful in the primary or secondary prevention of CHD. However, a growing body of evidence suggests that initiation of treatment with different HRT preparations in the perimenopausal period may have beneficial effects on the vasculature that may delay the progression of CVD and

**6.2 Types and routes of administration of postmenopausal estrogen replacement** 

dose and shortest duration of therapy, outweigh the risks of treatment.

ERT is only indicated for the treatment of moderate to severe menopausal symptoms, specifically vasomotor symptoms (eg. hot flushes). Contraindications to ERT include known CHD, breast cancer, a previous venous thromboembolic event or stroke, active liver disease, or high risk for these conditions. ERT should be initiated as close to the time of menopause as possible, typically beginning in the late forties to early fifties. Initiation of therapy beyond age 59 is controversial due to increased risk of CHD events. Most physicians now agree that the benefit of estrogen treatment in healthy, early menopausal women, using the lowest

Systemic ERT can be given orally or non-orally in the form of transdermal patches and topical creams, gels, and mists. Estrogen can also be given vaginally via tablets, topical formulations and vaginal rings. However, vaginal therapy is only indicated for the treatment of vaginal atrophy and not for systemic/vasomotor symptoms. Assuming that equivalent doses of replacement estrogen are given, the different routes of administration

poorly understood and warrants further study.

function, bone health, and cognitive function.

**6.1 Age- and sex-specific trends** 

prevent ischemic events.

**therapy** 

**6. Reproductive aging, sex hormones, and women's health** 

are equivalent in ameliorating menopausal symptoms. Among the oral estrogens, E2 is considered to be the most potent estrogen and estrone is reported to be 50-70% less active. Estriol is the least potent of the three estrogens, with a potency one-tenth that of E2.

While both oral and transdermal estrogens are absorbed systemically, oral estrogens are unique in that they undergo the "first-pass effect" in the liver. Intestinal absorption of estrogens leads to high concentrations of hormone in the portal vein, stimulating hepatic production of thyroxine-binding globulin, corticosteroid-binding globulin, SHBG, triglycerides, HDL, triglycerides, and clotting factors. Transdermal administration of estrogen does not have this effect and there is no resulting increased hepatic production of the above proteins.

Multiple oral estrogen preparations are available, including CEE, E2, esterified estrogen, and conjugated synthetic estrogens. (Table 1)


Sex Hormones and Vascular Function 13

**Progesterone Formulations** 

**Oral Therapy** 

**Vaginal Therapy** 

**Intrauterine Device (IUD)** 

**Combination Estrogen-Progesterone Formulations Oral Therapy** 

**Transdermal Patches**  Estradiol/norethindrone Combi-Patch Novartis 0.05/0.14, 0.05/0.25 mg

Micronized progesterone is the major natural progesterone available, and while it has been less well studied than MPA, it appears to be similar in efficacy and is widely prescribed. Synthetic progestins, including MPA, norgestrel, and norethindrone acetate, appear to increase hepatic lipase activity and attenuate the beneficial effects of estrogen on HDL levels (Stevenson 2009), while natural progesterone appears to have no adverse effect on HDL. MPA also opposes the NO-dependent vasodilator effect of E2, while natural progesterone

The slope of the age-related rise in incidence of CVD in women increases in the postmenopausal period, suggesting that withdrawal of ovarian hormones, particularly E2, has an adverse effect on cardiovascular health. (Lloyd-Jones et al, 2010) This increase is thought

Levonorgestrel IUD Mirena Bayer 52 mg/5 yrs

Estradiol/norgestimate Prefest Duramed 1/0.9 mg

Estradiol/drosperinone Angeliq Berlex 1/0.5 mg

Estradiol/levonorgestrel Climara Pro Berlex 0.045/0.015 mg Table 2. Available Progesterone Preparations. (*Modified from Martin and Barbieri, 2011*)

CEE/medroxyprogesterone Prempro Wyeth 0.3/1.5, 0.45/1.5,

Pharmaceuticals

Laboratories

Laboratories

Activella Novo Nordisk 1/0.5 mg

FemHRT Warner-Chilcot 5 mcg/1mg

2.5, 5, 10 mg

90 mg/applicator

90 mg/applicator

0.625/2.5, 0.625/5 mg

**Generic Name Brand Name Company Dose** 

Norethindrone acetate Aygestin Teva

Progesterone Cream Crinone Columbia

Progesterone Gel Prochieve Columbia

was found to have no effect. (Williams AK et al 1994)

**7. Clinical research in hormone replacement therapy** 

Estradiol/norethindonr

estradiol/norethindrone

**7.1 Observational studies** 

acetate

Ethinyl

Micronized Progesterone Prometrium Abbott 100, 200 mg Medroxyprogesterone Acetate Provera Pfizer 2.5, 5, 10 mg


Table 1. Available Estrogen Formulations (*Modified from Martin and Barbieri, 2011*)

CEE is one of the most commonly used preparations and, derived from mare urine, is composed of up to 10 different estrogenic compounds, predominantly the sodium sulfated conjungates of estrone. (Lyman GW 1982) The metabolism of CEE is a complex and still poorly understood process which occurs in the liver. After oral ingestion of CEEs, the compounds are rapidly absorbed by the gastrointestinal tract, then may become conjugated by hepatocytes or excreted in the feces. (Pan CC, 1985) Following oral intake of CEEs, mean serum estrone levels (152 pg/mL) are far higher than estradiol levels (31 pg/mL). (Powers MS et al 1985) However, the estrone component is largely inactive because it is albumin-bound. The clinical response to CEE is hypothesized to be mediated through a mechanism involving conversion of circulating, bound estrone to E2 in the liver. (Barnes RB et al 1987)

E2 is another commonly prescribed oral form of postmenopausal ERT. Native E2 is poorly absorbed; therefore it is manufactured in micronized, sulfated, and esterified forms to improve absorption. (Krantz JC et al 1958) Similar to metabolism of CEE, the majority of E2 is converted to estrone. However, following oral administration of E2, mean circulating levels of estrone (200 pg/mL) and E2 (50 pg/mL) are higher compared to serum levels following ingestion of CEE when equivalent doses are given. (Lobo RA and Cassidenti DL 1992) E2 also induces hepatic production of proteins, but this effect is much less than that of CEEs. (Maschak CA et al 1982).

Esterified estrogens result in serum E2 and estrone levels similar to those seen with CEEs. Synthetic conjugated estrogens are derived from plant sources. They are similar but not identical to CEEs and contain fewer molecular forms of estrogen. (Lobo et al, 2000)

#### **6.3 Types and routes of administration of progesterone replacement therapy**

Progesterone is indicated in addition to estrogen as part of a HRT regimen in postmenopausal women with an intact uterus (who have not undergone hysterectomy). Progesterone opposes the effects of estrogen on the endometrial lining and prevents development of endometrial hyperplasia and malignancy which occur in women treated with unopposed estrogen. Both natural and synthetic progestins are available. (Table 2)

pharmaceutical

1.5 mg/spray

**Rings** *Spray* 

Chilcott 0.05 mg/day

Vagifem Novo Nordisk 0.025 mg/tablet

Chilcot 0.1 mg/gram

Premarin Wyeth-Ayerst 0.625 mg/gram

Table 1. Available Estrogen Formulations (*Modified from Martin and Barbieri, 2011*)

CEE is one of the most commonly used preparations and, derived from mare urine, is composed of up to 10 different estrogenic compounds, predominantly the sodium sulfated conjungates of estrone. (Lyman GW 1982) The metabolism of CEE is a complex and still poorly understood process which occurs in the liver. After oral ingestion of CEEs, the compounds are rapidly absorbed by the gastrointestinal tract, then may become conjugated by hepatocytes or excreted in the feces. (Pan CC, 1985) Following oral intake of CEEs, mean serum estrone levels (152 pg/mL) are far higher than estradiol levels (31 pg/mL). (Powers MS et al 1985) However, the estrone component is largely inactive because it is albumin-bound. The clinical response to CEE is hypothesized to be mediated through a mechanism involving conversion of circulating, bound estrone to E2 in the

E2 is another commonly prescribed oral form of postmenopausal ERT. Native E2 is poorly absorbed; therefore it is manufactured in micronized, sulfated, and esterified forms to improve absorption. (Krantz JC et al 1958) Similar to metabolism of CEE, the majority of E2 is converted to estrone. However, following oral administration of E2, mean circulating levels of estrone (200 pg/mL) and E2 (50 pg/mL) are higher compared to serum levels following ingestion of CEE when equivalent doses are given. (Lobo RA and Cassidenti DL 1992) E2 also induces hepatic production of proteins, but this effect is much less than that of

Esterified estrogens result in serum E2 and estrone levels similar to those seen with CEEs. Synthetic conjugated estrogens are derived from plant sources. They are similar but not

Progesterone is indicated in addition to estrogen as part of a HRT regimen in postmenopausal women with an intact uterus (who have not undergone hysterectomy). Progesterone opposes the effects of estrogen on the endometrial lining and prevents development of endometrial hyperplasia and malignancy which occur in women treated with unopposed estrogen. Both natural and synthetic progestins are available. (Table 2)

identical to CEEs and contain fewer molecular forms of estrogen. (Lobo et al, 2000)

**6.3 Types and routes of administration of progesterone replacement therapy** 

Tablet

**Estradiol** 

**Conjugated equine estrogen** 

Femring Warner-

Estrace Warner-

liver. (Barnes RB et al 1987)

CEEs. (Maschak CA et al 1982).

**Cream** 

Estring Pharmacia 0.0075 mg/day EvaMist KV


Table 2. Available Progesterone Preparations. (*Modified from Martin and Barbieri, 2011*)

Micronized progesterone is the major natural progesterone available, and while it has been less well studied than MPA, it appears to be similar in efficacy and is widely prescribed. Synthetic progestins, including MPA, norgestrel, and norethindrone acetate, appear to increase hepatic lipase activity and attenuate the beneficial effects of estrogen on HDL levels (Stevenson 2009), while natural progesterone appears to have no adverse effect on HDL. MPA also opposes the NO-dependent vasodilator effect of E2, while natural progesterone was found to have no effect. (Williams AK et al 1994)

#### **7. Clinical research in hormone replacement therapy**

#### **7.1 Observational studies**

The slope of the age-related rise in incidence of CVD in women increases in the postmenopausal period, suggesting that withdrawal of ovarian hormones, particularly E2, has an adverse effect on cardiovascular health. (Lloyd-Jones et al, 2010) This increase is thought

Sex Hormones and Vascular Function 15

et al., 2004) and in vitro studies found that MPA signals differently from native progesterone in endothelial cells (Simoncini et al., 2004). The surprising outcomes of the estrogen-alone (EA) component of WHI (WHI SC, 2004) added further evidence that MPA might be a problem and that unopposed estrogen benefits younger post-menopausal women. This trial, which was stopped early, showed no significant effect of unopposed CEE on the primary CHD outcome and a surprising tendency for benefit in the primary safety

The advanced age (63 years in WHI, 67 years in HERS) and long period of hormone deprivation prior to starting HRT may account for deleterious outcomes of hormone treatment in WHI and HERS. Based on a review of pre-clinical studies, as well as observational studies and clinical trials in women, including those with intermediate endpoints and CVD outcomes, the "timing hypothesis" was developed (Phillips & Langer, 2005). The timing hypothesis states that the effects of HRT on the vasculature are dependent on the time of initiation of treatment. The timing hypothesis predicts that HRT initiated at the time of or prior to menopause should produce a decrease in CHD over time, while HRT begun years after menopause should produce an increase in CHD events shortly after therapy is begun, followed by later benefit. This hypothesis attributes the complex CHD responses to HRT in human trials to a combination of early erosion/rupture of 'vulnerable' coronary plaque, which is made worse by HRT; long-term reduction in plaque formation, which is improved by HRT; and modulation of the vasoprotective actions of estrogens by

Indirect support for the timing hypothesis has come from the report of final results from the EA trial in WHI, which included detailed analyses of primary and secondary coronary outcomes and subgroup analyses of participants by age and years since hysterectomy with no menopausal hormone therapy (Hsia J, et al). During the active intervention period, 201 coronary events were confirmed among women assigned to CEE compared with 217 events among women assigned to placebo (HR=0.95%; 95% CI 0.79-1.16). Among women aged 50- 59 years at baseline, the HR for the primary outcome (nonfatal myocardial infarction or coronary death) was 0.63 (95% CI 0.36-1.08). In that younger age group, coronary revascularization was less frequent among women assigned to CEE (HR=0.55; 95% CI 0.35- 0.86), as were several composite outcomes. Further analyses of the E+P arm of the WHI demonstrated a non-significant trend towards cardioprotection in women who began HRT less than 10 years after menopause (HR = 0.89; 95% CI 0.5-1.5), while women who initiated HRT more than 20 years after menopause had a significantly elevated risk of coronary events (HR = 1.71; 95% CI, 1.1-2.5). (Manson et al, 2003) When the EA and E+P arms from

Another consideration in the relationship between HRT and CVD risk is the duration of HRT. A recent post-hoc analysis of the E+P trial in WHI showed that in women less than 10 years since menopause, HRT resulted in a slightly lower event-free survival rate during the first 5 years of therapy compared to placebo; however, at 6 years, the two curves crossed each other and showed a non-significant trend towards a higher rate of event-free survival in the group using HRT. (Toh et al, 2010) Further analysis of women less than 10 years since menopause in the WHI E+P arm showed an increased risk in the first 2 years of HRT, followed by a decreased risk in the next 2 years, and an overall risk reduction over 8 years.

the WHI were combined, a similar trend was seen. (Rossouw et al, 2007)

(invasive breast cancer) outcome.

**7.3 The timing hypothesis** 

systemic progestins.

to be a consequence of the loss of the multiple protective effects of E2 on the vascular system. Multiple observational studies have suggested that HRT may protect against CVD in postmenopausal women. In a meta-analysis of 16 prospective observational trials, the relative risk of CVD for postmenopausal women who ever used any form of estrogen vs. those who had never used estrogen was 0.70 (95% CI, 0.63-0.77). (Grodstein et al, 1995) The relative risk in current users, calculated from 6 prospective studies, was even more impressive at 0.55 (95% CI, 0.44-0.70). In the Nurses' Health Study, which followed more than 70,000 postmenopausal women for 20 years, the risk for major coronary events was lower among current users of HRT compared with never-users (multi-variate adjusted relative risk, 0.61 [95% CI, 0.52-0.71]). (Grodstein et al, 2000) Buoyed by observational studies suggesting that HRT, including various E2 preparations with or without a progestin (most commonly a synthetic one), reduced CVD risk by ~50%. (Psaty et al, 1994) HRT use increased dramatically during the 2 decades prior to the publication of the WHI. It is estimated that annual hormone therapy prescriptions increased from 58 million in 1995 to 90 million in 1999, representing ~15 million women per year. (Hersh et al, 2004) This rate remained stable until 2002, but after the publication of WHI and other randomized controlled trials that showed no benefit of HRT, fell sharply by 66% in a single year. Many of the studies that prompted the upswing in postmenopausal HRT suffered from the limitations of discordance of the two treatment groups: women who take HRT are on average better educated, have higher incomes and better access to health care and are healthier even before starting therapy. (Barrett-Connor et al, 1989; Matthews et al, 1996) In a meta-analysis that adjusted for socioeconomic status and other risk factors, HRT was not associated with CVD risk reduction. (Humphrey et al, 2002)

#### **7.2 Clinical trials**

Publication of the estrogen plus progestin clinical trial component of the Women's Health Initiative (WHI) (Rossouw et al., 2002; Manson et al., 2003) initially sounded a death knell for hormone use in post-menopausal women. This placebo-controlled trial of HRT (CEE 0.625 mg/day plus MPA 2.5 mg/day) in 16,608 post-menopausal women found significant increases in the risk of CHD, stroke, venous thromboembolism and invasive breast cancer in the HRT group. The reductions in colorectal cancer and hip fracture seen with HRT did not balance these increased CVD and cancer risks, and publication of the WHI results stimulated consensus panels to recommend against the use of HRT for chronic disease prevention in post-menopausal women (Mosca et al., 2004). Based on the widely publicized findings of harm in the estrogen plus progestin (E+P) trial of the WHI and a major secondary prevention study that used the same hormone regimen, the Heart and Estrogen/Progestin Replacement Study (HERS) (Hulley et al., 1998; Grady et al., 2002), prescribing of HRT fell drastically. (Hersh et al., 2004) Transdermal hormone preparations were less affected, and transvaginal and low-dose preparations gained somewhat, reflecting caution in the use of the full-dose oral regimens that had been used in WHI and HERS.

Attempts to explain the unanticipated deleterious effects of HRT gave consideration to whether the formulation, dose and route of administration of HRT might play a role (Dubey et al., 2004; Turgeon et al., 2004; Phillips & Langer, 2005). In particular, the progestin MPA was identified as having potential deleterious effects on the vasculature. Pre-clinical studies had shown that MPA negates the vasoprotective and anti-inflammatory effects of E2 in the setting of acute vascular injury (Levine et al., 1996; Oparil et al., 1997; Xing et al., 2004; Miller

to be a consequence of the loss of the multiple protective effects of E2 on the vascular system. Multiple observational studies have suggested that HRT may protect against CVD in postmenopausal women. In a meta-analysis of 16 prospective observational trials, the relative risk of CVD for postmenopausal women who ever used any form of estrogen vs. those who had never used estrogen was 0.70 (95% CI, 0.63-0.77). (Grodstein et al, 1995) The relative risk in current users, calculated from 6 prospective studies, was even more impressive at 0.55 (95% CI, 0.44-0.70). In the Nurses' Health Study, which followed more than 70,000 postmenopausal women for 20 years, the risk for major coronary events was lower among current users of HRT compared with never-users (multi-variate adjusted relative risk, 0.61 [95% CI, 0.52-0.71]). (Grodstein et al, 2000) Buoyed by observational studies suggesting that HRT, including various E2 preparations with or without a progestin (most commonly a synthetic one), reduced CVD risk by ~50%. (Psaty et al, 1994) HRT use increased dramatically during the 2 decades prior to the publication of the WHI. It is estimated that annual hormone therapy prescriptions increased from 58 million in 1995 to 90 million in 1999, representing ~15 million women per year. (Hersh et al, 2004) This rate remained stable until 2002, but after the publication of WHI and other randomized controlled trials that showed no benefit of HRT, fell sharply by 66% in a single year. Many of the studies that prompted the upswing in postmenopausal HRT suffered from the limitations of discordance of the two treatment groups: women who take HRT are on average better educated, have higher incomes and better access to health care and are healthier even before starting therapy. (Barrett-Connor et al, 1989; Matthews et al, 1996) In a meta-analysis that adjusted for socioeconomic status and other risk factors, HRT was not

Publication of the estrogen plus progestin clinical trial component of the Women's Health Initiative (WHI) (Rossouw et al., 2002; Manson et al., 2003) initially sounded a death knell for hormone use in post-menopausal women. This placebo-controlled trial of HRT (CEE 0.625 mg/day plus MPA 2.5 mg/day) in 16,608 post-menopausal women found significant increases in the risk of CHD, stroke, venous thromboembolism and invasive breast cancer in the HRT group. The reductions in colorectal cancer and hip fracture seen with HRT did not balance these increased CVD and cancer risks, and publication of the WHI results stimulated consensus panels to recommend against the use of HRT for chronic disease prevention in post-menopausal women (Mosca et al., 2004). Based on the widely publicized findings of harm in the estrogen plus progestin (E+P) trial of the WHI and a major secondary prevention study that used the same hormone regimen, the Heart and Estrogen/Progestin Replacement Study (HERS) (Hulley et al., 1998; Grady et al., 2002), prescribing of HRT fell drastically. (Hersh et al., 2004) Transdermal hormone preparations were less affected, and transvaginal and low-dose preparations gained somewhat, reflecting caution in the use of the full-dose oral regimens that had been used in WHI and HERS. Attempts to explain the unanticipated deleterious effects of HRT gave consideration to whether the formulation, dose and route of administration of HRT might play a role (Dubey et al., 2004; Turgeon et al., 2004; Phillips & Langer, 2005). In particular, the progestin MPA was identified as having potential deleterious effects on the vasculature. Pre-clinical studies had shown that MPA negates the vasoprotective and anti-inflammatory effects of E2 in the setting of acute vascular injury (Levine et al., 1996; Oparil et al., 1997; Xing et al., 2004; Miller

associated with CVD risk reduction. (Humphrey et al, 2002)

**7.2 Clinical trials** 

et al., 2004) and in vitro studies found that MPA signals differently from native progesterone in endothelial cells (Simoncini et al., 2004). The surprising outcomes of the estrogen-alone (EA) component of WHI (WHI SC, 2004) added further evidence that MPA might be a problem and that unopposed estrogen benefits younger post-menopausal women. This trial, which was stopped early, showed no significant effect of unopposed CEE on the primary CHD outcome and a surprising tendency for benefit in the primary safety (invasive breast cancer) outcome.

#### **7.3 The timing hypothesis**

The advanced age (63 years in WHI, 67 years in HERS) and long period of hormone deprivation prior to starting HRT may account for deleterious outcomes of hormone treatment in WHI and HERS. Based on a review of pre-clinical studies, as well as observational studies and clinical trials in women, including those with intermediate endpoints and CVD outcomes, the "timing hypothesis" was developed (Phillips & Langer, 2005). The timing hypothesis states that the effects of HRT on the vasculature are dependent on the time of initiation of treatment. The timing hypothesis predicts that HRT initiated at the time of or prior to menopause should produce a decrease in CHD over time, while HRT begun years after menopause should produce an increase in CHD events shortly after therapy is begun, followed by later benefit. This hypothesis attributes the complex CHD responses to HRT in human trials to a combination of early erosion/rupture of 'vulnerable' coronary plaque, which is made worse by HRT; long-term reduction in plaque formation, which is improved by HRT; and modulation of the vasoprotective actions of estrogens by systemic progestins.

Indirect support for the timing hypothesis has come from the report of final results from the EA trial in WHI, which included detailed analyses of primary and secondary coronary outcomes and subgroup analyses of participants by age and years since hysterectomy with no menopausal hormone therapy (Hsia J, et al). During the active intervention period, 201 coronary events were confirmed among women assigned to CEE compared with 217 events among women assigned to placebo (HR=0.95%; 95% CI 0.79-1.16). Among women aged 50- 59 years at baseline, the HR for the primary outcome (nonfatal myocardial infarction or coronary death) was 0.63 (95% CI 0.36-1.08). In that younger age group, coronary revascularization was less frequent among women assigned to CEE (HR=0.55; 95% CI 0.35- 0.86), as were several composite outcomes. Further analyses of the E+P arm of the WHI demonstrated a non-significant trend towards cardioprotection in women who began HRT less than 10 years after menopause (HR = 0.89; 95% CI 0.5-1.5), while women who initiated HRT more than 20 years after menopause had a significantly elevated risk of coronary events (HR = 1.71; 95% CI, 1.1-2.5). (Manson et al, 2003) When the EA and E+P arms from the WHI were combined, a similar trend was seen. (Rossouw et al, 2007)

Another consideration in the relationship between HRT and CVD risk is the duration of HRT. A recent post-hoc analysis of the E+P trial in WHI showed that in women less than 10 years since menopause, HRT resulted in a slightly lower event-free survival rate during the first 5 years of therapy compared to placebo; however, at 6 years, the two curves crossed each other and showed a non-significant trend towards a higher rate of event-free survival in the group using HRT. (Toh et al, 2010) Further analysis of women less than 10 years since menopause in the WHI E+P arm showed an increased risk in the first 2 years of HRT, followed by a decreased risk in the next 2 years, and an overall risk reduction over 8 years.

Sex Hormones and Vascular Function 17

older women, particularly those with established vascular disease, has a proinflammatory effect, perhaps leading to atherosclerotic plaque instability and neovascularization (Störk et al., 2004; Mendelsohn & Karas, 2005). The mechanisms of these altered vascular responses are not fully understood, but may relate to age-related deterioration in ER expression and signaling. Recent studies of the effects of HRT on blood pressure and vascular function support the age-dependence of the action of HRT on the vasculature. Beneficial effects of HRT appear to be realized only in younger, perimenopausal women in whom hormone response systems remain intact. However, further study of the timing, dose, duration, and

Anderson, G.L., Limacher, M., Assaf, A.R., Bassford, T., Beresford, S.A., Black, H., Bonds, D.,

Brunner, R., Brzyski, R., Caan, B., Chlebowski, R., Curb, D., Gass, M., Hays, J., Heiss, G., Hendrix, S., Howard, B.V., Hsia, J., Hubbell, A., Jackson, R., Johnson, K.C., Judd, H., Kotchen, J.M., Kuller, L., LaCroix, A.Z., Lane, D., Langer, R.D., Lasser, N., Lewis, C.E., Manson, J., Margolis, K., Ockene, J., O'Sullivan, M.J., Phillips, L., Prentice, R.L., Ritenbaugh, C., Robbins, J., Rossouw, J.E., Sarto, G., Stefanick, M.L., Van Horn, L., Wactawski-Wende, J., Wallace, R., & Wassertheil-Smoller, S.. Women's Health Initiative Steering Committee. (2004). Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women's Health Initiative randomized controlled trial. *Journal of the American Medical Association,* Vol. 291, No. 14, (April 2004), pp. 1701-1712, ISSN 0098-7484. Andersson, C., Lydrup, M., Ferno, M., Idvall, I., Gustafson, J. & Nilsson B. (2001).

Immunocytochemical demonstration of oestrogen receptor beta in blood vessels of the femal rat. *Journal of Endocrinology,* Vol.169, No. 2, (May 2001), pp. 241-247, ISSN

vasoprotection is estrogen-receptor dependent: evidence from the balloon-injured rat carotid artery model. Circulation, Vol.101, No.20, (May 2000), pp. 2342-2344,

estrogen replacement therapy on platelet aggregation and adenosine triphosphate release in postmenopausal women. *Obstetrics & Gynecology*, Vol.81, No.2, (Feb

platelet aggregation and adenosine triphosphate release in vitro by 17β-estradiol and medroxyprogesterone acetate in postmenopausal women. Journal of Thrombosis and

Pharmacology, Mishell, D.R., JR, pp. 301-315, Year Book Medical Publishers, Inc.,

heart disease risk factors in the 1980s. Rancho bernardo, calif, revisited. *Journal of the American Medical Association,* Vol. 261, No. 14, (April 1989), pp. 2095-2100, ISSN

Bakir, S., Mori T, Durand J, Chen YF, Thompson JA, & Oparil S. (2000). Estrogen-induced

Bar, J., Tepper, R., Fuchs, J., Pardo, Y., Goldberger, S., & Ovadia, J. (1993). The effect of

Bar, J., Lahav, J., Hod, M., Ben-Rafael, Z., Weinberger, I., & Brosens, J. (2000). Regulation of

Barnes, R.B., Lobo, R.A. (1987). Pharmacology of Estrogens, In: Menopause: Physiology and

Barrett-Connor, E., Wingard, D., & Criqui, M. (1989). Postmenopausal estrogen use and

Haemostasis, Vol.84, No.4, (Oct 2000), pp. 695–700, ISSN 0340-6245.

route of administration of HRT in postmenopausal women may be informative.

**9. References** 

1479-6805.

0098-7484.

ISSN: 0009-7322.

1993), pp. 261–264, ISSN 1873-233X.

ISBN 0815159145, Chicago, IL.

(Toh et al, 2010). Similar results were seen in the EA arm of the WHI, with a significant decrease in CHD risk after 6 years of CEE alone compared to placebo. (Harman et al, 2011) Among women in the EA arm of the WHI followed up over 10.7 years, there was no difference in CHD risk in those using CEE for a median of 5.9 years compared to placebo at the end of the active treatment period, or overall. (LaCroix et al, 2011) In the postintervention follow-up period, the annualized rate for CHD in the EA arm was 0.64% compared to 0.67% in the placebo group (HR 0.97, 95% CI, 0.75-1.25). Health outcomes, including CHD, were more favorable for younger women compared to older women (P=0.05 for interaction). These findings support the current clinical recommendations to treat postmenopausal women with HRT for the "shortest possible duration" and may lead to more individualized management.

The WHI was limited by use of only one type of ERT (CEE), by inclusion of women who initiated HRT late after many years of ovarian hormone deprivation, and by exclusion of women who were experiencing menopausal symptoms. Ongoing clinical trials are addressing these deficiencies by examining the timing hypothesis in perimenopausal women. The Kronos Early Estrogen Prevention Study (KEEPS) is a prospective, randomized, double-blind study of 900 healthy perimenopausalwomen aged 45-54 with menopausal symptoms. (Harman et al., 2005) The main hypotheses are 1) HRT initiated early in menopause (before development of atherosclerotic lesions) will prevent progression of atherosclerotic lesions, and 2) both oral CEE and transdermal E2 will be similarly efficacious. Participants were randomized to oral CEE and a placebo patch, oral placebo and a transdermal patch containing E2, or placebo in both pill and patch. The primary endpoints of KEEPS are carotid intimal medial thickness by ultrasound and the progression of coronary calcium by electron beam tomography, surrogates for CVD. Another ongoing prospective, randomized, controlled trial, the Early versus Late Intervention Trial with Estradiol (ELITE) randomized 643 women who were less than 6 years or more than 10 years since menopause to receive oral E2 versus placebo. (clinicaltrials.gov NCT00114517; Hodis and Mack, 2011) The primary endpoint is rate of change of carotid artery intima-media thickness. These two prospective studies will provide much-needed information regarding the timing hypothesis and use of HRT in reducing CVD risk.

#### **7.4 Oral versus transdermal HRT**

To date, few studies have examined the difference in CHD outcomes between postmenopausal women treated with oral versus transdermal therapy. The one existing trial in the literature suggests no difference in CHD outcomes with regard to route. (Clarke SC 2002) This study examined transdermal E2 with or without transdermal norethindrone acetate, and found similar CHD outcomes to the WHI. The literature indicates that dose may be more important than route.

#### **8. Conclusions**

Importantly, cellular and molecular studies are urgently needed to elucidate the differential effects of HRT and its components on young, healthy arteries and on older, diseased arteries. Emerging evidence suggests that HRT administered to young healthy women has anti-inflammatory and vasodilator effects that tend to lower blood pressure and slow the progression of atherosclerotic lesions, while the same HRT preparation administered to older women, particularly those with established vascular disease, has a proinflammatory effect, perhaps leading to atherosclerotic plaque instability and neovascularization (Störk et al., 2004; Mendelsohn & Karas, 2005). The mechanisms of these altered vascular responses are not fully understood, but may relate to age-related deterioration in ER expression and signaling. Recent studies of the effects of HRT on blood pressure and vascular function support the age-dependence of the action of HRT on the vasculature. Beneficial effects of HRT appear to be realized only in younger, perimenopausal women in whom hormone response systems remain intact. However, further study of the timing, dose, duration, and route of administration of HRT in postmenopausal women may be informative.

#### **9. References**

16 Sex Hormones

(Toh et al, 2010). Similar results were seen in the EA arm of the WHI, with a significant decrease in CHD risk after 6 years of CEE alone compared to placebo. (Harman et al, 2011) Among women in the EA arm of the WHI followed up over 10.7 years, there was no difference in CHD risk in those using CEE for a median of 5.9 years compared to placebo at the end of the active treatment period, or overall. (LaCroix et al, 2011) In the postintervention follow-up period, the annualized rate for CHD in the EA arm was 0.64% compared to 0.67% in the placebo group (HR 0.97, 95% CI, 0.75-1.25). Health outcomes, including CHD, were more favorable for younger women compared to older women (P=0.05 for interaction). These findings support the current clinical recommendations to treat postmenopausal women with HRT for the "shortest possible duration" and may lead

The WHI was limited by use of only one type of ERT (CEE), by inclusion of women who initiated HRT late after many years of ovarian hormone deprivation, and by exclusion of women who were experiencing menopausal symptoms. Ongoing clinical trials are addressing these deficiencies by examining the timing hypothesis in perimenopausal women. The Kronos Early Estrogen Prevention Study (KEEPS) is a prospective, randomized, double-blind study of 900 healthy perimenopausalwomen aged 45-54 with menopausal symptoms. (Harman et al., 2005) The main hypotheses are 1) HRT initiated early in menopause (before development of atherosclerotic lesions) will prevent progression of atherosclerotic lesions, and 2) both oral CEE and transdermal E2 will be similarly efficacious. Participants were randomized to oral CEE and a placebo patch, oral placebo and a transdermal patch containing E2, or placebo in both pill and patch. The primary endpoints of KEEPS are carotid intimal medial thickness by ultrasound and the progression of coronary calcium by electron beam tomography, surrogates for CVD. Another ongoing prospective, randomized, controlled trial, the Early versus Late Intervention Trial with Estradiol (ELITE) randomized 643 women who were less than 6 years or more than 10 years since menopause to receive oral E2 versus placebo. (clinicaltrials.gov NCT00114517; Hodis and Mack, 2011) The primary endpoint is rate of change of carotid artery intima-media thickness. These two prospective studies will provide much-needed information regarding

To date, few studies have examined the difference in CHD outcomes between postmenopausal women treated with oral versus transdermal therapy. The one existing trial in the literature suggests no difference in CHD outcomes with regard to route. (Clarke SC 2002) This study examined transdermal E2 with or without transdermal norethindrone acetate, and found similar CHD outcomes to the WHI. The literature indicates that dose

Importantly, cellular and molecular studies are urgently needed to elucidate the differential effects of HRT and its components on young, healthy arteries and on older, diseased arteries. Emerging evidence suggests that HRT administered to young healthy women has anti-inflammatory and vasodilator effects that tend to lower blood pressure and slow the progression of atherosclerotic lesions, while the same HRT preparation administered to

to more individualized management.

**7.4 Oral versus transdermal HRT** 

may be more important than route.

**8. Conclusions** 

the timing hypothesis and use of HRT in reducing CVD risk.


Sex Hormones and Vascular Function 19

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**1. Introduction** 

**2** 

Gorazd Drevenšek

*Slovenia* 

**The Role of Sex Hormones** 

*University of Ljubljana, Faculty of Medicine,* 

**in the Cardiovascular System** 

*Institute of Pharmacology and Experimental Toxicology* 

In developed countries heart disease is the primary cause of death in men and in women over the age of 60. While premenopausal women have a low incidence of cardiovascular disease as compared to men, the mortality among post-menopausal women rises to the same frequency or even exceeds the rates of men (Adams et al., 1995; Fraser et al., 2000; Gray et al., 2001; Wild & Bartholemew, 1988). This significant gender difference is mostly attributed to the beneficial role of estrogens (Collins et al., 1993; Gray et al., 2001). Many studies have suggested that females have reduced incidence of cardiovascular diseases due to the beneficial effects of estrogen on both the lipid profile and on the vasculature. Lately, many new mechanisms are discovered in cardiovascular diseases and research has been focused on the role of both estrogen and testosterone, as well as some other androgens, but also on the estrogen receptor GPER, which shows an important role in the cardioprotection

The sex hormones in the cardiovascular system might be viewed at as biomarkers for cardiovascular health status, as well as by itself, as protective agents against myocardial diseases. The estrogens in premenopausal women are modulating health in the regular menstrual cycle. Testosterone is lacking such cycle activity and is probably more expressed in the physically active population. The effects of testosterone are increasing muscle mass induced by higher physical activity, and higher adrenal and hypophysis activity resulting in potential cardiovascular system damage. However, when testosterone or its derivatives are missused, ventricular hypertrophy, diastolic dysfunction and myocardial stiffening appear

The sex hormones, i.e. estrogen, progesterone and androgens and their receptors, ERs, PRs and ARs, have been studied as candidates to mediate sex-specific effects observed in gender related responses of the cardiovascular system and related diseases. Above all estrogen has received major attention, while testosterone is at present studied for its potential beneficial and cardioprotective mechanism of action. However, within several cardiovascular diseases like myocardial infarction, coronary artery disease and other ischemia related diseases, heart failure, ECG gender specific differences, the focus was already turned to the gender related sex hormones differences. These studies presented new approaches and specificities in

of both, males and females (Deschamps &Murphy, 2009).

and the potential risk for infarction increases (Malkin et al., 2010).


### **The Role of Sex Hormones in the Cardiovascular System**

Gorazd Drevenšek

*University of Ljubljana, Faculty of Medicine, Institute of Pharmacology and Experimental Toxicology Slovenia* 

#### **1. Introduction**

30 Sex Hormones

Xing, D., Hage, F., Chen, Y., McCrory, M., Feng, W., Skibinski, G., Majid-Hassan, E., Oparil, S.,

Yamada, S., Gotoh, T., Nakashima, Y., Kayaba, K., Ishikawa, S., Nago, N., Nakamura, Y.,

Yee, D.L., & Bray, P.F. Platelet hyperreactivity: risk factors and the effects of hormones. Adv

Yoshimura, T., Ohshige, A., Maeda, T., Ito, M., & Okamura, H. (1999). Estrogen replacement

Zhang, R., Zhang, Y., Huang, X., Wu, Y., Chung, A., Wu, E., Szalai, A., Wong, B., Lau, C., &

Zhu, Y., Bian, Z., Lu, P., Karas, R.H., Bao, L., Cox, D., Hodgin, J., Shaul, P.W., Thoren, P.,

*of Pathology,* Vol. 172, No. 1, (January 2008), pp. 22-30, ISSN 0002-9440. Xing D., Nozell, S., Chen, Y.F., Hage, F., Oparil, S. (2009). Estrogen and mechanisms of

(March 2009), pp. 289-295, ISSN 1524-4636.

Stud Med, Vol.4, No.2, (February 2004), pp. 72-78.

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No.5554, (January 2002), pp.505-508, ISSN 1095-9203.

pp. 1183-1190, ISSN 0002-9262.

0378-5122.

& Szalai, A. (2008). Exaggerated neointima formation in human c-reactive protein transgenic mice is IgG FC receptor type I (fc gamma rI)-dependent. *American Journal* 

vascular protection. *Arteriosclerosis, Thrombosis, and Vascular Biology*., Vol.29, No.3

Itoh, Y., & Kajii, E. (2001). Distribution of serum c-reactive protein and its association with atherosclerotic risk factors in a japanese population : Jichi medical school cohort study. *American Journal Of Epidemiology*, Vol. 153, No. 12, (June 2001),

therapy decreases platelet activating factor acetylhydrolase activity in postmenopausal women. *Maturitas*, Vol.31, No.2, (January 1999), pp. 149-153, ISSN

Lan, H. (2010). C-reactive protein promotes cardiac fibrosis and inflammation in angiotensin II-induced hypertensive cardiac disease. *Hypertension*, Vol. 55, No. 4,

Smithies, O., Gustafsson, J.A., & Mendelsohn, M.E. (2002). Abnormal vascular function and hypertension in mice deficient in estrogen receptor β. *Science*, Vol.295, In developed countries heart disease is the primary cause of death in men and in women over the age of 60. While premenopausal women have a low incidence of cardiovascular disease as compared to men, the mortality among post-menopausal women rises to the same frequency or even exceeds the rates of men (Adams et al., 1995; Fraser et al., 2000; Gray et al., 2001; Wild & Bartholemew, 1988). This significant gender difference is mostly attributed to the beneficial role of estrogens (Collins et al., 1993; Gray et al., 2001). Many studies have suggested that females have reduced incidence of cardiovascular diseases due to the beneficial effects of estrogen on both the lipid profile and on the vasculature. Lately, many new mechanisms are discovered in cardiovascular diseases and research has been focused on the role of both estrogen and testosterone, as well as some other androgens, but also on the estrogen receptor GPER, which shows an important role in the cardioprotection of both, males and females (Deschamps &Murphy, 2009).

The sex hormones in the cardiovascular system might be viewed at as biomarkers for cardiovascular health status, as well as by itself, as protective agents against myocardial diseases. The estrogens in premenopausal women are modulating health in the regular menstrual cycle. Testosterone is lacking such cycle activity and is probably more expressed in the physically active population. The effects of testosterone are increasing muscle mass induced by higher physical activity, and higher adrenal and hypophysis activity resulting in potential cardiovascular system damage. However, when testosterone or its derivatives are missused, ventricular hypertrophy, diastolic dysfunction and myocardial stiffening appear and the potential risk for infarction increases (Malkin et al., 2010).

The sex hormones, i.e. estrogen, progesterone and androgens and their receptors, ERs, PRs and ARs, have been studied as candidates to mediate sex-specific effects observed in gender related responses of the cardiovascular system and related diseases. Above all estrogen has received major attention, while testosterone is at present studied for its potential beneficial and cardioprotective mechanism of action. However, within several cardiovascular diseases like myocardial infarction, coronary artery disease and other ischemia related diseases, heart failure, ECG gender specific differences, the focus was already turned to the gender related sex hormones differences. These studies presented new approaches and specificities in

The Role of Sex Hormones in the Cardiovascular System 33

shows that the protective mechanism in the cardiovascular system is linked to the estrogen

In the past the action of androgens on the cardiovascular system has received relatively little attention and authors disagree about possible detrimental or protective effects of testosterone on the heart (Pugh et al., 2000). Later studies showed that cardioprotective effects of testosterone are mediated by a yet not identified androgen-dependent pathway, but only after chronic administration of testosterone (Kuhar at al., 2007; Borst et al., 2010).

Direct application as well as estradiol pretreatment increased the coronary flow in isolated female hearts after the onset of reperfusion as well as testosterone pretreated male hearts (which was equally effective to female hearts), showing the vasodilatative effects of sex hormones (Kuhar et al., 2007). The coronary flow was increased after direct application of estradiol, while direct testosterone administration lacked such a vasodilatatory effect (Kuhar et al., 2007). Estradiol is reported to possess a direct artery relaxant action and also a direct effect on myocardium. Similarly, testosterone influenced coronary flow directly and the effects were both beneficial and deleterious (Pugh et al., 2000). Estradiol improved coronary flow of rats directly through the stimulation of NO release from endothelial cells (Dai et al., 2004; Fraser et al., 2000; Santos et al., 2004; Woodman et al., 2004). The vasodilatatory effects of testosterone are mediated by opening the large conductance, calcium-activated potassium channels (Deenadayalu et al., 2001). The short-term administration of testosterone induces a beneficial effect on exercise-induced myocardial ischemia in men with coronary artery disease, which may be related to its direct coronary

In physiological conditions, both estrogens as well as androgens may elicit very rapid effects to keep the homeostasis balanced, without manifesting RNA and protein synthesis (Dechering et al., 2000; Revelli et al., 1998). For example, the plasma membrane estrogen receptor was shown to respond rapidly to estrogen (Peitras & Szego, 1977), while there is no

The sex hormones in the endothelium were generally believed to have mainly non-genomic effects. The endothelial estrogen receptor-α (ERα) in the endothelium as a whole, is considered one of the most important targets of cardioprotection. The endothelium and, in particular, the endothelial ER-α appear to be a key cellular and molecular targets of the protective actions of estradiol against ischemia/reperfusion (I/R)-induced coronary endothelial dysfunction. The activation of the endothelial estrogen receptor (ER) by estradiol, triggers a protective action on the coronary endothelial structure and function, which, in turn, limits the size of the infarct. This protection may be in part due to reduced cardiac oxidative stress, demonstrated by the decreased production of reactive oxygen species observed during early reperfusion. The signaling mechanisms of cardioprotection are to a great extent dependent on NO, which signals in cardiomyocytes via protein kinases and may possibly protect mitochondria, resulting in decreased cardiomyocyte death. Another indirect effect the reduced neutrophil-mediated cardiomyocyte injury may also

play a role as endothelial protection and thus in cardioprotection (Favre et al., 2010).

activation of ER receptors (Arnal et al., 2010).

relaxing effect (Rosano et al., 1999).

clear evidence yet of a similar effect for androgens.

**2.2 Endothelium, estrogen and the heart** 

**2.1 Coronary flow, acute and chronic effects of sex hormones** 

gender related pathways responses. Recently, most of the approaches of cardiovascular diseases focused on non-genomic action of sex hormones.

#### **1.1 Non-genomic estrogen action**

Since estrogens in premenopausal women with regular menstrual cycle are established as a natural protection against cardivoascular diseases, nuclear and non-nuclear mechanisms are evaluated for their mode of action. The non-nuclear modality of estrogens action is for example ascribed to their direct vascular effects, antioxidative activity and a new pathway discovered in the last years shows that the plasma membrane G protein estrogen receptor (GPER1) is involved in cardioprotection of both in females as in males.

#### **1.2 Testosterone and its non-genomic role**

For decades, the research reports proposed only cardiotoxic and deleterious effect of testosterone in the cardiovascular system. This was based on epidemiologic human studies, where direct testosterone treatment, especially in supra-physiological androgen abuse, showed an increase in left ventricular mass and hypertrophy, causing myocardial stiffness and diastolic disfunction. Animal studies with castrates showed similar deleterious effects from adrogen receptor antagonist studies. However, later, with the use of better designed studies it was shown that testosterone, in optimal levels, may enable significant and profound cardioprotection expressed in e.g., diminished reperfusion injuries, decreased arrhythmias. It is now obvious that testosterone must posses the comparable non-nuclear activity, at cytoplasmatic level, similar to the non-genomic action of estrogen, through yet unknown mechanism(s).

#### **2. Sex hormones and heart protection**

In the cardiovascular tissues the protective effects of estrogen and testosterone are manifested through the instantly responsive arteries of the coronary artery system, which estrogen directly relaxes via the endothelial nitric oxide (NO) mechanism (Dai et al., 2004; Santos et al., 2004; Woodman et al., 2004). It is probable that testosterone relaxes these arteries via a different mechanism, maybe even a non-genomic pathway, which does not involve the nuclear androgen receptors and is independent of the vascular endothelium. This testosterone response is initiated at specific binding sites in the cell membranes of smooth muscles. For example, testosterone directly inhibits voltage-gated calcium channels, with an additional inhibitory action of calcium store-operated calcium channels (Jones et al., 2004; Yildiz et al., 2005). In the myocardium the estrogens reduce the incidence of postischemic ventricular arrhythmias by reducing the accumulation of intracellular Ca2+, protecting mitochondrial structures, inhibiting apoptosis, having antioxidant action and interacting with heat stress proteins, thus protecting the heart from injuries (Fraser et al., 2000; Kim et al., 1998; Knowlton & Sun, 2001; Zhai et al., 2000). Further, both the mitogen activated protein kinase (MAPK) and phosphoinositide-3-kinase (PI3K) are believed to be involved in the regulation of NO synthesis by estrogen (Gray et al., 2001). The acetylcholineinduced and flow-dependent vasodilation are preserved or potentiated by estradiol by increasing the endothelial production of NO and prostacyclin. Estradiol also promotes the endothelial healing and angiogenesis through the activation of estrogen receptor-α, which

gender related pathways responses. Recently, most of the approaches of cardiovascular

Since estrogens in premenopausal women with regular menstrual cycle are established as a natural protection against cardivoascular diseases, nuclear and non-nuclear mechanisms are evaluated for their mode of action. The non-nuclear modality of estrogens action is for example ascribed to their direct vascular effects, antioxidative activity and a new pathway discovered in the last years shows that the plasma membrane G protein estrogen receptor

For decades, the research reports proposed only cardiotoxic and deleterious effect of testosterone in the cardiovascular system. This was based on epidemiologic human studies, where direct testosterone treatment, especially in supra-physiological androgen abuse, showed an increase in left ventricular mass and hypertrophy, causing myocardial stiffness and diastolic disfunction. Animal studies with castrates showed similar deleterious effects from adrogen receptor antagonist studies. However, later, with the use of better designed studies it was shown that testosterone, in optimal levels, may enable significant and profound cardioprotection expressed in e.g., diminished reperfusion injuries, decreased arrhythmias. It is now obvious that testosterone must posses the comparable non-nuclear activity, at cytoplasmatic level, similar to the non-genomic action of estrogen, through yet

In the cardiovascular tissues the protective effects of estrogen and testosterone are manifested through the instantly responsive arteries of the coronary artery system, which estrogen directly relaxes via the endothelial nitric oxide (NO) mechanism (Dai et al., 2004; Santos et al., 2004; Woodman et al., 2004). It is probable that testosterone relaxes these arteries via a different mechanism, maybe even a non-genomic pathway, which does not involve the nuclear androgen receptors and is independent of the vascular endothelium. This testosterone response is initiated at specific binding sites in the cell membranes of smooth muscles. For example, testosterone directly inhibits voltage-gated calcium channels, with an additional inhibitory action of calcium store-operated calcium channels (Jones et al., 2004; Yildiz et al., 2005). In the myocardium the estrogens reduce the incidence of postischemic ventricular arrhythmias by reducing the accumulation of intracellular Ca2+, protecting mitochondrial structures, inhibiting apoptosis, having antioxidant action and interacting with heat stress proteins, thus protecting the heart from injuries (Fraser et al., 2000; Kim et al., 1998; Knowlton & Sun, 2001; Zhai et al., 2000). Further, both the mitogen activated protein kinase (MAPK) and phosphoinositide-3-kinase (PI3K) are believed to be involved in the regulation of NO synthesis by estrogen (Gray et al., 2001). The acetylcholineinduced and flow-dependent vasodilation are preserved or potentiated by estradiol by increasing the endothelial production of NO and prostacyclin. Estradiol also promotes the endothelial healing and angiogenesis through the activation of estrogen receptor-α, which

diseases focused on non-genomic action of sex hormones.

(GPER1) is involved in cardioprotection of both in females as in males.

**1.1 Non-genomic estrogen action** 

**1.2 Testosterone and its non-genomic role** 

**2. Sex hormones and heart protection** 

unknown mechanism(s).

shows that the protective mechanism in the cardiovascular system is linked to the estrogen activation of ER receptors (Arnal et al., 2010).

In the past the action of androgens on the cardiovascular system has received relatively little attention and authors disagree about possible detrimental or protective effects of testosterone on the heart (Pugh et al., 2000). Later studies showed that cardioprotective effects of testosterone are mediated by a yet not identified androgen-dependent pathway, but only after chronic administration of testosterone (Kuhar at al., 2007; Borst et al., 2010).

#### **2.1 Coronary flow, acute and chronic effects of sex hormones**

Direct application as well as estradiol pretreatment increased the coronary flow in isolated female hearts after the onset of reperfusion as well as testosterone pretreated male hearts (which was equally effective to female hearts), showing the vasodilatative effects of sex hormones (Kuhar et al., 2007). The coronary flow was increased after direct application of estradiol, while direct testosterone administration lacked such a vasodilatatory effect (Kuhar et al., 2007). Estradiol is reported to possess a direct artery relaxant action and also a direct effect on myocardium. Similarly, testosterone influenced coronary flow directly and the effects were both beneficial and deleterious (Pugh et al., 2000). Estradiol improved coronary flow of rats directly through the stimulation of NO release from endothelial cells (Dai et al., 2004; Fraser et al., 2000; Santos et al., 2004; Woodman et al., 2004). The vasodilatatory effects of testosterone are mediated by opening the large conductance, calcium-activated potassium channels (Deenadayalu et al., 2001). The short-term administration of testosterone induces a beneficial effect on exercise-induced myocardial ischemia in men with coronary artery disease, which may be related to its direct coronary relaxing effect (Rosano et al., 1999).

In physiological conditions, both estrogens as well as androgens may elicit very rapid effects to keep the homeostasis balanced, without manifesting RNA and protein synthesis (Dechering et al., 2000; Revelli et al., 1998). For example, the plasma membrane estrogen receptor was shown to respond rapidly to estrogen (Peitras & Szego, 1977), while there is no clear evidence yet of a similar effect for androgens.

#### **2.2 Endothelium, estrogen and the heart**

The sex hormones in the endothelium were generally believed to have mainly non-genomic effects. The endothelial estrogen receptor-α (ERα) in the endothelium as a whole, is considered one of the most important targets of cardioprotection. The endothelium and, in particular, the endothelial ER-α appear to be a key cellular and molecular targets of the protective actions of estradiol against ischemia/reperfusion (I/R)-induced coronary endothelial dysfunction. The activation of the endothelial estrogen receptor (ER) by estradiol, triggers a protective action on the coronary endothelial structure and function, which, in turn, limits the size of the infarct. This protection may be in part due to reduced cardiac oxidative stress, demonstrated by the decreased production of reactive oxygen species observed during early reperfusion. The signaling mechanisms of cardioprotection are to a great extent dependent on NO, which signals in cardiomyocytes via protein kinases and may possibly protect mitochondria, resulting in decreased cardiomyocyte death. Another indirect effect the reduced neutrophil-mediated cardiomyocyte injury may also play a role as endothelial protection and thus in cardioprotection (Favre et al., 2010).

The Role of Sex Hormones in the Cardiovascular System 35

ERα knockouts were reduced to half of the values in control group. This leads to the conclusion that VEGF is supposed to act mainly via ERα to regulate VEGF transcription and elements of basic VEGF signaling, which makes it is crucial in the development of

The inability of the heart to supply sufficient cardiac output and blood flow to meet the needs of the body and lungs is defined as a heart failure. The causes of heart failure are myocardial infarction, ischemic heart disease in general, hypertension, valvular heart disease, and cardiomyopathy, with symptoms being shortness of breath, leg swelling,

Fig. 1B. Non-nuclear estrogen receptor signaling in vascular endothelial cell caveolae.

The non-nuclear estrogen receptors (ERs) localize to the endothelial cell membrane invaginations called caveolae by direct binding to the caveolar proteins, including the scaffold protein striatin, which is bound to the major caveolar structural protein, caveolin-1 (Cav-1). Upon estrogen binding (EDC), signaling complexes assemble that include the ERs and the G protein Gαi, Gβγ and sequentially activate the tyrosine kinase src (C-src), the serine/threonine kinase, phosphoinositide-3 protein kinase (PI3K), consisted of subunits p85a and p110a, that produce phosphatidylinositol (3,4,5)-triphosphate (PIP3), and the kinase Akt. Akt is serine/threonine protein kinase that plays a key role in multiple cellular processes such as cell proliferation, apoptosis, transcription, cell migration as well as in angiogenesis. Akt then directly phosphorylates endothelial nitric oxide synthase (eNOS) on serine 1,177, leading to its enzymatic activation and the production of nitric oxide (NO). Also, Akt activate extracellular signal-regulated kinases (Erk1/2) or MAP kinases, involved in differentiation of cells, but also in regulation of eNOS. The position of the assembling complex - on the internal or the external side of the cell membrane – remains unclear. This

non-nuclear ER dependent pathway confers protection against vascular injury.

microvasculatures in the heart (Jesmin et al., 2010).

**2.3 Heart failure** 

Adopted from Wu et al., (2011).

Fig. 1A. Endothelial estrogen-mediated responses. Adopted from Wu et al., (2011). *Left:* The first signaling pathway in vascular endothelial cells by estrogen receptor (ER) represent the cytosolic receptors, that are ligand-activated transcription factors, that regulate gene expression. After translocation to the nucleus receptor dimerizes and binds to specific DNA sequences called estrogen response elements (ERE), recruiting coactivator (CoA) proteins, displacing corepressors (CoR) from the DNA, and activating gene expression. *Center:* The second signaling pathway are the ERs that can be transcriptionally activated via ligand-independent pathways in which growth factor receptor (GFR) activation leads to activation of specific kinases that directly phosphorylate (circled P) the ER, again leading to altered gene expression, either directly by the ER or via ER interactions with other transcription factors (TFs).

*Right:* The third signaling pathway is mediated by non-nuclear ERs. In this pathway, estrogen induces a cell membrane–associated ERs to form a signaling complex that results in rapid activation of specific kinases, which in turn phosphorylate and enzymatically activate endothelial nitric oxide synthase (eNOS).

Another important endothelial, cardioprotection factor is the vascular endothelium growth factor (VEGF) and its basic signal molecules (VEGF receptor, Akt, eNOS). ERα knockout mice showed a marked decrease of capillary density, and the absence of receptor β has minimal effect, while the levels of the VEGF receptor, phosphorylated Akt and eNOS in the ERα knockouts were reduced to half of the values in control group. This leads to the conclusion that VEGF is supposed to act mainly via ERα to regulate VEGF transcription and elements of basic VEGF signaling, which makes it is crucial in the development of microvasculatures in the heart (Jesmin et al., 2010).

#### **2.3 Heart failure**

34 Sex Hormones

Fig. 1A. Endothelial estrogen-mediated responses. Adopted from Wu et al., (2011). *Left:* The first signaling pathway in vascular endothelial cells by estrogen receptor (ER) represent the cytosolic receptors, that are ligand-activated transcription factors, that regulate gene expression. After translocation to the nucleus receptor dimerizes and binds to specific DNA sequences called estrogen response elements (ERE), recruiting coactivator (CoA) proteins, displacing corepressors (CoR) from the DNA, and activating gene expression. *Center:* The second signaling pathway are the ERs that can be transcriptionally activated via ligand-independent pathways in which growth factor receptor (GFR) activation leads to activation of specific kinases that directly phosphorylate (circled P) the ER, again leading to

altered gene expression, either directly by the ER or via ER interactions with other

*Right:* The third signaling pathway is mediated by non-nuclear ERs. In this pathway,

estrogen induces a cell membrane–associated ERs to form a signaling complex that results in rapid activation of specific kinases, which in turn phosphorylate and enzymatically activate

Another important endothelial, cardioprotection factor is the vascular endothelium growth factor (VEGF) and its basic signal molecules (VEGF receptor, Akt, eNOS). ERα knockout mice showed a marked decrease of capillary density, and the absence of receptor β has minimal effect, while the levels of the VEGF receptor, phosphorylated Akt and eNOS in the

transcription factors (TFs).

endothelial nitric oxide synthase (eNOS).

The inability of the heart to supply sufficient cardiac output and blood flow to meet the needs of the body and lungs is defined as a heart failure. The causes of heart failure are myocardial infarction, ischemic heart disease in general, hypertension, valvular heart disease, and cardiomyopathy, with symptoms being shortness of breath, leg swelling,

Fig. 1B. Non-nuclear estrogen receptor signaling in vascular endothelial cell caveolae. Adopted from Wu et al., (2011).

The non-nuclear estrogen receptors (ERs) localize to the endothelial cell membrane invaginations called caveolae by direct binding to the caveolar proteins, including the scaffold protein striatin, which is bound to the major caveolar structural protein, caveolin-1 (Cav-1). Upon estrogen binding (EDC), signaling complexes assemble that include the ERs and the G protein Gαi, Gβγ and sequentially activate the tyrosine kinase src (C-src), the serine/threonine kinase, phosphoinositide-3 protein kinase (PI3K), consisted of subunits p85a and p110a, that produce phosphatidylinositol (3,4,5)-triphosphate (PIP3), and the kinase Akt. Akt is serine/threonine protein kinase that plays a key role in multiple cellular processes such as cell proliferation, apoptosis, transcription, cell migration as well as in angiogenesis. Akt then directly phosphorylates endothelial nitric oxide synthase (eNOS) on serine 1,177, leading to its enzymatic activation and the production of nitric oxide (NO). Also, Akt activate extracellular signal-regulated kinases (Erk1/2) or MAP kinases, involved in differentiation of cells, but also in regulation of eNOS. The position of the assembling complex - on the internal or the external side of the cell membrane – remains unclear. This non-nuclear ER dependent pathway confers protection against vascular injury.

The Role of Sex Hormones in the Cardiovascular System 37

Fig. 2. Cardioprotective signalling pathways induced by estrogen receptor activation.

shock proteins (HSP27/60/70), and cytosol like creatine kinase (CK), and malate

**2.4.1 Amelioration of ischemia and reperfusion induced myocardial injuries** 

Following after the activation of both PI3K/Akt, nitric oxide (NO) signaling and the chronic activation of ERβ comes a significant increase in S-nitrosylated proteins (SNO) in the mitochondria including F1F0-ATPase, aconitase (Ac), cytochrome c oxidase (Cyto C), heat

dehydrogenase (MD). The acute and moreover chronic activation of these pathways leads to

The anti-ischemic effects of sex hormones are considered to protect due to improvement of the coronary flow and due to the reduced incidence of arrhythmias and established cytoprotection during reperfusion. Both estradiol and testosterone in supraphysiological levels significantly decreased ischemia-reperfusion injuries of isolated rat hearts. The reperfusion injuries were reduced both by the direct application of sex hormones, and pretreatment prior to the isolated heart experiment. However, protective effects against ischemia-reperfusion injuries were only observed in the male hearts of animals pretreated with testosterone in a comparable effective way as in estradiol pretreated female hearts. The direct testosterone application was not comparable to the protection by the directly applied

Adopted from Deschamps, Murphy & Sun (2010).

enhanced cell survival resulting in cardioprotection.

estradiol (Kuhar et al., 2007).

exercise intolerance and the overall body becomes congested with fluid. Heart failure is a complex state of progressive multisystem diseases with significant morbidity and mortality and its clinical picture is defined by pathology of the cardiovascular system and is influenced by peripheral cytokine, hormonal, and musculoskeletal dysfunction. The cytokines, catecholamines, and hormones during heart failure have a maladaptive response that leads to a proinflammatory state tipping the metabolic balance toward catabolism. In addition to this procatabolic combination, chronically high levels of catecholamines, angiotensin II, and aldosterone eventually may contribute to testosterone deficiency, which blunts the anabolic compensatory pathways.

Present medical treatments for heart failure have proven to decrease mortality and include treatment with β-adrenergic receptor antagonists, which target adrenergic hyperstimulation of the failing myocardium; angiotensin converting enzyme inhibitors and angiotensin receptor blockers, which attenuate left ventricular remodeling; and aldosterone blockers, which blunt myocardial fibrosis.

Several studies have shown that more than ¼ of men is affected with chronic heart failure are deficient in testosterone. Thus testosterone is directly or indirectly involved in its pathology. Androgen deficient men without heart failure often report similar typical symptoms, such as shortness of breath, fatigue, deterioration of muscle mass, decline in strenght and endurance (Naghi et al., 2011). In male, but not female mice, G protein estrogen receptor GPR30-deficient mice manifested impaired left-ventricular cardiac function, indicating that non-genomic estradiol signalling is important in heart failure. Further, also regulation of vascular tone by GPER1 is indicated to play a role in heart failure (Delbeck et al., 2011).

#### **2.4 Ischemic heart disease and acute myocardial injuries**

The protective action of estradiol in cardiac ischemia/reperfusion (I/R) was demonstrated in several animal studies, but most of the cellular targets involved in this protection still need to be defined. Usually, in control animals after cardiac I/R the following are evoked: structural endothelium injuries, including necrosis, associated with altered coronary endothelial NO production. The long-term activation of the endothelial ERα by estradiol protected both the coronary endothelial and myocardial layers and endothelial structures associated with the NO-mediated coronary endothelial response (Favre et al., 2007). However, this was not the case for ovariectomized female mice and male mice, which during I/R showed endothelial dysfunction (Favre et al., 2007). More precisely, the ERα deficiency worsens global I/R-induced alteration in coronary flow and cardiac NO release in male mice and also abolishes the endogenous cardiac protection displayed in intact female mice (Favre et al., 2007).

Genomic pathway for cardioprotection by estrogen receptor (ER) activation is upregulated with chronic ERβ stimulation. This pathway is ligand-activated by transcription factors that, after translocation to the nucleus, bind to DNA sequences and regulate gene expression, resulting in for example modulators of both the NO system and apoptosis processes.

Stimulation of the nuclear G-protein coupled membrane bound estrogen receptor (GPER1) by tyrosine kinase src (C-src) results in activation of matrix metalloproteinase (MMP) leading to the release of epidermal growth factor (EGF) that can transactivate epidermal growth factor receptors (EGFRs). EGFR activation leads to multiple downstream events including activation of kinases within the phosphoinositide-3 protein kinase/ Akt pathways (PI3K/Akt).

exercise intolerance and the overall body becomes congested with fluid. Heart failure is a complex state of progressive multisystem diseases with significant morbidity and mortality and its clinical picture is defined by pathology of the cardiovascular system and is influenced by peripheral cytokine, hormonal, and musculoskeletal dysfunction. The cytokines, catecholamines, and hormones during heart failure have a maladaptive response that leads to a proinflammatory state tipping the metabolic balance toward catabolism. In addition to this procatabolic combination, chronically high levels of catecholamines, angiotensin II, and aldosterone eventually may contribute to testosterone deficiency, which

Present medical treatments for heart failure have proven to decrease mortality and include treatment with β-adrenergic receptor antagonists, which target adrenergic hyperstimulation of the failing myocardium; angiotensin converting enzyme inhibitors and angiotensin receptor blockers, which attenuate left ventricular remodeling; and aldosterone blockers,

Several studies have shown that more than ¼ of men is affected with chronic heart failure are deficient in testosterone. Thus testosterone is directly or indirectly involved in its pathology. Androgen deficient men without heart failure often report similar typical symptoms, such as shortness of breath, fatigue, deterioration of muscle mass, decline in strenght and endurance (Naghi et al., 2011). In male, but not female mice, G protein estrogen receptor GPR30-deficient mice manifested impaired left-ventricular cardiac function, indicating that non-genomic estradiol signalling is important in heart failure. Further, also regulation of vascular tone by GPER1 is indicated to play a role in heart failure (Delbeck et

The protective action of estradiol in cardiac ischemia/reperfusion (I/R) was demonstrated in several animal studies, but most of the cellular targets involved in this protection still need to be defined. Usually, in control animals after cardiac I/R the following are evoked: structural endothelium injuries, including necrosis, associated with altered coronary endothelial NO production. The long-term activation of the endothelial ERα by estradiol protected both the coronary endothelial and myocardial layers and endothelial structures associated with the NO-mediated coronary endothelial response (Favre et al., 2007). However, this was not the case for ovariectomized female mice and male mice, which during I/R showed endothelial dysfunction (Favre et al., 2007). More precisely, the ERα deficiency worsens global I/R-induced alteration in coronary flow and cardiac NO release in male mice and also abolishes the endogenous cardiac protection displayed in intact

Genomic pathway for cardioprotection by estrogen receptor (ER) activation is upregulated with chronic ERβ stimulation. This pathway is ligand-activated by transcription factors that, after translocation to the nucleus, bind to DNA sequences and regulate gene expression,

Stimulation of the nuclear G-protein coupled membrane bound estrogen receptor (GPER1) by tyrosine kinase src (C-src) results in activation of matrix metalloproteinase (MMP) leading to the release of epidermal growth factor (EGF) that can transactivate epidermal growth factor receptors (EGFRs). EGFR activation leads to multiple downstream events including activation

resulting in for example modulators of both the NO system and apoptosis processes.

of kinases within the phosphoinositide-3 protein kinase/ Akt pathways (PI3K/Akt).

blunts the anabolic compensatory pathways.

**2.4 Ischemic heart disease and acute myocardial injuries** 

which blunt myocardial fibrosis.

female mice (Favre et al., 2007).

al., 2011).

Following after the activation of both PI3K/Akt, nitric oxide (NO) signaling and the chronic activation of ERβ comes a significant increase in S-nitrosylated proteins (SNO) in the mitochondria including F1F0-ATPase, aconitase (Ac), cytochrome c oxidase (Cyto C), heat shock proteins (HSP27/60/70), and cytosol like creatine kinase (CK), and malate dehydrogenase (MD). The acute and moreover chronic activation of these pathways leads to enhanced cell survival resulting in cardioprotection.

#### **2.4.1 Amelioration of ischemia and reperfusion induced myocardial injuries**

The anti-ischemic effects of sex hormones are considered to protect due to improvement of the coronary flow and due to the reduced incidence of arrhythmias and established cytoprotection during reperfusion. Both estradiol and testosterone in supraphysiological levels significantly decreased ischemia-reperfusion injuries of isolated rat hearts. The reperfusion injuries were reduced both by the direct application of sex hormones, and pretreatment prior to the isolated heart experiment. However, protective effects against ischemia-reperfusion injuries were only observed in the male hearts of animals pretreated with testosterone in a comparable effective way as in estradiol pretreated female hearts. The direct testosterone application was not comparable to the protection by the directly applied estradiol (Kuhar et al., 2007).

The Role of Sex Hormones in the Cardiovascular System 39

possible to elicit direct effects that can not be observed in cardiomyocites. Second, the mechanisms of reperfusion injury to cardiomyocytes in vivo markedly differ from those involved in vitro. The immediate inflammatory response associated with severe oxidative stress appears to be operative in vivo but not in vitro and this phenomenon centrally involves the endothelium as both a target and a trigger of the inflammatory response. Third, another important aspect of reperfusion injury is the no-reflow phenomenon that may worsen I/R injury and that is likely to be reduced by estradiol secondary to endothelial

Cardioprotection also can be achieved by preconditioning, a process that can be assesed pharmacologically, by ischemia, or by other stressors. Preconditioning increases the resistance to subsequent longer stress. Among the most important benefits are reduction of reperfusion injuries, diminished arrhythmia, prevention of myocardial stunning and postischemic contractile dysfunction, marked limitation and decrease of infarct size, and reduction of endothelial injury (Geršak & Drevenšek, 2002). The receptors involved in preconditioning are mainly coupled to protein kinase C. In mice with removed testicles, the immediate cardioprotection of ischemic preconditioning is abolished, thus testosterone is needed for the acute cardioprotection of preconditioning. In the absence of testosterone, the preconditioning with metabolic inhibition in vitro or k-opioid agonist in vivo, failed to establish delayed cardioprotection against ischemic insult in ventricular myocytes or isolated perfused male rat hearts, respectively (Liu et al., 2006). This was the first evidence that testosterone at physiological concentrations is needed for the delayed cardioprotection

Estrogen is known to relax arteries directly via endothelial NO and thus exert potential antiatherogenic effects (Dai et al., 2004; Santos et al., 2004; Woodman et al., 2004). Also, estradiol prevents early atheroma through endothelial-mediated mechanisms (Arnal et al., 2010). Androgens on the other hand, have been associated with possible proatherogenic effects and an increased cardiovascular risk by adversely affecting the plasma lipid and lipoprotein profile, increasing the risk of thrombosis and cardiac hypertrophy (Adams et al., 1995). On the contrary, short-term administration of testosterone causes vasodilatation in a range of species including humans (Costarella et al., 1996; Crews & Khalil, 1999; Honda et al., 1999; Perusquia et al., 1996; Yue et al., 1995). However these beneficial early on atherogenesis effects of testosterone were not explained by changes in lipid levels. Besides, estradiol administration to orchidectomized males attenuated lesion formation to the same extent as testosterone administration. These results indicate that testosterone attenuates early atherogenesis and that this is most likely be caused by being converted to estrogens by the enzyme aromatase expressed in the vessel wall (Nathan et al., 2000). The latest study in an androgen receptor knockout mice on apolipoprotein E-deficient basis, showed acceleration of atherosclerosis, while testosterone treatment reduced this atherosclerosis. In conclusion, the male mice showed testosterone atheroprotection which has both androgen receptor-dependent and androgen receptor-independent components (Bourghardt et al.,

protection (Favre et al., 2010).

of preconditioning.

2010).

**2.4.2 Delayed cardioprotection by testosterone** 

**3. Atherogenesis, sex hormones and gender differences** 

Further, in female animals direct administration of estradiol showed protection against cell injuries, and no protection was shown in estradiol pretreatment. In male hearts cytoprotective effects were found only in testosterone pretreated animals, while estradiol lacks direct cytoprotective effects in isolated heart (Kuhar et al., 2007). Amelioration of ischemia and reperfusion induced myocardial injuries has also been demonstrated in some experimental animal models (Delyani et al., 1996). For estradiol a profound protective effect against stroke-like ischemic injuries in female rats was found (Wise et al., 2001), while also the cytoprotective effect of estradiol to hypoxia–reoxygenation induced injuries in cardiac cells has been reported (Jovanovic et al., 2000). Chronic estradiol treatment does show some cardioprotective effects which can be attributed to over expression of heat-shock proteins (HSPs), which is generally regarded as protective against cardiac injury. HSP90 is known to bind the intracellular hormone receptors and therefore it was suggested that the interaction between HSP90, the receptors, and heat-shock factor-1 (HSF-1) was an important element in the activation of HSF-1 by hormones (Knowlton & Sun, 2001). It is known that after treatment of cardiac myocytes with 17β-estradiol or progesterone the HSP90 redistributes. However, testosterone did not effect HSP levels and pretreatment of males with testosterone did not elicit protective effects (Knowlton & Sun, 2001). This is in accordance with the discovery that androgen receptors are absent in cardiac myocytes.

Morover, testosterone acutely and directly depolarizes and oxidizes cardiac mitochondria in a K-dependent, ATP-sensitive, and testosterone receptor-independent manner, by activation of mitochondrial K+ channels, while it does not activate sarcolemal K+ ATP channels. Thus, mitochondrial K+ channels play a key role in cardioprotection during ischemia and via this mechanism testosterone protects cardiomyocytes from ischemic cell death (Er et al., 2004). In contrast, cell injury tests did not confirm the direct protective effects of testosterone in ischemia/reperfusion induced myocardial injuries, which may be due to the limited electrolyte capacity of the mitochondria. To conclude, the action of the sex hormones is attributable not only due to its direct action on coronary arteries, but also due to other nongenomic properties.

The demonstration that estrogen exert a cardioprotective effect in male animals showed, that in vivo supplemental estrogen treatment of male mice reduces the prevalence of cardiac rupture during the acute phase of myocardial infarction (Cao et al., 2011). In other shortterm (acute) and long-term (chronic) cardiac function study, myocardial infarction-induced male mice treated with estrogen and female mice treated with testosterone, showed opposing chronic cardiac remodeling and function effects, with favorable (protective) effects exerted by estrogen and detrimental effects exerted by testosterone (Cavasin et al., 2003). During the acute phase of myocardial infarction, however, estrogen appeared to offer no or little protection against acute myocardial infarction-induced cardiac rupture. The castration alone could slightly reduce the prevalence of cardiac rupture in male mice. While a lower prevalence of cardiac rupture was observed in estrogen-treated mice as compared to placebo-treated ones (Cao et al., 2011), no difference was observed in another study (Cavasin et al., 2003).

The observation on the obligatory role of the endothelium for cardiomyocyte protection may appear contradictory to a direct protective action of estradiol on hypoxia/reoxygenationmediated death of isolated cardiomyocytes. First, the in vitro data used large amounts of immediately administered estradiol and thus with these pharmacological doses, it is

Further, in female animals direct administration of estradiol showed protection against cell injuries, and no protection was shown in estradiol pretreatment. In male hearts cytoprotective effects were found only in testosterone pretreated animals, while estradiol lacks direct cytoprotective effects in isolated heart (Kuhar et al., 2007). Amelioration of ischemia and reperfusion induced myocardial injuries has also been demonstrated in some experimental animal models (Delyani et al., 1996). For estradiol a profound protective effect against stroke-like ischemic injuries in female rats was found (Wise et al., 2001), while also the cytoprotective effect of estradiol to hypoxia–reoxygenation induced injuries in cardiac cells has been reported (Jovanovic et al., 2000). Chronic estradiol treatment does show some cardioprotective effects which can be attributed to over expression of heat-shock proteins (HSPs), which is generally regarded as protective against cardiac injury. HSP90 is known to bind the intracellular hormone receptors and therefore it was suggested that the interaction between HSP90, the receptors, and heat-shock factor-1 (HSF-1) was an important element in the activation of HSF-1 by hormones (Knowlton & Sun, 2001). It is known that after treatment of cardiac myocytes with 17β-estradiol or progesterone the HSP90 redistributes. However, testosterone did not effect HSP levels and pretreatment of males with testosterone did not elicit protective effects (Knowlton & Sun, 2001). This is in accordance with the

Morover, testosterone acutely and directly depolarizes and oxidizes cardiac mitochondria in a K-dependent, ATP-sensitive, and testosterone receptor-independent manner, by activation of mitochondrial K+ channels, while it does not activate sarcolemal K+ ATP channels. Thus, mitochondrial K+ channels play a key role in cardioprotection during ischemia and via this mechanism testosterone protects cardiomyocytes from ischemic cell death (Er et al., 2004). In contrast, cell injury tests did not confirm the direct protective effects of testosterone in ischemia/reperfusion induced myocardial injuries, which may be due to the limited electrolyte capacity of the mitochondria. To conclude, the action of the sex hormones is attributable not only due to its direct action on coronary arteries, but also due to other non-

The demonstration that estrogen exert a cardioprotective effect in male animals showed, that in vivo supplemental estrogen treatment of male mice reduces the prevalence of cardiac rupture during the acute phase of myocardial infarction (Cao et al., 2011). In other shortterm (acute) and long-term (chronic) cardiac function study, myocardial infarction-induced male mice treated with estrogen and female mice treated with testosterone, showed opposing chronic cardiac remodeling and function effects, with favorable (protective) effects exerted by estrogen and detrimental effects exerted by testosterone (Cavasin et al., 2003). During the acute phase of myocardial infarction, however, estrogen appeared to offer no or little protection against acute myocardial infarction-induced cardiac rupture. The castration alone could slightly reduce the prevalence of cardiac rupture in male mice. While a lower prevalence of cardiac rupture was observed in estrogen-treated mice as compared to placebo-treated ones (Cao et al., 2011), no difference was observed in another study

The observation on the obligatory role of the endothelium for cardiomyocyte protection may appear contradictory to a direct protective action of estradiol on hypoxia/reoxygenationmediated death of isolated cardiomyocytes. First, the in vitro data used large amounts of immediately administered estradiol and thus with these pharmacological doses, it is

discovery that androgen receptors are absent in cardiac myocytes.

genomic properties.

(Cavasin et al., 2003).

possible to elicit direct effects that can not be observed in cardiomyocites. Second, the mechanisms of reperfusion injury to cardiomyocytes in vivo markedly differ from those involved in vitro. The immediate inflammatory response associated with severe oxidative stress appears to be operative in vivo but not in vitro and this phenomenon centrally involves the endothelium as both a target and a trigger of the inflammatory response. Third, another important aspect of reperfusion injury is the no-reflow phenomenon that may worsen I/R injury and that is likely to be reduced by estradiol secondary to endothelial protection (Favre et al., 2010).

#### **2.4.2 Delayed cardioprotection by testosterone**

Cardioprotection also can be achieved by preconditioning, a process that can be assesed pharmacologically, by ischemia, or by other stressors. Preconditioning increases the resistance to subsequent longer stress. Among the most important benefits are reduction of reperfusion injuries, diminished arrhythmia, prevention of myocardial stunning and postischemic contractile dysfunction, marked limitation and decrease of infarct size, and reduction of endothelial injury (Geršak & Drevenšek, 2002). The receptors involved in preconditioning are mainly coupled to protein kinase C. In mice with removed testicles, the immediate cardioprotection of ischemic preconditioning is abolished, thus testosterone is needed for the acute cardioprotection of preconditioning. In the absence of testosterone, the preconditioning with metabolic inhibition in vitro or k-opioid agonist in vivo, failed to establish delayed cardioprotection against ischemic insult in ventricular myocytes or isolated perfused male rat hearts, respectively (Liu et al., 2006). This was the first evidence that testosterone at physiological concentrations is needed for the delayed cardioprotection of preconditioning.

#### **3. Atherogenesis, sex hormones and gender differences**

Estrogen is known to relax arteries directly via endothelial NO and thus exert potential antiatherogenic effects (Dai et al., 2004; Santos et al., 2004; Woodman et al., 2004). Also, estradiol prevents early atheroma through endothelial-mediated mechanisms (Arnal et al., 2010). Androgens on the other hand, have been associated with possible proatherogenic effects and an increased cardiovascular risk by adversely affecting the plasma lipid and lipoprotein profile, increasing the risk of thrombosis and cardiac hypertrophy (Adams et al., 1995). On the contrary, short-term administration of testosterone causes vasodilatation in a range of species including humans (Costarella et al., 1996; Crews & Khalil, 1999; Honda et al., 1999; Perusquia et al., 1996; Yue et al., 1995). However these beneficial early on atherogenesis effects of testosterone were not explained by changes in lipid levels. Besides, estradiol administration to orchidectomized males attenuated lesion formation to the same extent as testosterone administration. These results indicate that testosterone attenuates early atherogenesis and that this is most likely be caused by being converted to estrogens by the enzyme aromatase expressed in the vessel wall (Nathan et al., 2000). The latest study in an androgen receptor knockout mice on apolipoprotein E-deficient basis, showed acceleration of atherosclerosis, while testosterone treatment reduced this atherosclerosis. In conclusion, the male mice showed testosterone atheroprotection which has both androgen receptor-dependent and androgen receptor-independent components (Bourghardt et al., 2010).

The Role of Sex Hormones in the Cardiovascular System 41

lead to ventricular fibrilation, is associated with long QT syndrome and is more common in females than in males. The long QT syndrome can be inherited as congenital mutations of the ion channels that carry the cardiac action potential or acquired as a result of drugs that block these cardiac ion currents. The higher male incidence of the Brugada syndrome is associated with the early phase of ventricular repolarization, that is larger in the right ventricular epicardium of males than in females, resulting in a characteristic ST elevation in males. The early repolarization syndrome is characterized by a prominent J wave and by elevation of the ST-segment in the left precordial leads; it is most commonly seen in young

The evaluation of arrhythmias during ischemic-reperfusion induced injuries using high doses of hormones, showed that cardioprotection with testosterone is established only after pretreatment (Kuhar et al., 2007), but no protective effect was detected when testosterone was applied directly to the isolated animal hearts. Animals pretreated with testosterone as well as with estradiol showed high level of cardioprotection against post-ischemic injuries. Most of the beneficial effects shown in post-ischemic hearts were expressed as improved coronary flow, decreased release of lactate dehydrogenase rate and shorter lasting arrhythmias (Kuhar et al., 2007). The increased coronary flow in the hearts of pretreated animals of both sexes with estradiol and testosterone may be the result of induced NO production. The diminished arrhythmias in chronically treated hearts in rats of both sexes could be the consequence of both, vasodilatation and the direct cardioprotective effects of the hormones. The reduced lactate dehydrogenase release rate in the estradiol pretreated group showed more complex activity; cardioprotection might be induced also due to HSP

During reperfusion in the hearts of the animals pretreated with both testosterone and estradiol, all types of arrhythmias were reduced compared to the directly treated groups (Kuhar et al., 2007). The heart arrest was most severely decreased followed by, decreasing intensities in ventricular fibrilation, and ventricular premature complexes (Kuhar et al., 2007). The protective effect of sex hormones against the appearance of arrhythmias, fibrillation and ventricular tachycardia, was proposed by some other studies (Kim et al.,

The QT interval in the electrocardiogram (ECG) is defined as the interval from the onset of the QRS complex to the end of the T wave (Figure 4), it is the sum of ventricular myocardial action potential duration and the ventricular repolarization. The ST segment of the ECG represents the duration of ventricular repolarization only, with -J, -M, and –E segment representing the right and left ventricles and are measured by the ECG leads V2 and V5 respectively. The sex hormones, like estrogens, progesterones and androgens, can modulate a variety of ionic currents and are reported to influence the duration of the ECG (James et

The QTc represents the rate-corrected QT interval that is calculated using the method of Fridericia (QTc=QT/RR1/3, Schwartz et al., 2011). On molecular level, the duration of the QT interval is the net effect of the activity of multiple ion-channels and transporters. The

males (Ezaki et al., 2010).

activation with estradiol.

1998; Zhai et al., 2000).

al., 2007).

**4.1 Electrocardiogram, QTc and ST** 

**4.1.1 Molecular basis for QTc differences** 

Fig. 3. Acute ischemia/reperfusion induced testosterone signalling pathways in cardiomyocites during androgen receptor activation. Adopted from Huang et al. (2010). The activation by myocardial Akt in PI3K/Akt downstream signaling after activation of androgen receptor (AR) due to ischemia reperfusion causes increased release of cell death signals (Bad, Bcl-2, FOXO3a) and decrease apoptotic mediator FasL. The Akt protein at normal sex hormone status is more active in female then in male hearts and thus in this pathway testosterone is a negative factor in cardioprotection processes.

The genomic actions of the AR activation consist out of changes in gene expression involved in cardiac protection/injury, like modulators of the superoxide dismutase (MnSOD) system, apoptotic and death genes. Besides, testosterone in acute ischemia down-regulates FOXO3a, a trigger protein for apoptosis, and decreases antiapoptotic activator targets as Bim and FasL that are probably post-translational products with little or no influence on acute ischemia/reperfusion injuries. Overall, genomic pathway probably leads to testosterone induced cardioprotection.

#### **4. Arrhythmias**

The ventricular arrhythmias, mostly being the malignant ones of all arrhythmias, show important gender differences. Torsades de pointes, a ventricular tachyarrhythmia that can

Fig. 3. Acute ischemia/reperfusion induced testosterone signalling pathways in

pathway testosterone is a negative factor in cardioprotection processes.

induced cardioprotection.

**4. Arrhythmias** 

cardiomyocites during androgen receptor activation. Adopted from Huang et al. (2010). The activation by myocardial Akt in PI3K/Akt downstream signaling after activation of androgen receptor (AR) due to ischemia reperfusion causes increased release of cell death signals (Bad, Bcl-2, FOXO3a) and decrease apoptotic mediator FasL. The Akt protein at normal sex hormone status is more active in female then in male hearts and thus in this

The genomic actions of the AR activation consist out of changes in gene expression involved in cardiac protection/injury, like modulators of the superoxide dismutase (MnSOD) system, apoptotic and death genes. Besides, testosterone in acute ischemia down-regulates FOXO3a, a trigger protein for apoptosis, and decreases antiapoptotic activator targets as Bim and FasL that are probably post-translational products with little or no influence on acute ischemia/reperfusion injuries. Overall, genomic pathway probably leads to testosterone

The ventricular arrhythmias, mostly being the malignant ones of all arrhythmias, show important gender differences. Torsades de pointes, a ventricular tachyarrhythmia that can lead to ventricular fibrilation, is associated with long QT syndrome and is more common in females than in males. The long QT syndrome can be inherited as congenital mutations of the ion channels that carry the cardiac action potential or acquired as a result of drugs that block these cardiac ion currents. The higher male incidence of the Brugada syndrome is associated with the early phase of ventricular repolarization, that is larger in the right ventricular epicardium of males than in females, resulting in a characteristic ST elevation in males. The early repolarization syndrome is characterized by a prominent J wave and by elevation of the ST-segment in the left precordial leads; it is most commonly seen in young males (Ezaki et al., 2010).

The evaluation of arrhythmias during ischemic-reperfusion induced injuries using high doses of hormones, showed that cardioprotection with testosterone is established only after pretreatment (Kuhar et al., 2007), but no protective effect was detected when testosterone was applied directly to the isolated animal hearts. Animals pretreated with testosterone as well as with estradiol showed high level of cardioprotection against post-ischemic injuries. Most of the beneficial effects shown in post-ischemic hearts were expressed as improved coronary flow, decreased release of lactate dehydrogenase rate and shorter lasting arrhythmias (Kuhar et al., 2007). The increased coronary flow in the hearts of pretreated animals of both sexes with estradiol and testosterone may be the result of induced NO production. The diminished arrhythmias in chronically treated hearts in rats of both sexes could be the consequence of both, vasodilatation and the direct cardioprotective effects of the hormones. The reduced lactate dehydrogenase release rate in the estradiol pretreated group showed more complex activity; cardioprotection might be induced also due to HSP activation with estradiol.

During reperfusion in the hearts of the animals pretreated with both testosterone and estradiol, all types of arrhythmias were reduced compared to the directly treated groups (Kuhar et al., 2007). The heart arrest was most severely decreased followed by, decreasing intensities in ventricular fibrilation, and ventricular premature complexes (Kuhar et al., 2007). The protective effect of sex hormones against the appearance of arrhythmias, fibrillation and ventricular tachycardia, was proposed by some other studies (Kim et al., 1998; Zhai et al., 2000).

#### **4.1 Electrocardiogram, QTc and ST**

The QT interval in the electrocardiogram (ECG) is defined as the interval from the onset of the QRS complex to the end of the T wave (Figure 4), it is the sum of ventricular myocardial action potential duration and the ventricular repolarization. The ST segment of the ECG represents the duration of ventricular repolarization only, with -J, -M, and –E segment representing the right and left ventricles and are measured by the ECG leads V2 and V5 respectively. The sex hormones, like estrogens, progesterones and androgens, can modulate a variety of ionic currents and are reported to influence the duration of the ECG (James et al., 2007).

#### **4.1.1 Molecular basis for QTc differences**

The QTc represents the rate-corrected QT interval that is calculated using the method of Fridericia (QTc=QT/RR1/3, Schwartz et al., 2011). On molecular level, the duration of the QT interval is the net effect of the activity of multiple ion-channels and transporters. The

The Role of Sex Hormones in the Cardiovascular System 43

(Schwartz et al., 2011). Male hypogonadism is associated with an increased prevalence of prolonged QT interval, over 2.5%, as compared to the control and the healthy population (Schwartz et al., 1993), and hence, a higher risk for fatal ventricular arrhythmias. The QTc interval in the hypogonadal state is prolonged and by hormone replacement therapy normalized (Pecori Giraldi et al., 2010). This evidence led to the conclusion that testosterone is the main determinant of gender-related differences in the ventricular refractory periods

In vitro and data on animals showed that sex hormones increased the QTc intervals in females, or conversely, decreased the QTc intervals in males (Fülöp et al., 2006). Women with excess androgen secretion present shorter and faster repolarization (Bidoggia et al., 2000b). In orchiectomized rabbits testosterone administration shortens the QT interval and

Gender differences in the ST segment are known for healthy subjects and the ST level is elevated in young males compared to females (Ezaki et al., 2010). Also females showed longer JT intervals than males. These differences of ventricular repolarization are not observed in early childhood, but they become apparent after puberty (Ezaki et al., 2010), suggesting an important role of sex hormones. After puberty the leads that represent the right and left ventricles show that the J point amplitude is higher and besides that the ST

Brugada syndrome has higher incidence in young males than in females and causes sudden death by ventricular fibrillation: in the early phase of the ventricular repolarization of males, the transient outward potassium current Ito is stronger and contributes more largely to the repolarisation of the right ventricular epicardium. In the early repolarization syndrome an important elevation of the J wave and ST-segment is detected in males (Ezaki et al., 2010). In pre-pubescent subjects of age 5–12 years, there is no significant gender differences in the ST levels. In males, the ST levels from both leads increase significantly after puberty and reach a maximum after 20 to 29 years of age and decrease during the 3rd decade of life (Nankin & Calkins, 1986). While for females, with increasing age there was a reduction in lead V5 only and irrespective of female age, from lead V2 the ST levels remained low and almost constant. For both sexes, all 3 ST segments were significantly higher in lead V2, which shows a more potent left ventricle. After puberty, the ST levels from both leads V2 and V5 were significantly higher in males than in females, suggesting that the effect of sex hormones on ST levels might be smaller in females than in males (Ezaki et al., 2010). In males, androgen-deprivation therapy significantly lowered all 3 ST segments and they closely resembled the ST segments of age-matched control females (Ezaki et al., 2010). Further, the J point amplitude was significantly lower in males with secondary

These significant age- and gender differences in the ST segment suggest that sex hormones modulate the early phase of ventricular repolarization (Ezaki et al., 2010). For example, in healthy adults, estrogen prolongs the repolarization (ST segment), while testosterone shortens it (James et al., 2007). Sex differences in the ST segment elevation showed an important role for the male hormone testosterone. Since the plasma testosterone concentration increases around puberty, reaches peaks at 20–30 years of age, and decreases gradually due to the physiologic effects of aging in both males and females, the ST segment

(James et al., 2007), and several experimental studies support this hypothesis.

drug-induced QT prolongation (Liu et al., 2002).

**4.1.3 Gender related ST differences** 

segment and angle is steeper in males.

hypogonadism and in castrated males.

combined activity of two delayed rectifier currents IKr (rapid delayed rectifier potassium channel) and IKs (slow delayed potassium channel) account for the majority of phase 3 repolarization of the ventricles. Several mutations in genes regulating these channels are responsible for the more common forms of inherited long QT syndromes. But also acquired conditions such as cardiac disease, electrolyte derangements (e.g. hypokalemia, hypocalcemia), and renal insufficiency (Genovesi et al., 2008), and iatrogenic causes, as cardiac as well as non-cardiac drugs are known to prolong the QT interval (Roden, 2004). Moreover, Iks, but not Ikr, is highly influenced by β-adrenergic stimulation and blockade.

#### **4.1.2 Gender related QTc differences**

In men left ventricular mass is greater than in women (Hayward et al., 2001) and besides, the hearts from a small number of young and middle-aged non-diseased women showed reduced expression of a variety of K+ channel subunits (HERG, mink, Kir2.3, Kv1.4, Kir6.2, KchIP2, SUR2) as compared to male hearts (Gaborit et al., 2010). In general, the QT interval durations of men are generally shorter than of women. The androgen receptors expressed in the heart muscle cells might play an important role in gender-dependent heart function differences, in particular the electrical activity of the left ventriculum. The QT interval at birth is comparable between genders (Stramba-Badiale et al., 1995) and up to 10 years of age, then at puberty it shortens by some 20 msec in young males (Pham & Rosen, 2002). The normal upper limit for QTc in men is 440 msec (Schwartz et al., 2011) and the shorter the QT interval the more it protects men from developing malignant ventricular arrhythmias such as Torsade de pointes (Abi-Gerges et al., 2004). Women which have a faster heart rate and thus a shorter QT interval show a higher incidence of Torsade de pointes. So the QT differences can be attributed to gender.

In women, repolarization lasts longer and proceeds slower compared with men and, indeed, surface ECG reveals longer QT interval and lower T-wave amplitude in adult women of all ages as compared with men (Bidoggia et al., 2000a). Also, the method of Fridericia to correct the QT intervals for heart rate (QTc) showed longer QTc intervals in women (about 1%) compared with men (Kadish et al., 2004; Schwartz et al., 2011). Moreover, the gender difference in QT interval is larger at long cardiac cycle lengths (Genovesi et al., 2007). The gender-related differences in QTc interval and T-wave amplitude are not present at birth (Stramba-Badiale et al., 1995) and during childhood, but appear during the teenage years (Pham & Rosen, 2002; Surawicz & Parikh, 2002), and decrease at older ages suggesting that this gender related QT difference of cardiac repolarization can be attributed to the life cycle: first an increase followed by a decrease of the sex hormones. The average sex differences in QTc intervals range from 10–15 ms (approximately 2-6 %). Besides, the longitudinal assessment of QT interval is independent of the menstrual cycle of women (Burke et al., 1996; Surawicz & Parikh, 2002), whereas in men the QT intervals shorten by some 20 msec at puberty (Surawicz & Parikh, 2002), and in both men and women testosterone levels directly shorten the QT interval independently of the heart rate (Schwartz et al., 2011), with differences in men of about 4% between high and low testosterone levels (Charbit et al., 2009; Pecori Giraldi et al., 2010). Further, these differences were larger than expected in older men and women during decreased hormonal status, where for women, testosterone decreased QT and QTc intervals but were longer in comparison with men (Schwartz et al., 2011). Moreover, a direct shortening of QT intervals by testosterone in older men and older women or hypogonadal status is known to exist independent of heart rate changes

combined activity of two delayed rectifier currents IKr (rapid delayed rectifier potassium channel) and IKs (slow delayed potassium channel) account for the majority of phase 3 repolarization of the ventricles. Several mutations in genes regulating these channels are responsible for the more common forms of inherited long QT syndromes. But also acquired conditions such as cardiac disease, electrolyte derangements (e.g. hypokalemia, hypocalcemia), and renal insufficiency (Genovesi et al., 2008), and iatrogenic causes, as cardiac as well as non-cardiac drugs are known to prolong the QT interval (Roden, 2004). Moreover, Iks, but not Ikr, is highly influenced by β-adrenergic stimulation and blockade.

In men left ventricular mass is greater than in women (Hayward et al., 2001) and besides, the hearts from a small number of young and middle-aged non-diseased women showed reduced expression of a variety of K+ channel subunits (HERG, mink, Kir2.3, Kv1.4, Kir6.2, KchIP2, SUR2) as compared to male hearts (Gaborit et al., 2010). In general, the QT interval durations of men are generally shorter than of women. The androgen receptors expressed in the heart muscle cells might play an important role in gender-dependent heart function differences, in particular the electrical activity of the left ventriculum. The QT interval at birth is comparable between genders (Stramba-Badiale et al., 1995) and up to 10 years of age, then at puberty it shortens by some 20 msec in young males (Pham & Rosen, 2002). The normal upper limit for QTc in men is 440 msec (Schwartz et al., 2011) and the shorter the QT interval the more it protects men from developing malignant ventricular arrhythmias such as Torsade de pointes (Abi-Gerges et al., 2004). Women which have a faster heart rate and thus a shorter QT interval show a higher incidence of Torsade de pointes. So the QT

In women, repolarization lasts longer and proceeds slower compared with men and, indeed, surface ECG reveals longer QT interval and lower T-wave amplitude in adult women of all ages as compared with men (Bidoggia et al., 2000a). Also, the method of Fridericia to correct the QT intervals for heart rate (QTc) showed longer QTc intervals in women (about 1%) compared with men (Kadish et al., 2004; Schwartz et al., 2011). Moreover, the gender difference in QT interval is larger at long cardiac cycle lengths (Genovesi et al., 2007). The gender-related differences in QTc interval and T-wave amplitude are not present at birth (Stramba-Badiale et al., 1995) and during childhood, but appear during the teenage years (Pham & Rosen, 2002; Surawicz & Parikh, 2002), and decrease at older ages suggesting that this gender related QT difference of cardiac repolarization can be attributed to the life cycle: first an increase followed by a decrease of the sex hormones. The average sex differences in QTc intervals range from 10–15 ms (approximately 2-6 %). Besides, the longitudinal assessment of QT interval is independent of the menstrual cycle of women (Burke et al., 1996; Surawicz & Parikh, 2002), whereas in men the QT intervals shorten by some 20 msec at puberty (Surawicz & Parikh, 2002), and in both men and women testosterone levels directly shorten the QT interval independently of the heart rate (Schwartz et al., 2011), with differences in men of about 4% between high and low testosterone levels (Charbit et al., 2009; Pecori Giraldi et al., 2010). Further, these differences were larger than expected in older men and women during decreased hormonal status, where for women, testosterone decreased QT and QTc intervals but were longer in comparison with men (Schwartz et al., 2011). Moreover, a direct shortening of QT intervals by testosterone in older men and older women or hypogonadal status is known to exist independent of heart rate changes

**4.1.2 Gender related QTc differences** 

differences can be attributed to gender.

(Schwartz et al., 2011). Male hypogonadism is associated with an increased prevalence of prolonged QT interval, over 2.5%, as compared to the control and the healthy population (Schwartz et al., 1993), and hence, a higher risk for fatal ventricular arrhythmias. The QTc interval in the hypogonadal state is prolonged and by hormone replacement therapy normalized (Pecori Giraldi et al., 2010). This evidence led to the conclusion that testosterone is the main determinant of gender-related differences in the ventricular refractory periods (James et al., 2007), and several experimental studies support this hypothesis.

In vitro and data on animals showed that sex hormones increased the QTc intervals in females, or conversely, decreased the QTc intervals in males (Fülöp et al., 2006). Women with excess androgen secretion present shorter and faster repolarization (Bidoggia et al., 2000b). In orchiectomized rabbits testosterone administration shortens the QT interval and drug-induced QT prolongation (Liu et al., 2002).

#### **4.1.3 Gender related ST differences**

Gender differences in the ST segment are known for healthy subjects and the ST level is elevated in young males compared to females (Ezaki et al., 2010). Also females showed longer JT intervals than males. These differences of ventricular repolarization are not observed in early childhood, but they become apparent after puberty (Ezaki et al., 2010), suggesting an important role of sex hormones. After puberty the leads that represent the right and left ventricles show that the J point amplitude is higher and besides that the ST segment and angle is steeper in males.

Brugada syndrome has higher incidence in young males than in females and causes sudden death by ventricular fibrillation: in the early phase of the ventricular repolarization of males, the transient outward potassium current Ito is stronger and contributes more largely to the repolarisation of the right ventricular epicardium. In the early repolarization syndrome an important elevation of the J wave and ST-segment is detected in males (Ezaki et al., 2010).

In pre-pubescent subjects of age 5–12 years, there is no significant gender differences in the ST levels. In males, the ST levels from both leads increase significantly after puberty and reach a maximum after 20 to 29 years of age and decrease during the 3rd decade of life (Nankin & Calkins, 1986). While for females, with increasing age there was a reduction in lead V5 only and irrespective of female age, from lead V2 the ST levels remained low and almost constant. For both sexes, all 3 ST segments were significantly higher in lead V2, which shows a more potent left ventricle. After puberty, the ST levels from both leads V2 and V5 were significantly higher in males than in females, suggesting that the effect of sex hormones on ST levels might be smaller in females than in males (Ezaki et al., 2010). In males, androgen-deprivation therapy significantly lowered all 3 ST segments and they closely resembled the ST segments of age-matched control females (Ezaki et al., 2010). Further, the J point amplitude was significantly lower in males with secondary hypogonadism and in castrated males.

These significant age- and gender differences in the ST segment suggest that sex hormones modulate the early phase of ventricular repolarization (Ezaki et al., 2010). For example, in healthy adults, estrogen prolongs the repolarization (ST segment), while testosterone shortens it (James et al., 2007). Sex differences in the ST segment elevation showed an important role for the male hormone testosterone. Since the plasma testosterone concentration increases around puberty, reaches peaks at 20–30 years of age, and decreases gradually due to the physiologic effects of aging in both males and females, the ST segment

2002).

1997).

**5.1.1 ERα**

women (Lizotte et al., 2009).

protection is dedicated to direct estrogen effects.

**5.1 Estrogen receptors** 

The Role of Sex Hormones in the Cardiovascular System 45

against cell death during ischemia and decreases the extent of cell death (Wise et al., 2001). Testosterone is known to act via nuclear receptors and regulate protein synthesis (Reid et al., 2003), and experimental data also indicates a non-genomic pathway of testosterone action on the cardiovascular system, i.e. direct testosterone mediated vasodilatation (English et al.,

At present, there are three known estrogen receptors. The two »classical« nuclear estrogen receptors, ERα and ERβ, are ligand-activated nuclear transcription factors that bind regulatory response elements in the promoters of genes (Carroll & Brown, 2006). The third estrogen receptor, GPER1 or GPER (previously GPR30) was identified as an orphan 7 transmembrane G protein-coupled receptor (GPCR) with low homology to other known GPCRs (Carmeci et al., 1997; Kvingedal & Smeland, 1997; O'Dowd et al., 1998; Takada et al.,

Although the distinction between the modes of cellular activation, rapid signaling versus transcription, there exists extensive overlap between these artificially defined categories. Classical estrogen receptors are traditionally thought of as regulators of transcription, however, there is extensive evidence of their ability to mediate rapid signaling events (Moriarty et al., 2006). In addition, rapid signaling events, whether initiated by nuclear steroid receptors, growth factor receptors or GPCRs, result in the modification of transcriptional activity of conventional transcription factors (Ma & Pei, 2007). Thus the cellular effects of estrogen will depend on the specific receptors expressed and the

In cardiomyocytes ERα is distributed in the cytosolic, nuclear and membrane compartments (Lizotte et al., 2009), in T-tubular membranes (Ropero et al., 2006) and in the caveolae (Chung et al., 2009), which suggests that the ERα is localized in these complexes as an estrogen non-genomic rapid signaling, and its cytosolic and nuclear distribution suggest a genomic signaling. Moreover, ERα is more densely expressed in ventricular tissue as compared to the atrium, having higher densities in men than in

Estradiol induces the translocation of ERα to the PI3K regulatory domain and results in endothelial NOS (eNOS) activation (Simoncini et al., 2000). In an in vivo rabbit ischemia/reperfusion (I/R) model, acute treatment with estradiol significantly decreases the infarct size in female hearts after ischemia (Booth et al., 2005), suggesting that activation of ERα is required for the acute cardioprotective effects of estrogen. Also an in vivo study with ovariectomized female rats found that acute estrogen-mediated cardioprotection, following I/R, is mimicked by pretreatment with an ERα agonist and unaffected by ERβ antagonist pretreatment (Jeanes et al., 2008). In a model of ERα knockout mice cardioprotection in the ischemia model was blocked in female animals (Zhai et al., 2000). Subchronical estradiol pretreated female rats showed cardioprotection comparable, but lower, than the protection of directly applied estradiol (Kuhar et al., 2007). In conclusion, the importance of ERα in cardioprotection is confirmed in many models, despite being controversial and more

integration of their stimulatory and inhibitory signaling pathways.

fluctuation with age is correlated to the level of testosterone. In contrast, the female sex hormone, which also increases around puberty, has little effect on the ST levels because it does not change the ST segment in pubescent females (Ezaki et al., 2010).

**ST-J**: The J point level at the end of the QRS complex with respect to the baseline. The middle or **ST-M** level is the level at 1/16th of the preceding RR interval of the following ST segment. **ST-E**: The level at 2/16th of the preceding RR interval of the following ST segment. T-wave amplitude is defined as the absolute distance from the baseline from the S point to the apex of the T-wave. In men the level of repolarizarion (ST) is elevated in all 3 segments and the duration is shortened (blue hatched line), while testosterone deprivation therapy in men decreases the elevation of these segments. The duration of the repolarization (ST) is prolonged by estrogens (red hatched line). The method of Fridericia for calculating the ratecorrected QT interval: QTc=QT/RR1/3 is described in text. Adopted from Ezaki et al. (2010) and James et al. (2007).

#### **5. Sex hormone receptors and their role in the cardiovascular system**

Androgen receptors are expressed both in the atria and the ventricles, while estrogen receptors are more pronounced in atrial myocytes (Lizotte et al., 2009). Anti-androgen drugs inhibit the androgen receptors of cardiac ventricular myocytes and decrease the hormonal modulation of ventricular repolarization (Ezaki et al., 2010). The actions of estrogen are mediated through (1) receptor-α and –β, (2) an unknown cytosolic membrane receptor and (3) another membrane based GPER1. ERα directly modulates transcription of target genes through two activation functions, AF1 and AF2. ERα demonstrates to have a prominent role in vascular biology, i.e., an AF1-deficient ERα isoform can be physiologically expressed in the endothelium and appears sufficient to mediate most of the vasculoprotective actions of estradiol (Arnal et al., 2010). Pretreatment of cardiac myocytes with estradiol protects against cell death during ischemia and decreases the extent of cell death (Wise et al., 2001). Testosterone is known to act via nuclear receptors and regulate protein synthesis (Reid et al., 2003), and experimental data also indicates a non-genomic pathway of testosterone action on the cardiovascular system, i.e. direct testosterone mediated vasodilatation (English et al., 2002).

#### **5.1 Estrogen receptors**

44 Sex Hormones

fluctuation with age is correlated to the level of testosterone. In contrast, the female sex hormone, which also increases around puberty, has little effect on the ST levels because it

**ST-J**: The J point level at the end of the QRS complex with respect to the baseline. The middle or **ST-M** level is the level at 1/16th of the preceding RR interval of the following ST segment. **ST-E**: The level at 2/16th of the preceding RR interval of the following ST segment. T-wave amplitude is defined as the absolute distance from the baseline from the S point to the apex of the T-wave. In men the level of repolarizarion (ST) is elevated in all 3 segments and the duration is shortened (blue hatched line), while testosterone deprivation therapy in men decreases the elevation of these segments. The duration of the repolarization (ST) is prolonged by estrogens (red hatched line). The method of Fridericia for calculating the ratecorrected QT interval: QTc=QT/RR1/3 is described in text. Adopted from Ezaki et al. (2010)

**5. Sex hormone receptors and their role in the cardiovascular system** 

Androgen receptors are expressed both in the atria and the ventricles, while estrogen receptors are more pronounced in atrial myocytes (Lizotte et al., 2009). Anti-androgen drugs inhibit the androgen receptors of cardiac ventricular myocytes and decrease the hormonal modulation of ventricular repolarization (Ezaki et al., 2010). The actions of estrogen are mediated through (1) receptor-α and –β, (2) an unknown cytosolic membrane receptor and (3) another membrane based GPER1. ERα directly modulates transcription of target genes through two activation functions, AF1 and AF2. ERα demonstrates to have a prominent role in vascular biology, i.e., an AF1-deficient ERα isoform can be physiologically expressed in the endothelium and appears sufficient to mediate most of the vasculoprotective actions of estradiol (Arnal et al., 2010). Pretreatment of cardiac myocytes with estradiol protects

does not change the ST segment in pubescent females (Ezaki et al., 2010).

Fig. 4. Electrocardiogram of the heart

and James et al. (2007).

At present, there are three known estrogen receptors. The two »classical« nuclear estrogen receptors, ERα and ERβ, are ligand-activated nuclear transcription factors that bind regulatory response elements in the promoters of genes (Carroll & Brown, 2006). The third estrogen receptor, GPER1 or GPER (previously GPR30) was identified as an orphan 7 transmembrane G protein-coupled receptor (GPCR) with low homology to other known GPCRs (Carmeci et al., 1997; Kvingedal & Smeland, 1997; O'Dowd et al., 1998; Takada et al., 1997).

Although the distinction between the modes of cellular activation, rapid signaling versus transcription, there exists extensive overlap between these artificially defined categories. Classical estrogen receptors are traditionally thought of as regulators of transcription, however, there is extensive evidence of their ability to mediate rapid signaling events (Moriarty et al., 2006). In addition, rapid signaling events, whether initiated by nuclear steroid receptors, growth factor receptors or GPCRs, result in the modification of transcriptional activity of conventional transcription factors (Ma & Pei, 2007). Thus the cellular effects of estrogen will depend on the specific receptors expressed and the integration of their stimulatory and inhibitory signaling pathways.

#### **5.1.1 ERα**

In cardiomyocytes ERα is distributed in the cytosolic, nuclear and membrane compartments (Lizotte et al., 2009), in T-tubular membranes (Ropero et al., 2006) and in the caveolae (Chung et al., 2009), which suggests that the ERα is localized in these complexes as an estrogen non-genomic rapid signaling, and its cytosolic and nuclear distribution suggest a genomic signaling. Moreover, ERα is more densely expressed in ventricular tissue as compared to the atrium, having higher densities in men than in women (Lizotte et al., 2009).

Estradiol induces the translocation of ERα to the PI3K regulatory domain and results in endothelial NOS (eNOS) activation (Simoncini et al., 2000). In an in vivo rabbit ischemia/reperfusion (I/R) model, acute treatment with estradiol significantly decreases the infarct size in female hearts after ischemia (Booth et al., 2005), suggesting that activation of ERα is required for the acute cardioprotective effects of estrogen. Also an in vivo study with ovariectomized female rats found that acute estrogen-mediated cardioprotection, following I/R, is mimicked by pretreatment with an ERα agonist and unaffected by ERβ antagonist pretreatment (Jeanes et al., 2008). In a model of ERα knockout mice cardioprotection in the ischemia model was blocked in female animals (Zhai et al., 2000). Subchronical estradiol pretreated female rats showed cardioprotection comparable, but lower, than the protection of directly applied estradiol (Kuhar et al., 2007). In conclusion, the importance of ERα in cardioprotection is confirmed in many models, despite being controversial and more protection is dedicated to direct estrogen effects.

The Role of Sex Hormones in the Cardiovascular System 47

reduced activation of the PI3K/Akt proteins (Wang et al., 2009). In ovariectomized female mice long-term treatment with ERβ agonists has been shown to be cardioprotective and reduce I/R injury. The gene profiling of this experimental model showed that a number of protective genes were upregulated, i.e., encoding the NO biosynthesis and the antiapoptotic proteins. Through activation of ERβ, estrogen plays a cardioprotective role against I/R injury (Nikolic et al., 2007). Other reported that estrogen-mediated cardioprotection following I/R is unaffected by an ERβ antagonist and is activated by ERα agonists (Jeanes et al., 2008). The chronic treatment with estradiol and/or ERβ activation leads to activation of protein-S nitrosylation and cardioprotection, which could be blocked by NOS inhibition (Lin et al., 2009), suggesting that chronic estrogen exposure protects the hearts largely via

The G protein-coupled estrogen receptor (GPR30 or now GPER-1) was initially identified as an orphan G-protein coupled receptor (GPCR), and were traditionally recognized as mediating rapid changes in the levels of second messengers and to regulate various pathways of kinases (Luttrell, 2006). Then estrogen was identified as an endogenous ligand, and estradiol binding to GPER1 was found to result in Gbg activation of Src and resulting in the matrix metalloproteinase (MMP) cleavage of heparan-bound epidermal growth factor (EGF). Subsequently, the activated EGF receptor results in acute PI3K and ERK activation (Filardo et al., 2000). Thus, as a transmembrane estrogen receptor, GPER1 activation may mediate rapid cell signaling (Prossnitz et al., 2008). GPER1 is expressed in both the ventricles and the atria of the human heart, although more in ventricles than in atria, and besides, in the atrioventricular sinus and aorta. It is absent in the atrioventricular node and

GPER1 appears to be present in all tissues where ischemic injuries takes place. Under hypoxic conditions the up-regulation of GPER1 in estrogen receptor-negative HL-1 cardiomyocytes is activated by HIF-1-responsive elements located within the promoter region of GPER1 (Recchia et al., 2011), and required for this cardiomyocyte pathway is the ROS-induced activation of EGFR/ERK signaling. Thus the adaptive cell responses to hypoxia induced by estrogen are most likely GPER mediated (Recchia et al., 2011). In Langendoff perfused male and female rat hearts, the acute activation of GPER1 by its specific agonist G-1 reduced the myocardial infarct size and improved the functional recovery of contractility when compared to control (Deschamps & Murphy, 2009). Besides, less myocardial inflammation was found, indicated by decreased levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6 (Wang et al., 2006). Also increased phosphorylation of both Akt and ERK were found, which could be reversed by the use of the PI3K/Akt inhibitor (Filardo et al., 2000). Moreover, the mitochondria permeability transition pore opening was inhibited through the activation of the extracellular signal regulated kinase (ERK) pathway (Bopassa et al., 2010). Further, administration of G1 prior to global ischemia also improved the cardiac contractility function, while the improvements were abolished both by co-administration of GPER1 specific antibodies, or an inhibitor of the PI3K pathway (Deschamps & Murphy, 2009). Further, the acute administration of G-1 causes the activation of

the ERK pathway, inducing phosphorylation of eNOS (Filice et al., 2009).

activation of ERβ and NO signaling.

in the heart apex (Lizotte et al., 2009).

**5.2.1 GPER1 and ischemia** 

**5.2 GPER receptors** 

Fig. 5. Overview of genomic and non-genomic action of estrogen receptors Estradiol stimulates cytoplasmic as well as nuclear signaling. Estradiol (E2) binds to the estrogen receptors (ERα, ERβ), stabilizes ER dimers, and stimulates direct interaction with growth factor receptors (GFR), association with proto-oncogenic tyrosine kinase Src (c-Src) and adaptor molecules: modulator of non-genomic activity of the estrogen receptor (MNAR), apoptotic protein (Shc), cellular apoptosis susceptibility protein, an exportin (Cas), and stimulation of common cytoplasmic signaling pathways via phosphoinositide-3 protein kinase (PI3K). ER can also initiate gene transcription in the absence of estradiol via phosphorylation (circled P) and activation of receptors and coactivators (cAMP-response element-binding protein - CBP), by growth factor signaling cascades, or by a ligandstimulated mechanism. Testosterone stimulates, probably similarly to estrogens, both cytoplasmic and nuclear androgen receptors. Adopted from Fox et al. (2009) and Huang et al. (2010) and Wu et al. (2011).

#### **5.1.2 ERβ**

ERβ is predominantly localized in the nucleus and cytosol of adult murine cardiomyocytes (Lizotte et al., 2009), and has been reported to be localized in the mitochondria (Yang et al., 2004). Being primarily found in the sarcolemma, the possible ERβ-mediated effects will depend mostly on gene transcription. ERβ is evenly distributed in the heart (Lizotte et al., 2009), with females showing a higher density than males. Direct application of a specific ERβ agonist showed no antiischemic effects (Booth et al., 2005) thus suggesting that acute activation of ERβ is lacking cardioprotection.

Most studies have found that female ERβ knockout mice had more I/R injury than control (Wang et al., 2008). However, the knockouts showed increased damage, i.e., decreased gene expression of fatty acids and nitric oxide (NO) production (Gabel et al., 2005), and further reduced activation of the PI3K/Akt proteins (Wang et al., 2009). In ovariectomized female mice long-term treatment with ERβ agonists has been shown to be cardioprotective and reduce I/R injury. The gene profiling of this experimental model showed that a number of protective genes were upregulated, i.e., encoding the NO biosynthesis and the antiapoptotic proteins. Through activation of ERβ, estrogen plays a cardioprotective role against I/R injury (Nikolic et al., 2007). Other reported that estrogen-mediated cardioprotection following I/R is unaffected by an ERβ antagonist and is activated by ERα agonists (Jeanes et al., 2008). The chronic treatment with estradiol and/or ERβ activation leads to activation of protein-S nitrosylation and cardioprotection, which could be blocked by NOS inhibition (Lin et al., 2009), suggesting that chronic estrogen exposure protects the hearts largely via activation of ERβ and NO signaling.

#### **5.2 GPER receptors**

46 Sex Hormones

Fig. 5. Overview of genomic and non-genomic action of estrogen receptors

Estradiol stimulates cytoplasmic as well as nuclear signaling. Estradiol (E2) binds to the estrogen receptors (ERα, ERβ), stabilizes ER dimers, and stimulates direct interaction with growth factor receptors (GFR), association with proto-oncogenic tyrosine kinase Src (c-Src) and adaptor molecules: modulator of non-genomic activity of the estrogen receptor

kinase (PI3K). ER can also initiate gene transcription in the absence of estradiol via phosphorylation (circled P) and activation of receptors and coactivators (cAMP-response element-binding protein - CBP), by growth factor signaling cascades, or by a ligandstimulated mechanism. Testosterone stimulates, probably similarly to estrogens, both cytoplasmic and nuclear androgen receptors. Adopted from Fox et al. (2009) and Huang et

al. (2010) and Wu et al. (2011).

activation of ERβ is lacking cardioprotection.

**5.1.2 ERβ**

(MNAR), apoptotic protein (Shc), cellular apoptosis susceptibility protein, an exportin (Cas), and stimulation of common cytoplasmic signaling pathways via phosphoinositide-3 protein

ERβ is predominantly localized in the nucleus and cytosol of adult murine cardiomyocytes (Lizotte et al., 2009), and has been reported to be localized in the mitochondria (Yang et al., 2004). Being primarily found in the sarcolemma, the possible ERβ-mediated effects will depend mostly on gene transcription. ERβ is evenly distributed in the heart (Lizotte et al., 2009), with females showing a higher density than males. Direct application of a specific ERβ agonist showed no antiischemic effects (Booth et al., 2005) thus suggesting that acute

Most studies have found that female ERβ knockout mice had more I/R injury than control (Wang et al., 2008). However, the knockouts showed increased damage, i.e., decreased gene expression of fatty acids and nitric oxide (NO) production (Gabel et al., 2005), and further The G protein-coupled estrogen receptor (GPR30 or now GPER-1) was initially identified as an orphan G-protein coupled receptor (GPCR), and were traditionally recognized as mediating rapid changes in the levels of second messengers and to regulate various pathways of kinases (Luttrell, 2006). Then estrogen was identified as an endogenous ligand, and estradiol binding to GPER1 was found to result in Gbg activation of Src and resulting in the matrix metalloproteinase (MMP) cleavage of heparan-bound epidermal growth factor (EGF). Subsequently, the activated EGF receptor results in acute PI3K and ERK activation (Filardo et al., 2000). Thus, as a transmembrane estrogen receptor, GPER1 activation may mediate rapid cell signaling (Prossnitz et al., 2008). GPER1 is expressed in both the ventricles and the atria of the human heart, although more in ventricles than in atria, and besides, in the atrioventricular sinus and aorta. It is absent in the atrioventricular node and in the heart apex (Lizotte et al., 2009).

#### **5.2.1 GPER1 and ischemia**

GPER1 appears to be present in all tissues where ischemic injuries takes place. Under hypoxic conditions the up-regulation of GPER1 in estrogen receptor-negative HL-1 cardiomyocytes is activated by HIF-1-responsive elements located within the promoter region of GPER1 (Recchia et al., 2011), and required for this cardiomyocyte pathway is the ROS-induced activation of EGFR/ERK signaling. Thus the adaptive cell responses to hypoxia induced by estrogen are most likely GPER mediated (Recchia et al., 2011). In Langendoff perfused male and female rat hearts, the acute activation of GPER1 by its specific agonist G-1 reduced the myocardial infarct size and improved the functional recovery of contractility when compared to control (Deschamps & Murphy, 2009). Besides, less myocardial inflammation was found, indicated by decreased levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6 (Wang et al., 2006). Also increased phosphorylation of both Akt and ERK were found, which could be reversed by the use of the PI3K/Akt inhibitor (Filardo et al., 2000). Moreover, the mitochondria permeability transition pore opening was inhibited through the activation of the extracellular signal regulated kinase (ERK) pathway (Bopassa et al., 2010). Further, administration of G1 prior to global ischemia also improved the cardiac contractility function, while the improvements were abolished both by co-administration of GPER1 specific antibodies, or an inhibitor of the PI3K pathway (Deschamps & Murphy, 2009). Further, the acute administration of G-1 causes the activation of the ERK pathway, inducing phosphorylation of eNOS (Filice et al., 2009).

The Role of Sex Hormones in the Cardiovascular System 49

Fig. 6. Signaling pathways activated by estrogens and androgens. Adopted from Meyer et al.

Specific cellular actions of estradiol (E2) are activated through both genomic and nongenomic transduction pathways. In the primary genomic pathway (left), activated estrogen receptors (ER) influence the nuclear transcription as well as rapid signaling by nitric oxide (NO) and phosphoinositide-3 protein kinase (PI3K) /Akt activation. In the secondary (center) non-genomic pathway, stimulation of membrane bound G protein-coupled estrogen receptor (GPER1) activates G proteins, which trigger multiple effectors. Both pathways activate Gαi/o proteins and activate adenylate cyclase (AC) to either, positively or negatively

regulate the cAMP level and the subsequent cAMP-dependent protein kinase (PKA) activity. Also, the stimulation of Gαi/o leads to activation of PI3K and subsequent Akt/PKB protein kinase (Akt/PKB). Activation of c-Src protein kinase, forming the complex with adaptor protein, activaties matrix metalloproteinase (MMP), that liberate heparin-bound epidermal growth factor (HB-EGF) and activates the EGF receptor (EGFR). The EGFR activation leads to multiple downstream events including activation of mitogen activated protein kinases (MAPK) and PI3 kinases (PI3K), which increase expression of transcription factors (TFs). Besides, the GPER1 stimulation also leads to elevation of intracellular Ca2+ through unknown mechanisms that involve either primary signaling through G proteins or secondary signaling through EGFR transactivation. Moreover, the estrogen stimulation can lead to the expression of target genes whose promoters do not contain steroid response elements (nonEREs). The combined effects of these signaling and transcriptional events

The androgen receptors (AR, right) are known to activate hypoxia induced factors (HIF), vascular endothelium growth factor (VEGF) and effects resulting in activation of protein kinase C (PKC), and besides AR activation leads to deactivation of PI3K, thus leading also to diminished cardioprotection, and direct effects on calcium metabolism is also described.

often lead to cell cycle progression and cell proliferation.

(2009) and Nilsson et al. (2011).

In ovariectomized rats, G-1 was able to prevent an elevation in systolic blood pressure that occurs due to estrogen depletion, and in a GPER1 deficient mouse model, female mice develop a significant increase in mean arterial blood pressure (Mårtensson et al., 2009).

#### **5.2.2 GPER1 and other pathways**

Under hypoxic conditions the GPER1 may also mediate additional effects that are separate from those of both genomic and nongenomic classic ERs signaling. From isolated female mouse hearts with knockout mutations for ERα or ERβ, the role for both of these receptors in mediating the protective effects of estrogen through GPER1 against I/R injury was established (Wang et al., 2009; Weil et al., 2010). The upregulation of protein kinase A and the following inhibition of apoptosis was contributed to GPER1 and not to the ER. The acute administration of estradiol in the isolated rat heart was protective against acute I/R injury, and both ERα and/or ERβ may mediate these effects. ERβ mediates the upregulation of extracellular receptor kinase (ERK)-signaling and the antiapoptotic PI3K/Akt pathways and ERα mediates the downregulation of proapoptotic c-Jun N-terminal kinase (JNK) pathways. The GPER1 activation provides cardioprotection by decreasing inflammation, including activation of proinflammatory cellular pathways, upregulation of protective mitogen activated kinase (p38 MAPK) and/or JNK pathways (Weil et al., 2010), in addition to decreasing cellular apoptosis, and to promote survival.

#### **5.2.3 GPER mediates cardioprotection**

These results show that the G protein estrogen receptor GPER1 plays an important role in mediating the acute cardioprotective effects of estrogen against global ischemia/reperfusion. They can be upregulated by ischemia and mediate protection through adaptation to low oxygen and reactive oxygen species generation conditions and may contribute to progression of disease in the metabolic function, impulse cell proliferation and improve the contractility of myocites (Patel et al., 2010; Recchia et al., 2011). It is suggested that these mechanisms of protection by GPER1 activation are mediated through the EGF receptor/extracellular signal regulated kinase (ERK) and the PI3K/AKT, and eNOS signaling pathways (Filardo et al., 2000; Filice et al., 2009).

Just like ERα and ERβ the membrane estrogen receptor GPER1 is involved in estradiol induced cardiac activity. The cardiotropic effects induced by estrogen include the ERK, PI3K and PKA transduction cascades. A potential functional interactivity between GPER1, ERα and ERβ, might exert their combined cardioprotection, and involves all of the ERK, PI3K, PKA pathways and converge downstream on the eNOS transduction pathway, suggesting that NO production plays a central role in the response of male heart to estrogen stimulation (Filice et al., 2009).

#### **5.3 Androgens**

#### **5.3.1 Angiogenesis**

The androgen receptors have independent of their genomic function, which changes gene transcription, a second mode of action, in which cytoplasmic androgen binding to androgen receptors causes rapid changes in cell function, such as changes in ion transport. The most potent natural androgen is dihydrotestosterone (DHT), that in contrary to testosterone, cannot be aromatised to estradiol and thus no secondary estrogen receptor mediated effects can be produced. Endothelial cells exposed to DHT produce a dose-dependent increase in

In ovariectomized rats, G-1 was able to prevent an elevation in systolic blood pressure that occurs due to estrogen depletion, and in a GPER1 deficient mouse model, female mice develop a significant increase in mean arterial blood pressure (Mårtensson et al., 2009).

Under hypoxic conditions the GPER1 may also mediate additional effects that are separate from those of both genomic and nongenomic classic ERs signaling. From isolated female mouse hearts with knockout mutations for ERα or ERβ, the role for both of these receptors in mediating the protective effects of estrogen through GPER1 against I/R injury was established (Wang et al., 2009; Weil et al., 2010). The upregulation of protein kinase A and the following inhibition of apoptosis was contributed to GPER1 and not to the ER. The acute administration of estradiol in the isolated rat heart was protective against acute I/R injury, and both ERα and/or ERβ may mediate these effects. ERβ mediates the upregulation of extracellular receptor kinase (ERK)-signaling and the antiapoptotic PI3K/Akt pathways and ERα mediates the downregulation of proapoptotic c-Jun N-terminal kinase (JNK) pathways. The GPER1 activation provides cardioprotection by decreasing inflammation, including activation of proinflammatory cellular pathways, upregulation of protective mitogen activated kinase (p38 MAPK) and/or JNK pathways (Weil et al., 2010), in addition to

These results show that the G protein estrogen receptor GPER1 plays an important role in mediating the acute cardioprotective effects of estrogen against global ischemia/reperfusion. They can be upregulated by ischemia and mediate protection through adaptation to low oxygen and reactive oxygen species generation conditions and may contribute to progression of disease in the metabolic function, impulse cell proliferation and improve the contractility of myocites (Patel et al., 2010; Recchia et al., 2011). It is suggested that these mechanisms of protection by GPER1 activation are mediated through the EGF receptor/extracellular signal regulated kinase (ERK) and the PI3K/AKT, and eNOS

Just like ERα and ERβ the membrane estrogen receptor GPER1 is involved in estradiol induced cardiac activity. The cardiotropic effects induced by estrogen include the ERK, PI3K and PKA transduction cascades. A potential functional interactivity between GPER1, ERα and ERβ, might exert their combined cardioprotection, and involves all of the ERK, PI3K, PKA pathways and converge downstream on the eNOS transduction pathway, suggesting that NO production plays a central role in the response of male heart to estrogen stimulation

The androgen receptors have independent of their genomic function, which changes gene transcription, a second mode of action, in which cytoplasmic androgen binding to androgen receptors causes rapid changes in cell function, such as changes in ion transport. The most potent natural androgen is dihydrotestosterone (DHT), that in contrary to testosterone, cannot be aromatised to estradiol and thus no secondary estrogen receptor mediated effects can be produced. Endothelial cells exposed to DHT produce a dose-dependent increase in

**5.2.2 GPER1 and other pathways** 

decreasing cellular apoptosis, and to promote survival.

signaling pathways (Filardo et al., 2000; Filice et al., 2009).

**5.2.3 GPER mediates cardioprotection** 

(Filice et al., 2009).

**5.3 Androgens 5.3.1 Angiogenesis** 

Fig. 6. Signaling pathways activated by estrogens and androgens. Adopted from Meyer et al. (2009) and Nilsson et al. (2011).

Specific cellular actions of estradiol (E2) are activated through both genomic and nongenomic transduction pathways. In the primary genomic pathway (left), activated estrogen receptors (ER) influence the nuclear transcription as well as rapid signaling by nitric oxide (NO) and phosphoinositide-3 protein kinase (PI3K) /Akt activation. In the secondary (center) non-genomic pathway, stimulation of membrane bound G protein-coupled estrogen receptor (GPER1) activates G proteins, which trigger multiple effectors. Both pathways activate Gαi/o proteins and activate adenylate cyclase (AC) to either, positively or negatively regulate the cAMP level and the subsequent cAMP-dependent protein kinase (PKA) activity. Also, the stimulation of Gαi/o leads to activation of PI3K and subsequent Akt/PKB protein kinase (Akt/PKB). Activation of c-Src protein kinase, forming the complex with adaptor protein, activaties matrix metalloproteinase (MMP), that liberate heparin-bound epidermal growth factor (HB-EGF) and activates the EGF receptor (EGFR). The EGFR activation leads to multiple downstream events including activation of mitogen activated protein kinases (MAPK) and PI3 kinases (PI3K), which increase expression of transcription factors (TFs). Besides, the GPER1 stimulation also leads to elevation of intracellular Ca2+ through unknown mechanisms that involve either primary signaling through G proteins or secondary signaling through EGFR transactivation. Moreover, the estrogen stimulation can lead to the expression of target genes whose promoters do not contain steroid response elements (nonEREs). The combined effects of these signaling and transcriptional events often lead to cell cycle progression and cell proliferation.

The androgen receptors (AR, right) are known to activate hypoxia induced factors (HIF), vascular endothelium growth factor (VEGF) and effects resulting in activation of protein kinase C (PKC), and besides AR activation leads to deactivation of PI3K, thus leading also to diminished cardioprotection, and direct effects on calcium metabolism is also described.

The Role of Sex Hormones in the Cardiovascular System 51

Most of the present cardioprotective effects are dedicated to the NO-related mechanisms that play a role in the regulation of cardiovascular function. Beside the activation of cyclic guanosine monophosphate (cGMP)-dependent pathway, NO also regulates cell function through protein S-nitrosylation. This is a reversible redox-sensitive posttranslational protein modification, which involves the attachment of a NO moiety to the nucleophilic protein sulfhydryl, resulting in S-nitrosothiol (SNO) formation. It is very likely that the protein S-

Estrogen-induced protein S-nitrosylation has been shown to be involved in a murine model of I/R resulting in a cardioprotection (Lin et al., 2009). During the Langendorff model of I/R, hearts of ovariectomized female mice pretreated with estradiol and/or ERβ-selective agonists, showed increased post-ischemic recovery. This protection was blocked by a NOS inhibitor (Lin et al., 2009), suggesting that increased NO signaling contributes to the cardioprotection. These chonic estradiol or ERβ agonist exposed hearts showed increased Snitrosylated proteins and this protein S-nitrosylation could be abolished by pretreating hearts with the NOS inhibitor (Lin et al., 2009). These data suggest that chronic estrogen treatment and activation of ERβ would indeed lead to increased NO/SNO signaling,

Many of the S-nitrosylated proteins found in ERβ-selective agonist-treated hearts (Lin et al., 2009) have also been shown to be increased in preconditioned hearts (Sun et al., 2007), including the mitochondrial F1F0-ATPase, aconitase, malate dehydrogenase, creatine kinase, cytochrome c oxidase and heat shock proteins (HSP 27, 60, and 70). The protein Snitrosylation might also elicit cardioprotective effects by regulating intracellular Ca2+

Estradiol, the naturally occuring major form of estrogen, increases protein S-nitrosylation levels in cultured endothelial cells and besides in intact endotheliums where, the estradiol was shown to act through ERα, activates eNOS and generates NO which leads to Snitrosylation (Sun & Murphy, 2010). The S-nitrosylation also mediates the inhibitory effects of estradiol on endothelial ICAM-1 expression by angiotensin II (Chakrabarti et al., 2010). It is a post-translational protein modification induced by both, endogenous NO, generated by NOS in endothelium, and exogenous NO in a variety of cells. Over 100 different cellular proteins have been shown to be S-nitrosylated in processes that reduce the generation of harmful free radicals on one hand, and activate the free radical scavengers on the other (Chakrabarti et al., 2010). Both processes contributing to a reduction in oxidative stress, but also reducing pro-inflamatory signaling, processes which are crucial in I/R injury protection

The Akt pathway is considered as one of the most important molecular kinase that mediates cardioprotection during ischemia reperfusion (Wang et al., 2009). Female hearts have higher level of Akt activity and thus, compared to male hearts, have better protection against I/R injuries and better heart recovery. Indeed, male hearts showed lower Akt activity and worse I/R recovery. In ovariectomized rat hearts the Akt activity is reduced, the recovery decreased (Huang et al., 2010), and supposedly the occurance of apoptosis during the

handling, apoptosis, and post-infarct myocardial remodeling (Sun & Murphy, 2010).

nitrosylation plays an important role in cardioprotection (Sun & Murphy, 2010).

**6.1 NO and protein S-nitrosylation** 

playing an essential role in cardioprotection.

(Chakrabarti et al., 2010).

**6.2 PI3K/Akt pathway and its activation by sex hormones** 

myocardial infarction is one of the potential reasons.

the production of vascular endothelial growth factor (VEGF), a key angiogenic growth factor and show increased messenger RNA expression of VEGF receptors 1 and 2 (Flt-1 and KDR respectively, Sieveking et al., 2010). This suggests that the proangiogenic effects of DHT in male endothelial cells are VEGF dependent. Besides, it is known that KDR is the main mediator of the mitogenic/angiogenic action of VEGF in endothelial cells, while Flt-1 is a negative regulator of VEGF action and Flt-1 mRNA is indeed expressed less upon DHT exposure. This upregulated anti-VEGF action, and also the activated inhibitor of phosphoinositol 3-kinase (PI3K), a key enzyme in the PI3K– AKT pathway of VEGF signaling, both inhibited DHT-mediated tubulogenesis genes (Sieveking et al., 2010). In comparison, estrogen receptor α and β are both involved in the cerebral VEGF/Akt/NO pathway in angiogenesis in female mice, while VEGF signaling is disrupted in the hearts of mice lacking estrogen receptor α (Jesmin et al., 2010a, b).

Also, orchidectomy markedly decreased in vivo vascularization in males, and in females this angiogenesis is not dependent on the presence of dihydrotestosterone (Sieveking et al., 2010). In ischemia induced angiogenesis, the endogeneous androgens modulate recovery in ischemic hindlimbs, and in orchidectomized animals DHT enhances recovery from ischemia. The orchidectomy significantly reduced the expression of hypoxia-inducible factor 1α (HIF-1α), which is the key subunit to HIF-1, a critical, genome-wide transcription regulator responsible for oxygen homeostasis and responsive to hypoxic stress. HIF-1 drives the expression of more than a hundred genes, including the genes associated with angiogenesis (e.g., VEGF and its receptors). In conclusion, the endogenous androgens play an important role in the coordination of ischemia-mediated angiogenesis by the regulation of key angiogenesis-related genes (Sieveking et al., 2010).

#### **5.3.2 Vascular tone**

The castration of animals causes reduced arterial pressure and reduced responses to angiotensin II (Song et al., 2010). In the castrated animals, treatment with testosterone restored the response to angiotensin II. It is concluded that long term effects of testosterone is pressor-related to angiotensin II responses. Treatment of the castrates with a protein kinase C (PKC) inhibitor attenuated the differences in arterial pressure to angiotensin II. Also, mRNA expression of PKCδ and PKCε are attenuated by castration, but are restored by testosterone. The expression of protein kinase C (CPI-17) and phospho-CPI-17 was decreased in the castrated group, whereas drug replacement of testosterone in castrated rats reversed this effect (Song et al., 2010). These findings suggest that in genetically hypertensive rats the PKC/CPI-17 pathway may contribute to androgenic potentiation of the pressor and renal vascular responses of angiotensin II (Song et al., 2010).

#### **6. Pathway basis of sex hormones action in cardiovascular system**

The estrogen binding to GPER1 activates downstream PI3K, MAPK, and NOS along a similar path as the non-genomic signaling of the classic ERs-mediated signaling. However, the GPER1 also may mediate additional effects that are separate from those of both genomic and non-genomic signaling mediated by the classic ERs. For example, it seems that GPER1 and not ERα is responsible for the upregulation of protein kinase A and the inhibition of apoptosis.

#### **6.1 NO and protein S-nitrosylation**

50 Sex Hormones

the production of vascular endothelial growth factor (VEGF), a key angiogenic growth factor and show increased messenger RNA expression of VEGF receptors 1 and 2 (Flt-1 and KDR respectively, Sieveking et al., 2010). This suggests that the proangiogenic effects of DHT in male endothelial cells are VEGF dependent. Besides, it is known that KDR is the main mediator of the mitogenic/angiogenic action of VEGF in endothelial cells, while Flt-1 is a negative regulator of VEGF action and Flt-1 mRNA is indeed expressed less upon DHT exposure. This upregulated anti-VEGF action, and also the activated inhibitor of phosphoinositol 3-kinase (PI3K), a key enzyme in the PI3K– AKT pathway of VEGF signaling, both inhibited DHT-mediated tubulogenesis genes (Sieveking et al., 2010). In comparison, estrogen receptor α and β are both involved in the cerebral VEGF/Akt/NO pathway in angiogenesis in female mice, while VEGF signaling is disrupted in the hearts of

Also, orchidectomy markedly decreased in vivo vascularization in males, and in females this angiogenesis is not dependent on the presence of dihydrotestosterone (Sieveking et al., 2010). In ischemia induced angiogenesis, the endogeneous androgens modulate recovery in ischemic hindlimbs, and in orchidectomized animals DHT enhances recovery from ischemia. The orchidectomy significantly reduced the expression of hypoxia-inducible factor 1α (HIF-1α), which is the key subunit to HIF-1, a critical, genome-wide transcription regulator responsible for oxygen homeostasis and responsive to hypoxic stress. HIF-1 drives the expression of more than a hundred genes, including the genes associated with angiogenesis (e.g., VEGF and its receptors). In conclusion, the endogenous androgens play an important role in the coordination of ischemia-mediated angiogenesis by the regulation

The castration of animals causes reduced arterial pressure and reduced responses to angiotensin II (Song et al., 2010). In the castrated animals, treatment with testosterone restored the response to angiotensin II. It is concluded that long term effects of testosterone is pressor-related to angiotensin II responses. Treatment of the castrates with a protein kinase C (PKC) inhibitor attenuated the differences in arterial pressure to angiotensin II. Also, mRNA expression of PKCδ and PKCε are attenuated by castration, but are restored by testosterone. The expression of protein kinase C (CPI-17) and phospho-CPI-17 was decreased in the castrated group, whereas drug replacement of testosterone in castrated rats reversed this effect (Song et al., 2010). These findings suggest that in genetically hypertensive rats the PKC/CPI-17 pathway may contribute to androgenic potentiation of

the pressor and renal vascular responses of angiotensin II (Song et al., 2010).

**6. Pathway basis of sex hormones action in cardiovascular system** 

The estrogen binding to GPER1 activates downstream PI3K, MAPK, and NOS along a similar path as the non-genomic signaling of the classic ERs-mediated signaling. However, the GPER1 also may mediate additional effects that are separate from those of both genomic and non-genomic signaling mediated by the classic ERs. For example, it seems that GPER1 and not ERα is responsible for the upregulation of protein kinase A and the inhibition of

mice lacking estrogen receptor α (Jesmin et al., 2010a, b).

of key angiogenesis-related genes (Sieveking et al., 2010).

**5.3.2 Vascular tone** 

apoptosis.

Most of the present cardioprotective effects are dedicated to the NO-related mechanisms that play a role in the regulation of cardiovascular function. Beside the activation of cyclic guanosine monophosphate (cGMP)-dependent pathway, NO also regulates cell function through protein S-nitrosylation. This is a reversible redox-sensitive posttranslational protein modification, which involves the attachment of a NO moiety to the nucleophilic protein sulfhydryl, resulting in S-nitrosothiol (SNO) formation. It is very likely that the protein Snitrosylation plays an important role in cardioprotection (Sun & Murphy, 2010).

Estrogen-induced protein S-nitrosylation has been shown to be involved in a murine model of I/R resulting in a cardioprotection (Lin et al., 2009). During the Langendorff model of I/R, hearts of ovariectomized female mice pretreated with estradiol and/or ERβ-selective agonists, showed increased post-ischemic recovery. This protection was blocked by a NOS inhibitor (Lin et al., 2009), suggesting that increased NO signaling contributes to the cardioprotection. These chonic estradiol or ERβ agonist exposed hearts showed increased Snitrosylated proteins and this protein S-nitrosylation could be abolished by pretreating hearts with the NOS inhibitor (Lin et al., 2009). These data suggest that chronic estrogen treatment and activation of ERβ would indeed lead to increased NO/SNO signaling, playing an essential role in cardioprotection.

Many of the S-nitrosylated proteins found in ERβ-selective agonist-treated hearts (Lin et al., 2009) have also been shown to be increased in preconditioned hearts (Sun et al., 2007), including the mitochondrial F1F0-ATPase, aconitase, malate dehydrogenase, creatine kinase, cytochrome c oxidase and heat shock proteins (HSP 27, 60, and 70). The protein Snitrosylation might also elicit cardioprotective effects by regulating intracellular Ca2+ handling, apoptosis, and post-infarct myocardial remodeling (Sun & Murphy, 2010).

Estradiol, the naturally occuring major form of estrogen, increases protein S-nitrosylation levels in cultured endothelial cells and besides in intact endotheliums where, the estradiol was shown to act through ERα, activates eNOS and generates NO which leads to Snitrosylation (Sun & Murphy, 2010). The S-nitrosylation also mediates the inhibitory effects of estradiol on endothelial ICAM-1 expression by angiotensin II (Chakrabarti et al., 2010). It is a post-translational protein modification induced by both, endogenous NO, generated by NOS in endothelium, and exogenous NO in a variety of cells. Over 100 different cellular proteins have been shown to be S-nitrosylated in processes that reduce the generation of harmful free radicals on one hand, and activate the free radical scavengers on the other (Chakrabarti et al., 2010). Both processes contributing to a reduction in oxidative stress, but also reducing pro-inflamatory signaling, processes which are crucial in I/R injury protection (Chakrabarti et al., 2010).

#### **6.2 PI3K/Akt pathway and its activation by sex hormones**

The Akt pathway is considered as one of the most important molecular kinase that mediates cardioprotection during ischemia reperfusion (Wang et al., 2009). Female hearts have higher level of Akt activity and thus, compared to male hearts, have better protection against I/R injuries and better heart recovery. Indeed, male hearts showed lower Akt activity and worse I/R recovery. In ovariectomized rat hearts the Akt activity is reduced, the recovery decreased (Huang et al., 2010), and supposedly the occurance of apoptosis during the myocardial infarction is one of the potential reasons.

The Role of Sex Hormones in the Cardiovascular System 53

plays a crucial role in the cardioprotection against I/R injury through an acute gender-

At present, PI3K seems to be the most important activation pathway in the cardioprotection of sex hormones. This pathway involves further Akt and NOS activation. Both in vivo and in vitro studies show that acute estradiol treatment reduces cardiomyocyte apoptosis and elicits cardioprotection via ERα activation and PI3K/Akt signaling (Patten et al., 2004). In endothelial cells a direct protein-protein interaction between ligand-activated ERα and the regulatory subunit p85 of PI3K through a nongenomic mechanism is suggested (Simoncini et al., 2000), by which estradiol rapidly activates eNOS via the activation of PI3K/Akt.

Decreased testosterone in castrated animals showed increased myocardial Akt activation (Huang et al., 2010), so some researchers suggested its negative role in I/R induced injuries and others confirmed increased Akt activity. In isolated rat hearts, testosterone used in acute ischemia reperfusion caused gender differences in myocardial Akt activation and its dowstream signaling molecules (p-Bad, Bcl-2, p-FOXO3a). Bad and Bcl are triggers for apoptosis and once these levels increase, apoptosis is suppressed. FOXO, another downstream target of Akt pathway, enables cell survival by inducing death genes. Also, the use of the testosterone antagonist flutamide or castration of the animals prior to the experiment showed an increase in myocardial Akt pathway and increased all three markers (p-Bad, Bcl-2, p-FOXO3a) in the male hearts. Moreover, the effect of castration in the activation of the Akt pathway can be reversed by some agonists, but not by

The degradation of the extracellular matrix by metalloproteinases (MMPs) is involved in post-myocardial infarction processes of healing and remodeling. Knockout mice targeting the MMP-9 gene (the primary MMP protein functioning in post-myocardial infarction cardiac remodeling) were reported to have a reduced prevalence of cardiac rupture and attenuated left ventricular remodeling compared to control mice. Also a temporal change in the expression of MMP-9 and MMP-2 after myocardial infarction has been found (Tao et al., 2004). With estrogen treatment a significant reduction in MMP-9 expression was exerted regardless of castration status, and no reduction was observed in the MMP-2 protein. The decreased activity of matrix MMP-9 by estrogen induced cardioprotection in males after acute myocardial infarction was accompanied with increased Akt-Bcl-2 anti-apoptotic

Apoptosis of cardiomyocytes in infarct zones can be determined by the anti-apoptotic protein marker Bcl-2. Estrogen treated mice showed higher amounts of Bcl-2 expression during myocardial infarction as compared to control mice (Cao et al., 2011). This study has established a pivotal role for the Akt gene in estrogen-induced inhibition of apoptosis.

dependent ER-mediated and gender-independent GPER1 signaling.

**6.2.1 Estrogen** 

**6.2.2 Testosterone** 

dihydrotestosterone (Huang et al., 2010).

signaling (Cao et al., 2011).

**6.3 Matrix metalloproteinase in ischemia** 

**6.4 Apoptosis in cardiac ischemia-reperfusion** 

However, which of the ER isoforms (α or β) play a role is uncertain.

Fig. 7. Integrative model of putative mechanism of non-genomic estrogen signaling. Modified from Meyer et al. (2009) and Moriarty et al. (2006).

Caveolae, invaginations of the endothelial plasma membrane, are centers for signaling processing, providing localization for the molecules involved. Here, estradiol (E2) binds to the estrogen receptor α (ERα) and the "modulator of non-genomic activity of the ER" (MNAR) promotes complex formation with ERα, c-Src, and p85, the regulatory subunit of phosphoinositide-3 protein kinase (PI3K; with subunits p85 and p110), facilitating activation of the PI3K/Akt-signaling. Alternatively, c-Src activates the monomeric GTPase p21ras (ras), which is capable of directly recruiting downstream the mitogen activated protein kinases (MAPK) pathway. Essential for the activation of c-Src is the direct interaction of the G protein Gαi with ERα. Once activated, both PI3K/Akt- and MAPK-pathways can modulate gene transcription, and besides in endothelial cells, alternatively, the activation of PI3K/Aktsignaling leads to the phosphorylation of endothelial nitric oxide synthase (eNOS) protein, which is localized to caveolae through interaction with caveolin-1 (cav-1), a protein that also targets ERα. The molecular chaperone heat shock protein 90 (Hsp90) enhances the PI3K/AkteNOS interaction. Once eNOS is activated, the release of nitric oxide (NO) induces rapid cellular effects.

The ablation of ERβ significantly decreased postischemic functional recovery in female, but not in male hearts (Wang et al., 2009), and besides, a reduced activation of PI3K/Akt was noted in the female ERβ knockout hearts. Since, females show higher densities in cardiac ERβ expression in women than in men, the activation of the PI3K/Akt signaling cascade plays a crucial role in the cardioprotection against I/R injury through an acute genderdependent ER-mediated and gender-independent GPER1 signaling.

#### **6.2.1 Estrogen**

52 Sex Hormones

Fig. 7. Integrative model of putative mechanism of non-genomic estrogen signaling.

Caveolae, invaginations of the endothelial plasma membrane, are centers for signaling processing, providing localization for the molecules involved. Here, estradiol (E2) binds to the estrogen receptor α (ERα) and the "modulator of non-genomic activity of the ER" (MNAR) promotes complex formation with ERα, c-Src, and p85, the regulatory subunit of phosphoinositide-3 protein kinase (PI3K; with subunits p85 and p110), facilitating activation of the PI3K/Akt-signaling. Alternatively, c-Src activates the monomeric GTPase p21ras (ras), which is capable of directly recruiting downstream the mitogen activated protein kinases (MAPK) pathway. Essential for the activation of c-Src is the direct interaction of the G protein Gαi with ERα. Once activated, both PI3K/Akt- and MAPK-pathways can modulate gene transcription, and besides in endothelial cells, alternatively, the activation of PI3K/Aktsignaling leads to the phosphorylation of endothelial nitric oxide synthase (eNOS) protein, which is localized to caveolae through interaction with caveolin-1 (cav-1), a protein that also targets ERα. The molecular chaperone heat shock protein 90 (Hsp90) enhances the PI3K/AkteNOS interaction. Once eNOS is activated, the release of nitric oxide (NO) induces rapid

The ablation of ERβ significantly decreased postischemic functional recovery in female, but not in male hearts (Wang et al., 2009), and besides, a reduced activation of PI3K/Akt was noted in the female ERβ knockout hearts. Since, females show higher densities in cardiac ERβ expression in women than in men, the activation of the PI3K/Akt signaling cascade

Modified from Meyer et al. (2009) and Moriarty et al. (2006).

cellular effects.

At present, PI3K seems to be the most important activation pathway in the cardioprotection of sex hormones. This pathway involves further Akt and NOS activation. Both in vivo and in vitro studies show that acute estradiol treatment reduces cardiomyocyte apoptosis and elicits cardioprotection via ERα activation and PI3K/Akt signaling (Patten et al., 2004). In endothelial cells a direct protein-protein interaction between ligand-activated ERα and the regulatory subunit p85 of PI3K through a nongenomic mechanism is suggested (Simoncini et al., 2000), by which estradiol rapidly activates eNOS via the activation of PI3K/Akt.

#### **6.2.2 Testosterone**

Decreased testosterone in castrated animals showed increased myocardial Akt activation (Huang et al., 2010), so some researchers suggested its negative role in I/R induced injuries and others confirmed increased Akt activity. In isolated rat hearts, testosterone used in acute ischemia reperfusion caused gender differences in myocardial Akt activation and its dowstream signaling molecules (p-Bad, Bcl-2, p-FOXO3a). Bad and Bcl are triggers for apoptosis and once these levels increase, apoptosis is suppressed. FOXO, another downstream target of Akt pathway, enables cell survival by inducing death genes. Also, the use of the testosterone antagonist flutamide or castration of the animals prior to the experiment showed an increase in myocardial Akt pathway and increased all three markers (p-Bad, Bcl-2, p-FOXO3a) in the male hearts. Moreover, the effect of castration in the activation of the Akt pathway can be reversed by some agonists, but not by dihydrotestosterone (Huang et al., 2010).

#### **6.3 Matrix metalloproteinase in ischemia**

The degradation of the extracellular matrix by metalloproteinases (MMPs) is involved in post-myocardial infarction processes of healing and remodeling. Knockout mice targeting the MMP-9 gene (the primary MMP protein functioning in post-myocardial infarction cardiac remodeling) were reported to have a reduced prevalence of cardiac rupture and attenuated left ventricular remodeling compared to control mice. Also a temporal change in the expression of MMP-9 and MMP-2 after myocardial infarction has been found (Tao et al., 2004). With estrogen treatment a significant reduction in MMP-9 expression was exerted regardless of castration status, and no reduction was observed in the MMP-2 protein. The decreased activity of matrix MMP-9 by estrogen induced cardioprotection in males after acute myocardial infarction was accompanied with increased Akt-Bcl-2 anti-apoptotic signaling (Cao et al., 2011).

#### **6.4 Apoptosis in cardiac ischemia-reperfusion**

Apoptosis of cardiomyocytes in infarct zones can be determined by the anti-apoptotic protein marker Bcl-2. Estrogen treated mice showed higher amounts of Bcl-2 expression during myocardial infarction as compared to control mice (Cao et al., 2011). This study has established a pivotal role for the Akt gene in estrogen-induced inhibition of apoptosis. However, which of the ER isoforms (α or β) play a role is uncertain.

The Role of Sex Hormones in the Cardiovascular System 55

ERα demonstrates to have a more prominent role compared to ERβ, which might be genderrelated. ERα is widely distributed in cardiomyocytes (Lizotte et al., 2009), thus indicating its important role, and its membrane position suggests important estrogen non-genomic rapid signaling, while its cytosolic and nuclear distribution suggest genomic signaling. Among the rapid signalling effects, estradiol induces the translocation of ERα to the PI3K regulatory domain and shows NOS (eNOS) activation in endothelium (Simoncini et al., 2000) as one of the most important effector product. Also, GPER1 activation results in matrix metalloproteinase cleavage of heparin-bound epidermal growth factor, that is able to activate the EGF receptor, that subsequently results in acute PI3K and ERK activation (Filardo et al., 2000). Besides, being a transmembrane estrogen receptor, GPER1 activation mediates rapid cell signaling too (Prossnitz et al., 2008). GPER1 deactivation of the PI3K pathway was confirmed by abolishing the agonist-mediated protective effect, suggesting that the more important mechanism of protection by GPER1 activation is through the PI3K/AKT pathway. The activation of the PI3K pathway by GPER1-mediated transactivation of the EGF receptor (Filardo et al., 2000), leads not only to activation of PI3K but also to activation of ERK. Also, HIF- 1 in hypoxic conditions activates the up-regulation of GPER1 in cardiomyocytes and ROS-induced activation of EGFR/ERK signaling is required for this pathway. Hypoxia-induced expression of GPER1 may be included among the mechanisms involved in the anti-apoptotic effects elicited by estrogens. Blocking the PI3K activation resulted in reduced phosphorylation of Akt and in reduced recovery and larger infarct sizes compared to agonist-treated hearts. The acute activation of the estrogen receptor GPER1 is gender-independent cardioprotective (Deschamps & Murphy, 2009). Androgenes, mostly testosterone, have been known up till now for their deleterious effects in cardiovascular system, and are supposed to potentiate ischemic/reperfusion injuries; but those effects are present only in acute heart injuries. Rapid androgen receptor activation, analogous to estrogen receptor cytosolic activation, probably enable yet unknown cardioprotective pathways, which are expressed as diminished ischemic effects, and decreased arrhythmias. Most of such protective effects, beyond their nuclear cardioprotection pathway, are proposed to be mainly long-term activated. Also, endothelial cells exposed to dihydrotestosterone, produced a dose-dependent increase in the production of a key angiogenic growth factor - vascular endothelial growth factor, and further increased the expression of VEGF receptors Flt-1 and KDR. VEGF receptor KDR is the main mediator of the mitogenic/angiogenic action of VEGF in endothelial cells, and VEGF receptor Flt-1 is a negative regulator of VEGF action, so dihydrotestosterone plays a proangiogenic role for VEGF signaling. Anti-VEGF action inhibits tubulogenesis, and also the inhibitor of phosphoinositol 3-kinase (PI3K), a key enzyme in the PI3K–AKT pathway of VEGF

signaling, inhibited dihydrotestosterone-mediated tubulogenesis.

The main pathways for cardioprotection are known to be PI3K-mediated. Estradiol induces the translocation of ERα to the PI3K regulatory domain and results in endothelial NOS (eNOS) activation (Simoncini et al., 2000). ERβ activation leads to protein S-nitrosylation and thus cardioprotection, which could be blocked by NOS inhibition, suggesting that chronic estrogen exposure protects hearts largely via activation of ERβ and NO signaling (Lin et al., 2009). The role for the Akt gene in estrogen-induced inhibition of apoptosis is probably a crucial step. However, which ER isoform plays the major role, ERα or ERβ, and whether gender-dependent, is uncertain. In short, ERα and GPER1 might be more important in acute

protection, and ERβ in more long term, estrogen chronically exposed conditions.

To established the protective effects of estrogen on hypoxia-induced apoptosis cells with minimal ERα expression were used (Cao et al., 2011), and it was revealed that 17β-estradiol protects against apoptosis induced by H2O2-induced oxidative stress through the glutathione/glutaredoxin-dependent redox regulation of the Akt protein. After estrogen treatment, the activity of the pro-apoptotic Akt (P-Akt) began to decrease, while the expression of the anti-apoptotic Bcl-2 began to increase. Cell-cycle analyses indicated that hypoxia-induced apoptosis was efficiently inhibited through supplemental of 17β-estradiol, which shows that ERβ is at least partly involved in the estrogen-mediated cardioprotection (Cao et al., 2011).

#### **6.5 Gonadotrophin releasing hormone as a new target in the heart**

Among the other potential hormones acting in the heart, gonadotrophin releasing hormone is the potential cardiac marker, responsible for the release of luteinizing hormone and follicle-stimulating hormone from the anterior pituitary, that is synthesized and released from neurons in the hypothalamus. Treatment may be associated with an increased risk of cardiac dysfunction that is attributed to the accompanying androgen deprivation. Gonadotrophin releasing hormone by itself may be a major contributor to the cardiac pathology. The chronically administered agonists may prolong QT interval in men and in women and reduce cardiac index, and decreased blood pressure. The potential pathway of activation is the PKA pathway and not the PKC pathway, that leads to gonadotrophin releasing hormone-mediated increased contractility. The PKA-mediated pathway has targets for phosphorylation, promotes cardiomyocyte contractile function, including the Ltype Ca2+ channel on the sarcolemma and components of the contractile apparatus. Besides, PKA also phosphorylates the ryanodine receptor and Ca2+ release channels in the cardiomyocytes, which in turn regulates channel opening and leads to increased sensitivity to Ca2+-induced activation. Higher doses of gonadotrophin releasing hormone elevated resting intracellular Ca2+ and may be a reflection of increased sarcoplasmic reticulum Ca2+ release and cardiac contractility (Dong et al., 2011).

#### **6.6 Other potential mechanism of sex hormones mediated protection**

Some other potential mechanisms may contribute to the cardioprotective effects of sex hormones. For example, it has been reported that estrogen exerts cardioprotective effects by modulating the cardiac expression of tumor necrosis factor-α (TNFα) and its receptor (Xu et al., 2006). Additionally, nitric oxide synthase (NOS) has also been shown to mediate estrogen-induced cardioprotection (Lin et al., 2009). Therefore, the entire mechanism may be far beyond our current knowledge. Also, understanding the exact functional relationship between estrogen and androgen in the cardiovascular system should be the goal of future research.

#### **7. Conclusion**

Many studies of the last years focused not only on estrogen, but also on testosterone for a role in cardiovascular diseases. Importantly, as a new approach of non-nuclear modulation of cardioprotection the estrogen receptor GPER1 was mentioned.

The actions of estrogen are mediated by estrogen receptors -α and –β, by two cytosolic receptors and another, membrane bound receptor GPER1. In the cardiovascular system the

To established the protective effects of estrogen on hypoxia-induced apoptosis cells with minimal ERα expression were used (Cao et al., 2011), and it was revealed that 17β-estradiol protects against apoptosis induced by H2O2-induced oxidative stress through the glutathione/glutaredoxin-dependent redox regulation of the Akt protein. After estrogen treatment, the activity of the pro-apoptotic Akt (P-Akt) began to decrease, while the expression of the anti-apoptotic Bcl-2 began to increase. Cell-cycle analyses indicated that hypoxia-induced apoptosis was efficiently inhibited through supplemental of 17β-estradiol, which shows that ERβ is at least partly involved in the estrogen-mediated cardioprotection

Among the other potential hormones acting in the heart, gonadotrophin releasing hormone is the potential cardiac marker, responsible for the release of luteinizing hormone and follicle-stimulating hormone from the anterior pituitary, that is synthesized and released from neurons in the hypothalamus. Treatment may be associated with an increased risk of cardiac dysfunction that is attributed to the accompanying androgen deprivation. Gonadotrophin releasing hormone by itself may be a major contributor to the cardiac pathology. The chronically administered agonists may prolong QT interval in men and in women and reduce cardiac index, and decreased blood pressure. The potential pathway of activation is the PKA pathway and not the PKC pathway, that leads to gonadotrophin releasing hormone-mediated increased contractility. The PKA-mediated pathway has targets for phosphorylation, promotes cardiomyocyte contractile function, including the Ltype Ca2+ channel on the sarcolemma and components of the contractile apparatus. Besides, PKA also phosphorylates the ryanodine receptor and Ca2+ release channels in the cardiomyocytes, which in turn regulates channel opening and leads to increased sensitivity to Ca2+-induced activation. Higher doses of gonadotrophin releasing hormone elevated resting intracellular Ca2+ and may be a reflection of increased sarcoplasmic reticulum Ca2+

**6.5 Gonadotrophin releasing hormone as a new target in the heart** 

**6.6 Other potential mechanism of sex hormones mediated protection** 

of cardioprotection the estrogen receptor GPER1 was mentioned.

Some other potential mechanisms may contribute to the cardioprotective effects of sex hormones. For example, it has been reported that estrogen exerts cardioprotective effects by modulating the cardiac expression of tumor necrosis factor-α (TNFα) and its receptor (Xu et al., 2006). Additionally, nitric oxide synthase (NOS) has also been shown to mediate estrogen-induced cardioprotection (Lin et al., 2009). Therefore, the entire mechanism may be far beyond our current knowledge. Also, understanding the exact functional relationship between estrogen and androgen in the cardiovascular system should be the goal of future

Many studies of the last years focused not only on estrogen, but also on testosterone for a role in cardiovascular diseases. Importantly, as a new approach of non-nuclear modulation

The actions of estrogen are mediated by estrogen receptors -α and –β, by two cytosolic receptors and another, membrane bound receptor GPER1. In the cardiovascular system the

release and cardiac contractility (Dong et al., 2011).

(Cao et al., 2011).

research.

**7. Conclusion** 

ERα demonstrates to have a more prominent role compared to ERβ, which might be genderrelated. ERα is widely distributed in cardiomyocytes (Lizotte et al., 2009), thus indicating its important role, and its membrane position suggests important estrogen non-genomic rapid signaling, while its cytosolic and nuclear distribution suggest genomic signaling. Among the rapid signalling effects, estradiol induces the translocation of ERα to the PI3K regulatory domain and shows NOS (eNOS) activation in endothelium (Simoncini et al., 2000) as one of the most important effector product. Also, GPER1 activation results in matrix metalloproteinase cleavage of heparin-bound epidermal growth factor, that is able to activate the EGF receptor, that subsequently results in acute PI3K and ERK activation (Filardo et al., 2000). Besides, being a transmembrane estrogen receptor, GPER1 activation mediates rapid cell signaling too (Prossnitz et al., 2008). GPER1 deactivation of the PI3K pathway was confirmed by abolishing the agonist-mediated protective effect, suggesting that the more important mechanism of protection by GPER1 activation is through the PI3K/AKT pathway. The activation of the PI3K pathway by GPER1-mediated transactivation of the EGF receptor (Filardo et al., 2000), leads not only to activation of PI3K but also to activation of ERK. Also, HIF- 1 in hypoxic conditions activates the up-regulation of GPER1 in cardiomyocytes and ROS-induced activation of EGFR/ERK signaling is required for this pathway. Hypoxia-induced expression of GPER1 may be included among the mechanisms involved in the anti-apoptotic effects elicited by estrogens. Blocking the PI3K activation resulted in reduced phosphorylation of Akt and in reduced recovery and larger infarct sizes compared to agonist-treated hearts. The acute activation of the estrogen receptor GPER1 is gender-independent cardioprotective (Deschamps & Murphy, 2009).

Androgenes, mostly testosterone, have been known up till now for their deleterious effects in cardiovascular system, and are supposed to potentiate ischemic/reperfusion injuries; but those effects are present only in acute heart injuries. Rapid androgen receptor activation, analogous to estrogen receptor cytosolic activation, probably enable yet unknown cardioprotective pathways, which are expressed as diminished ischemic effects, and decreased arrhythmias. Most of such protective effects, beyond their nuclear cardioprotection pathway, are proposed to be mainly long-term activated. Also, endothelial cells exposed to dihydrotestosterone, produced a dose-dependent increase in the production of a key angiogenic growth factor - vascular endothelial growth factor, and further increased the expression of VEGF receptors Flt-1 and KDR. VEGF receptor KDR is the main mediator of the mitogenic/angiogenic action of VEGF in endothelial cells, and VEGF receptor Flt-1 is a negative regulator of VEGF action, so dihydrotestosterone plays a proangiogenic role for VEGF signaling. Anti-VEGF action inhibits tubulogenesis, and also the inhibitor of phosphoinositol 3-kinase (PI3K), a key enzyme in the PI3K–AKT pathway of VEGF signaling, inhibited dihydrotestosterone-mediated tubulogenesis.

The main pathways for cardioprotection are known to be PI3K-mediated. Estradiol induces the translocation of ERα to the PI3K regulatory domain and results in endothelial NOS (eNOS) activation (Simoncini et al., 2000). ERβ activation leads to protein S-nitrosylation and thus cardioprotection, which could be blocked by NOS inhibition, suggesting that chronic estrogen exposure protects hearts largely via activation of ERβ and NO signaling (Lin et al., 2009). The role for the Akt gene in estrogen-induced inhibition of apoptosis is probably a crucial step. However, which ER isoform plays the major role, ERα or ERβ, and whether gender-dependent, is uncertain. In short, ERα and GPER1 might be more important in acute protection, and ERβ in more long term, estrogen chronically exposed conditions.

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Of course, some clinical findings predicts even more potential clinical protection by sex hormones, by yet unknown mechanisms. A review analysis of physiologic responses to critical illnesses and injury as well as their relative rates of survival and recovery, proposed estrogen to have protective effects in numerous conditions ranging from global ischemic insults and massive systemic inflammatory responses to devastating focal injury and apoptosis in vital organs. Furthermore, the authors suggested even exogenous infusion of estrogen, not only as a direct therapeutic agent, that can benefit in some instances, but proposed estrogen administration for synergism with other resuscitative interventions (Wigginton et al., 2010).

#### **8. Acknowledgement**

This manuscript was proofread by Dr. Cécil J.W. Meulenberg. Figures were prepared by Mr. Jura Štok. Publication was made possible by Grant J3-0024 »Pharmacological protection against ischemic-reperfusion injuries in brain and cardiovascular tissues« from the Slovenian Research Agency. The author declare no conflicts of interest.

#### **9. References**


Cardioprotective effects induced by estrogen involved ERK, PI3K and PKA transduction cascades. In endothelial cells, ERα activation culminates in two major signal transduction events, one is the ERK pathway, and a second is increased PI3K/Akt activity. In both pathways, the final event involves a rapid NO production by eNOS. A potential functional interactivity between GPER1, ERα and ERβ leads to cardioprotective action, responsible for estrogen involvement in ERK, PI3K, PKA and eNOS. Gender-independent GPER1 activation of eNOS plays a central role in the response to estrogen stimulation (Filice et al., 2009). Testosterone on the other hand, is known to act via PKC; PKC inhibition attenuates the differences in arterial pressure to Ang II. Also, expression of PKCδ and PKCε are attenuated by castration, but are restored by testosterone. Undoubtly, testosterone cardioprotection is the focus of present studies and search for its protective pathways, independent from

Of course, some clinical findings predicts even more potential clinical protection by sex hormones, by yet unknown mechanisms. A review analysis of physiologic responses to critical illnesses and injury as well as their relative rates of survival and recovery, proposed estrogen to have protective effects in numerous conditions ranging from global ischemic insults and massive systemic inflammatory responses to devastating focal injury and apoptosis in vital organs. Furthermore, the authors suggested even exogenous infusion of estrogen, not only as a direct therapeutic agent, that can benefit in some instances, but proposed estrogen administration for synergism with other resuscitative interventions

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**3** 

Üner Tan

*Turkey* 

**Serum Free Testosterone and Estradiol Levels** 

Cognitive sex differences have been demonstrated in humans: men usually outperform women on spatial ability (Linn & Peterson, 1985; Gladue et al., 1990; Mann et al., 1990; Gouchie & Kimura, 1991; Gladue & Bailey, 1995; Halpern & Tan, 2001; Tan et al., 2003a, b; Herlitz & Lovén, 2009); women outperform men on verbal tasks (Hyde & Linn, 1988; Mann et al., 1990; Halpern & Tan, 2001; Tan et al., 2003a, 2003b; Herlitz & Lovén, 2009). The sex hormones were thought to be responsible for the observed sex differences in cognitive abilities, but the available scientific literature does not provide any generally acceptable conclusion on this topic. For instance, testosterone has been reported to be beneficial for visuospatial ability in men (Hier & Crowly, 1982; Gordon & Lee, 1986; Christiansen & Knussmann, 1987; Tan, 1990a, b; Tan & Akgun, 1992; Christiansen, 1993; Janowsky et al., 1994; Van Goozen et al., 1994; Van Goozen et al., 1995; Tan & Tan, 1998; Barrett-Connor et al., 1999a; Silverman et al., 1999; Aleman et al., 2001; Kutlu et al., 2001; Kenny et al., 2002; Yaffe et al., 2002; Azurmendi et al., 2005), and women (Shute et al., 1983; Resnick et al., 1986; McKeever, 1987; Gouchie & Kimura, 1991; Van Goozen et al., 1995; Moffat & Hampson, 1996; Barrett-Connor et al., 1999b; Celec et al., 2002; Ostatnikova, et al., 2002). On the other hand, testosterone has also been reported as having no benefit for spatial ability in either men or women (Shute et al., 1983; Gouchie & Kimura, 1991; Moffat & Hampson, 1996; Van Goozen et al., 1995). Contrarily, some authors did not accept any significant association between testosterone and spatial cognition (McKeever et al., 1987; Kampen & Sherwin, 1996; Herlitz & Lovén, 2009). An inverse U-shaped relation between testosterone and spatial cognition was also reported, that is, the optimal performance was found in moderately high levels of testosterone (Shute et al., 1983; Gouchie & Kimura, 1991; Moffat & Hampson, 1996; Alexander et al., 1998; Neave, et al., 1999). With regard to handedness, significant relationships have been found between testosterone and spatial ability in right-handers, but

Verbal abilities have also been studied in relation to sex-hormone concentrations in men and women. Some authors could not demonstrate any significant association between testosterone and verbal tasks (Gordon & Lee, 1986; Gouchie & Kimura, 1991; Neave, et al., 1999; Herlitz & Lovén, 2009), but others reported beneficial effects of testosterone on verbal ability (Cherrier, 1999; Alexander et al., 1998), but detrimental influence of androgen levels

**1. Introduction** 

not in left-handers (Moffat & Hampson, 1996).

**in Perceptual-Verbal and Spatial Abilities;** 

**Differences in Sex and Hand Preference** 

*Çukurova University, Medical School, Department of Physiology, Adana,* 


### **Serum Free Testosterone and Estradiol Levels in Perceptual-Verbal and Spatial Abilities; Differences in Sex and Hand Preference**

Üner Tan

*Çukurova University, Medical School, Department of Physiology, Adana, Turkey* 

#### **1. Introduction**

64 Sex Hormones

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Cognitive sex differences have been demonstrated in humans: men usually outperform women on spatial ability (Linn & Peterson, 1985; Gladue et al., 1990; Mann et al., 1990; Gouchie & Kimura, 1991; Gladue & Bailey, 1995; Halpern & Tan, 2001; Tan et al., 2003a, b; Herlitz & Lovén, 2009); women outperform men on verbal tasks (Hyde & Linn, 1988; Mann et al., 1990; Halpern & Tan, 2001; Tan et al., 2003a, 2003b; Herlitz & Lovén, 2009). The sex hormones were thought to be responsible for the observed sex differences in cognitive abilities, but the available scientific literature does not provide any generally acceptable conclusion on this topic. For instance, testosterone has been reported to be beneficial for visuospatial ability in men (Hier & Crowly, 1982; Gordon & Lee, 1986; Christiansen & Knussmann, 1987; Tan, 1990a, b; Tan & Akgun, 1992; Christiansen, 1993; Janowsky et al., 1994; Van Goozen et al., 1994; Van Goozen et al., 1995; Tan & Tan, 1998; Barrett-Connor et al., 1999a; Silverman et al., 1999; Aleman et al., 2001; Kutlu et al., 2001; Kenny et al., 2002; Yaffe et al., 2002; Azurmendi et al., 2005), and women (Shute et al., 1983; Resnick et al., 1986; McKeever, 1987; Gouchie & Kimura, 1991; Van Goozen et al., 1995; Moffat & Hampson, 1996; Barrett-Connor et al., 1999b; Celec et al., 2002; Ostatnikova, et al., 2002). On the other hand, testosterone has also been reported as having no benefit for spatial ability in either men or women (Shute et al., 1983; Gouchie & Kimura, 1991; Moffat & Hampson, 1996; Van Goozen et al., 1995). Contrarily, some authors did not accept any significant association between testosterone and spatial cognition (McKeever et al., 1987; Kampen & Sherwin, 1996; Herlitz & Lovén, 2009). An inverse U-shaped relation between testosterone and spatial cognition was also reported, that is, the optimal performance was found in moderately high levels of testosterone (Shute et al., 1983; Gouchie & Kimura, 1991; Moffat & Hampson, 1996; Alexander et al., 1998; Neave, et al., 1999). With regard to handedness, significant relationships have been found between testosterone and spatial ability in right-handers, but not in left-handers (Moffat & Hampson, 1996).

Verbal abilities have also been studied in relation to sex-hormone concentrations in men and women. Some authors could not demonstrate any significant association between testosterone and verbal tasks (Gordon & Lee, 1986; Gouchie & Kimura, 1991; Neave, et al., 1999; Herlitz & Lovén, 2009), but others reported beneficial effects of testosterone on verbal ability (Cherrier, 1999; Alexander et al., 1998), but detrimental influence of androgen levels

Serum Free Testosterone and Estradiol Levels in

Halpern & Tan, 2001; Tan et al., 2003b).

determined for each subject.

**2.4 Hormone tests** 

**2.2 Mental Rotation Test (MRT)** 

giving a maximum possible score of 25.

**2.3 Cattell nonverbal intelligence test (Test of "g": Culture Fair)** 

important factor with some loading on verbal and numerical abilities.

**2.1 Finding As test** 

Perceptual-Verbal and Spatial Abilities; Differences in Sex and Hand Preference 67

signs and symptoms. Their cognitive functions were measured using three tests: "Finding As

To measure the perceptual-verbal ability, the Finding As test from the ETS kit of Factor-Referenced Cognitive Tests (Ekstrom et al., 1976), classified as "feminine," was administered to the subjects. The test-retest correlation has been reported to be r = .87 for this test (Ekstrom et al., 1976). This test is usually used to assess the perceptual speed, and favors women, with very large effect size (Jensen, 1998 and e.g., Kimura & Hampson, 1994;

In each column of 40 words, the respondents were required to identify the five words containing the letter "a." In the Turkish version of this test (see Halpern & Tan, 2001), the subjects were asked to read the words in several columns as fast as possible, and cross out the letter "a" whenever it appears in the word list. This is a timed test of two minutes was allowed to read one page out of three pages, which the subjects should examine consequently. After completing the test, the number of the correctly chosen words was

To study mental rotation functions and the ability to solve spatial problems, the "Mental Rotation Test" (MRT) was used. This test was originally developed by Vandenberg and Kuse (1978), based on a design by Shepard and Metzler (1971). This test is a timed, groupadministered paper-and-pencil test comprising problems in which participants are required to say whether pairs of two- or three-dimensional drawings of cubes from different angles and perspectives were the same or mirror images of each other. There were 25 problems,

The Cattell's Culture Fair Intelligence Test was originally designed to be a general measure of inborn intelligence without utilizing verbal material (Cattell, 1973, 1987). We applied the Institute for Personality and Ability (IPAT) Culture Fair Intelligence Test, Scale 3A, Form A to university students. The test primarily measures fluid intelligence, with part-whole relations, similarities, causal and spatial relations, inductive reasoning, inferential relations, including series, classification, matrices, and topology. There were 13 items on Subset 1 (3 min), 14 items in Subset 2 (4 min), 13 items in Subset 3 (3 min), and 10 items in Subset 4 (4 min). Raw scores were converted to normalized scores expressed by age group, and scoring was performed by using score key overlays with the response forms. Factor analysis showed validity in the 1970s and 1980s when the general ability factor was correlated with the concepts featured in the subtests (see Cattell, 1987). The spatial ability seems to be the most

Following the tests for cognitive abilities, blood was taken from the cubital vein the same day, and stored in the deep freeze for later analysis of hormone concentrations. The serum free testosterone and estradiol concentrations were measured using a radioimmunoassay technique (Diagnostic Product Corporation, USA), which is commercially available. Because

Test", "Mental Rotation Test," and "Cattell's Culture Fair Intelligence Test.

pn the vocabulary ability in girls (Azurmendi et al., 2005) . In female-to-male transsexuals, verbal skills have been shown to dramatically decline within three months following high doses of testosterone (Van Goozen et al., 1995). In contrast, high estrogen levels have been reported to be advantageous for verbal skills in women, and vary according to their ovarian cycle (Kimura, 1996; Halpern & Tan, 2001).

Cattell`s Culture Fair Intelligence Test measuring the sex-neutral IQ (Tan et al., 1993; Tan & Tan, 1998; Halpern & Tan, 2001) shows menstrual cycle fluctuations, being lower during the pre-ovulatory and post-ovulatory phases; in fact, an inverse U-shaped relation has been found between serum estradiol concentration and Cattell IQ in women (see Halpern & Tan, 2001). Despite the sex-neutrality of this IQ test, a direct relation has been found between Cattell IQ and total serum testosterone level in left-handed men (Tan et al., 1993). An inverse U-shaped relation has also been demonstrated between the total serum testosterone level and Cattell IQ in men and women (Tan & Tan, 1998). Students successful in exams for university entrance have also been shown to have higher total testosterone than those unsuccessful in university exams (Kutlu et al., 2001). On the other hand, matrices taken from the Kaufman Brief Intelligence Test, measuring the fluid intelligence similar to the Cattell's Culture Fair Intelligence Test, showed negative correlation with serum testosterone levels in 5-year-old children (Azurmendi et al., 2005).

Tan et al. (2003a) recently reported that an observed sex difference in visuo-spatial ability was not a real sex difference but merely depended upon the body size, because with covariates of height, weight, and vital capacity, sex differences in mental rotation completely disappeared. Under a combined co-variation of height, weight, and testosterone, the sex difference in mental rotation ability reversed, women scoring better than men (Tan et al., 2003b). In perceptual-verbal ability studies (Finding As test), with covariates of estradiol and progesterone, the sex difference also disappeared, suggesting the prominent role of these sex hormones in different verbal abilities of men and women with related differences in height and weight associated with *nature vs nuture* (Tan et al., 2003b). In accord, the height and weight are correlated with brain size, which is also correlated with IQ (r = .40) (Rushton, 1992, 1997; Raz et al., 1993; Tan et al., 1999; McDaniel, 2005). These results suggest that the body size should also be considered as a co-variate in studies of sex-related differences in cognitive abilities.

Under light of the above mentioned controversial results about the relations of sex hormones, testosterone and estrogens, on the verbal and non-verbal, visuo-spatial abilities, the aim of the present work was to evaluate the relations of the serum free testosterone and estradiol levels to the verbal, and non-verbal, visuo-spatial ability scores in right- and lefthanded men and women. Although there are numerous reports on this subject with numerous controversial results, the present work will be the first, in the scientific literature, taking only the participants within the similar range of body size in male and female young adults, following the interrelationships among the body size, intelligence, and sex hormones. Controlling the body size could help to obtain a more reliable, homogenous sample of men and women within the same age range.

#### **2. Methods**

Participants in the study were 52 male and 24 female university students. All volunteers were between 18 and 20 years old, and willing to participate in this study since they wanted to know their IQs and sex-hormone levels. They were healthy and did not exhibit neurological signs and symptoms. Their cognitive functions were measured using three tests: "Finding As Test", "Mental Rotation Test," and "Cattell's Culture Fair Intelligence Test.

#### **2.1 Finding As test**

66 Sex Hormones

pn the vocabulary ability in girls (Azurmendi et al., 2005) . In female-to-male transsexuals, verbal skills have been shown to dramatically decline within three months following high doses of testosterone (Van Goozen et al., 1995). In contrast, high estrogen levels have been reported to be advantageous for verbal skills in women, and vary according to their ovarian

Cattell`s Culture Fair Intelligence Test measuring the sex-neutral IQ (Tan et al., 1993; Tan & Tan, 1998; Halpern & Tan, 2001) shows menstrual cycle fluctuations, being lower during the pre-ovulatory and post-ovulatory phases; in fact, an inverse U-shaped relation has been found between serum estradiol concentration and Cattell IQ in women (see Halpern & Tan, 2001). Despite the sex-neutrality of this IQ test, a direct relation has been found between Cattell IQ and total serum testosterone level in left-handed men (Tan et al., 1993). An inverse U-shaped relation has also been demonstrated between the total serum testosterone level and Cattell IQ in men and women (Tan & Tan, 1998). Students successful in exams for university entrance have also been shown to have higher total testosterone than those unsuccessful in university exams (Kutlu et al., 2001). On the other hand, matrices taken from the Kaufman Brief Intelligence Test, measuring the fluid intelligence similar to the Cattell's Culture Fair Intelligence Test, showed negative correlation with serum testosterone levels in

Tan et al. (2003a) recently reported that an observed sex difference in visuo-spatial ability was not a real sex difference but merely depended upon the body size, because with covariates of height, weight, and vital capacity, sex differences in mental rotation completely disappeared. Under a combined co-variation of height, weight, and testosterone, the sex difference in mental rotation ability reversed, women scoring better than men (Tan et al., 2003b). In perceptual-verbal ability studies (Finding As test), with covariates of estradiol and progesterone, the sex difference also disappeared, suggesting the prominent role of these sex hormones in different verbal abilities of men and women with related differences in height and weight associated with *nature vs nuture* (Tan et al., 2003b). In accord, the height and weight are correlated with brain size, which is also correlated with IQ (r = .40) (Rushton, 1992, 1997; Raz et al., 1993; Tan et al., 1999; McDaniel, 2005). These results suggest that the body size should also be considered as a co-variate in studies of sex-related

Under light of the above mentioned controversial results about the relations of sex hormones, testosterone and estrogens, on the verbal and non-verbal, visuo-spatial abilities, the aim of the present work was to evaluate the relations of the serum free testosterone and estradiol levels to the verbal, and non-verbal, visuo-spatial ability scores in right- and lefthanded men and women. Although there are numerous reports on this subject with numerous controversial results, the present work will be the first, in the scientific literature, taking only the participants within the similar range of body size in male and female young adults, following the interrelationships among the body size, intelligence, and sex hormones. Controlling the body size could help to obtain a more reliable, homogenous

Participants in the study were 52 male and 24 female university students. All volunteers were between 18 and 20 years old, and willing to participate in this study since they wanted to know their IQs and sex-hormone levels. They were healthy and did not exhibit neurological

cycle (Kimura, 1996; Halpern & Tan, 2001).

5-year-old children (Azurmendi et al., 2005).

differences in cognitive abilities.

**2. Methods** 

sample of men and women within the same age range.

To measure the perceptual-verbal ability, the Finding As test from the ETS kit of Factor-Referenced Cognitive Tests (Ekstrom et al., 1976), classified as "feminine," was administered to the subjects. The test-retest correlation has been reported to be r = .87 for this test (Ekstrom et al., 1976). This test is usually used to assess the perceptual speed, and favors women, with very large effect size (Jensen, 1998 and e.g., Kimura & Hampson, 1994; Halpern & Tan, 2001; Tan et al., 2003b).

In each column of 40 words, the respondents were required to identify the five words containing the letter "a." In the Turkish version of this test (see Halpern & Tan, 2001), the subjects were asked to read the words in several columns as fast as possible, and cross out the letter "a" whenever it appears in the word list. This is a timed test of two minutes was allowed to read one page out of three pages, which the subjects should examine consequently. After completing the test, the number of the correctly chosen words was determined for each subject.

#### **2.2 Mental Rotation Test (MRT)**

To study mental rotation functions and the ability to solve spatial problems, the "Mental Rotation Test" (MRT) was used. This test was originally developed by Vandenberg and Kuse (1978), based on a design by Shepard and Metzler (1971). This test is a timed, groupadministered paper-and-pencil test comprising problems in which participants are required to say whether pairs of two- or three-dimensional drawings of cubes from different angles and perspectives were the same or mirror images of each other. There were 25 problems, giving a maximum possible score of 25.

#### **2.3 Cattell nonverbal intelligence test (Test of "g": Culture Fair)**

The Cattell's Culture Fair Intelligence Test was originally designed to be a general measure of inborn intelligence without utilizing verbal material (Cattell, 1973, 1987). We applied the Institute for Personality and Ability (IPAT) Culture Fair Intelligence Test, Scale 3A, Form A to university students. The test primarily measures fluid intelligence, with part-whole relations, similarities, causal and spatial relations, inductive reasoning, inferential relations, including series, classification, matrices, and topology. There were 13 items on Subset 1 (3 min), 14 items in Subset 2 (4 min), 13 items in Subset 3 (3 min), and 10 items in Subset 4 (4 min). Raw scores were converted to normalized scores expressed by age group, and scoring was performed by using score key overlays with the response forms. Factor analysis showed validity in the 1970s and 1980s when the general ability factor was correlated with the concepts featured in the subtests (see Cattell, 1987). The spatial ability seems to be the most important factor with some loading on verbal and numerical abilities.

#### **2.4 Hormone tests**

Following the tests for cognitive abilities, blood was taken from the cubital vein the same day, and stored in the deep freeze for later analysis of hormone concentrations. The serum free testosterone and estradiol concentrations were measured using a radioimmunoassay technique (Diagnostic Product Corporation, USA), which is commercially available. Because

Serum Free Testosterone and Estradiol Levels in

**FREE TESTOSTERONE (ng/dL)** 2 4 6 8 10 12 14

**ESTRADIOL (pg/mL)** 0 10 20 30 40 50 60 70

**3.4 Cattell`s culture fair intelligence test** 

**A. RH MEN** 

**B. RH MEN**

**NUMBER CORRECT (MENTAL ROTATION)**

0

**NUMBER CORRECT (MENTAL ROTATION)**

0

**3.3.2 Left-handers** 

5

10

15

20

25

5

10

15

20

25

Perceptual-Verbal and Spatial Abilities; Differences in Sex and Hand Preference 69

**NUMBER CORRECT (MENTAL ROTATION)**

**NUMBER CORRECT (MENTAL ROTATION)**

Fig. 1. Relations of serum testosterone and estradiol concentrations (abscissa) to mental

estradiol was best described by a direct correlation, r = .68, F(1, 17) = 14.3, p < .001.

The scattergrams in Figure 2 illustrate the variations in mental rotation scores (ordinate) with various serum testosterone (left) and estradiol (right) levels in left-handed subjects. Since there were only a few left-handed females (N = 6), all left-handers were analyzed together (Figure 2), and the results were essentially similar to the right-handed male subjects. The relation of mental rotation ability versus testosterone could best be described by a quadratric equation, r = .49, F(1, 17) = 5.5, P < .05, with a slight decrease in mental rotation ability towards the higher testosterone levels. The relation of mental rotation to

There was no significant sex difference in Cattell IQ, F(1, 161) = 0.31, p > .55. Since there were only a few left-handed subjects who took the Cattell IQ test, only the right-handers will be analyzed in this section. Figure 3 illustrates the relation between the serum testosterone and estradiol levels (abscissa) to Cattell IQ (ordinate) in the right-handed male

rotation test scores (ordinate) in right-handed men (A, B) and women (C, D).

**C. RH WOMEN**

**D. RH WOMEN**

**FREE TESTOSTERONE (ng/dL)** 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6

**ESTRADIOL (pg/mL)** 0 50 100 150 200 250 300

testosterone shows diurnal and circadian variations (Kimura & Hampson, 1994; Moffat & Hampson, 1996), all the tests were completed and blood samples were taken before noon in the spring semester (May).

#### **3. Results**

#### **3.1 Sex hormone levels**

The mean testosterone levels were found to be 7.56±2.7 ng/dL and 5.2±3.7 ng/dL for the right-handed men (N = 33) and left-handed men (N = 19), respectively. The difference between these means was statistically significant: t = 2.67, df = 50, p = .01.

There was no significant difference between the mean estradiol levels of the right-handed (28.26±15.04 pg/mL)) and left-handed (29.16±14.93 pg/mL) male subjects, t = 0.21, df = 50, P > .80. The number of the left-handed females was not statistically suitable to make any comparison with right-handed women.


#### **3.2 Mental rotation ability**

Table 1. Mental rotation, Cattell IQ and Finding As Test results

#### **3.3 Sex hormones**

#### **3.3.1 Right-handers**

Figure 1 illustrates the relation between serum testosterone and estradiol levels (abscissa) and mental rotation ability (ordinate) in the right-handed men (A, B) and women (C, D). In right-handed men, the relation between serum testosterone level and the number of correct answers on the mental rotation test could be best described by a quadratic equation (Figure 1A), i.e., the lowest mental rotation scores were at both ends of the inverted U-shaped curve. This quadratic relation was statistically significant: r = .61, F(1, 24) = 7.7, p < .001. In righthanded women, there was a direct relation between serum testosterone level and mental rotation scores, r = .61, F(1, 22) = 13.2, p < .001 (Figure 1C).

The serum estradiol level directly correlated with mental rotation test scores in the righthanded men (Figure 1B); the relation was statistically significant, r = .61, F(1, 24) = 13.9, p < .001. Similar to testosterone versus mental rotation in right-handed men, there was a quadratic relation (inverse U) between serum estradiol level and mental rotation ability in right-handed women, r = .57, F(1, 22) = 5.3, p < .05 (Figure 1D).

Fig. 1. Relations of serum testosterone and estradiol concentrations (abscissa) to mental rotation test scores (ordinate) in right-handed men (A, B) and women (C, D).

#### **3.3.2 Left-handers**

68 Sex Hormones

testosterone shows diurnal and circadian variations (Kimura & Hampson, 1994; Moffat & Hampson, 1996), all the tests were completed and blood samples were taken before noon in

The mean testosterone levels were found to be 7.56±2.7 ng/dL and 5.2±3.7 ng/dL for the right-handed men (N = 33) and left-handed men (N = 19), respectively. The difference

There was no significant difference between the mean estradiol levels of the right-handed (28.26±15.04 pg/mL)) and left-handed (29.16±14.93 pg/mL) male subjects, t = 0.21, df = 50, P > .80. The number of the left-handed females was not statistically suitable to make any

Test N Mean Minimum Maximum 25%-75%

5.00 3.00 2.00

104 106

27 23

Figure 1 illustrates the relation between serum testosterone and estradiol levels (abscissa) and mental rotation ability (ordinate) in the right-handed men (A, B) and women (C, D). In right-handed men, the relation between serum testosterone level and the number of correct answers on the mental rotation test could be best described by a quadratic equation (Figure 1A), i.e., the lowest mental rotation scores were at both ends of the inverted U-shaped curve. This quadratic relation was statistically significant: r = .61, F(1, 24) = 7.7, p < .001. In righthanded women, there was a direct relation between serum testosterone level and mental

The serum estradiol level directly correlated with mental rotation test scores in the righthanded men (Figure 1B); the relation was statistically significant, r = .61, F(1, 24) = 13.9, p < .001. Similar to testosterone versus mental rotation in right-handed men, there was a quadratic relation (inverse U) between serum estradiol level and mental rotation ability in

20.00 16.00 8.00

128 133

47 59 8.00-13.25 5.50-10.00 6.00-10.75

108-120 114-124

33-42 35-46

between these means was statistically significant: t = 2.67, df = 50, p = .01.

10.55**±**3.40 8.17**±**3.12 8.16**±**3.18

115.8±6.6 117.9±8.3

37.9±5.4 40.7±.9

Table 1. Mental rotation, Cattell IQ and Finding As Test results

rotation scores, r = .61, F(1, 22) = 13.2, p < .001 (Figure 1C).

right-handed women, r = .57, F(1, 22) = 5.3, p < .05 (Figure 1D).

the spring semester (May).

**3.1 Sex hormone levels** 

**3.2 Mental rotation ability** 

Mental rotation RH males RH females LH males

Cattell RH men RH women

> RH men RH women

**3.3 Sex hormones 3.3.1 Right-handers** 

As

comparison with right-handed women.

29 24 19

25 18

25 20

**3. Results** 

The scattergrams in Figure 2 illustrate the variations in mental rotation scores (ordinate) with various serum testosterone (left) and estradiol (right) levels in left-handed subjects. Since there were only a few left-handed females (N = 6), all left-handers were analyzed together (Figure 2), and the results were essentially similar to the right-handed male subjects. The relation of mental rotation ability versus testosterone could best be described by a quadratric equation, r = .49, F(1, 17) = 5.5, P < .05, with a slight decrease in mental rotation ability towards the higher testosterone levels. The relation of mental rotation to estradiol was best described by a direct correlation, r = .68, F(1, 17) = 14.3, p < .001.

#### **3.4 Cattell`s culture fair intelligence test**

There was no significant sex difference in Cattell IQ, F(1, 161) = 0.31, p > .55. Since there were only a few left-handed subjects who took the Cattell IQ test, only the right-handers will be analyzed in this section. Figure 3 illustrates the relation between the serum testosterone and estradiol levels (abscissa) to Cattell IQ (ordinate) in the right-handed male

Serum Free Testosterone and Estradiol Levels in

**3.5 Perceptual-verbal ability ("Finding As Test")** 

**SERUM TESTOSTERONE (ng/dL)** 2 4 6 810 12 14

**SERUM ESTRADIOL (pg/mL)** 0 10 20 30 40 50 60

N = 16, r = .15, F = 0.4, p > .55).

male (A, B) and female (C, D) subjects.

**3.5.1 Right-handers** 

**NUMBER CORRECT ON As**

25

**NUMBER CORRECT ON As**

subjects.

**B. RH MEN**

30

35

40

45

50

Perceptual-Verbal and Spatial Abilities; Differences in Sex and Hand Preference 71

An opposite picture was found for the female subjects. Namely, there was a quadratic, inverse U-shaped relation between testosterone and IQ (Figure 3C; N = 16, r = .55, F = 7.3, p < .05), and there was no significant correlation between estradiol and Cattell IQ (Figure 3D;

Figure 4 illustrates the relation between the number of correct answers on the "Finding As Test" (ordinate) to the serum testosterone and estradiol levels (abscissa) in right-handed

There was an inverse relation between the serum free testosterone levels and the number of correct answers, and this was best described by a quadratic relation, r = .73, F = 12.9, p < .001 (see Figure 4A). The serum estradiol level exhibited an inverse U-shaped (quadratic) relation to the perceptual-verbal ability (N = 25, F = 19.3, p < .001). In right-handed female subjects, the serum testosterone level significantly correlated with the numbers of correct answers on the test (see Figure 4C; r = .53, F = 7.1, p < .05). The relation between serum estradiol level and the

**C. RH WOMEN**

**D. RH WOMEN**

**SERUM TESTOSTERONE (ng/dL)** 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5

**SERUM ESTRADIOL (pg/mL)** 0 50 100 150 200 250 300 350

number of correct answers was inverse U-shaped (Figure 4D; r = .57, F = 9.0, p < .01).

**NUMBER CORRECT ON As**

Fig. 4. Relations of number correct on As (ordinate, perceptual-verbal ability) to serum testorterone and estradiol levels (abscissa) in right-handed male (left) and female (right)

**NUMBER CORRECT ON As**

20

30

40

50

60

70

**A. CRH MEN**

Fig. 2. Relations of mental rotation test scores (ordinate) to serum testosterone (left) and estradiol (right) levels in left-handed subjects.

(A, B) and female (C, D) subjects. In males, there was no significant correlation between testosterone and IQ (Figure 3A; r = .00; N = 25). The relation between estradiol and IQ could best be described by a quadratic (inverse U shaped) function (Figure 3B). This relation was found to be statistically significant (r = .47, F = 7.2, p < .05).

Fig. 3. Relations of serum testosterone and estradiol levels (abscissa) to Cattell IQ (ordinate) in male (left) and female (right) right-handed subjects.

An opposite picture was found for the female subjects. Namely, there was a quadratic, inverse U-shaped relation between testosterone and IQ (Figure 3C; N = 16, r = .55, F = 7.3, p < .05), and there was no significant correlation between estradiol and Cattell IQ (Figure 3D; N = 16, r = .15, F = 0.4, p > .55).

### **3.5 Perceptual-verbal ability ("Finding As Test")**

#### **3.5.1 Right-handers**

70 Sex Hormones

**NUMBER CORRECT (MENTAL ROTATION)**

Fig. 2. Relations of mental rotation test scores (ordinate) to serum testosterone (left) and

(A, B) and female (C, D) subjects. In males, there was no significant correlation between testosterone and IQ (Figure 3A; r = .00; N = 25). The relation between estradiol and IQ could best be described by a quadratic (inverse U shaped) function (Figure 3B). This relation was

**CATTELL IQ** 

Fig. 3. Relations of serum testosterone and estradiol levels (abscissa) to Cattell IQ (ordinate)

90

100

110

120

130

140

**CATTELL IQ** 

**ESTRADIOL (pg/mL)**

**C. WOMEN**

**D. RH WOMEN**

0 10 20 30 40 50 60

**SERUM TESTOSTERONE** 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6

**SERUM ESTRADIOL (pg/mL)** 0 50 100 150 200 250 300 350

**TESTOSTERONE (ng/dL)** 0 2 4 6 8 10 12 14

estradiol (right) levels in left-handed subjects.

**SERUM TESTOSTERONE (ng/dL)** 2 4 6 8 10 12 14

**SERUM ESTRADIOL (pg/mL)** 0 10 20 30 40 50 60 70

in male (left) and female (right) right-handed subjects.

found to be statistically significant (r = .47, F = 7.2, p < .05).

**NUMBER CORRECT (MENTAL ROTATION)**

**A. RH MEN**

**B. RH MEN** 

**CATTELL IQ** 

**CATTELL IQ**

90

100

110

120

130

140

Figure 4 illustrates the relation between the number of correct answers on the "Finding As Test" (ordinate) to the serum testosterone and estradiol levels (abscissa) in right-handed male (A, B) and female (C, D) subjects.

There was an inverse relation between the serum free testosterone levels and the number of correct answers, and this was best described by a quadratic relation, r = .73, F = 12.9, p < .001 (see Figure 4A). The serum estradiol level exhibited an inverse U-shaped (quadratic) relation to the perceptual-verbal ability (N = 25, F = 19.3, p < .001). In right-handed female subjects, the serum testosterone level significantly correlated with the numbers of correct answers on the test (see Figure 4C; r = .53, F = 7.1, p < .05). The relation between serum estradiol level and the number of correct answers was inverse U-shaped (Figure 4D; r = .57, F = 9.0, p < .01).

Fig. 4. Relations of number correct on As (ordinate, perceptual-verbal ability) to serum testorterone and estradiol levels (abscissa) in right-handed male (left) and female (right) subjects.

Serum Free Testosterone and Estradiol Levels in

McKeever et al., 1987; McKeever & Deyo, 1990).

serum testosterone levels, as seen Figure 1A.

Perceptual-Verbal and Spatial Abilities; Differences in Sex and Hand Preference 73

preference. Consistent with a curvilinear relation, Beech (2001) found that moderate baldness reflecting high levels of blood dihydrotestosterone is related to better performance in the mental rotation test in males. Supporting the dominant beneficial effect of testosterone on spatial ability in men, Christiansen and Knussman (1987) found a positive linear correlation between testosterone (salivary and serum) and spatial performance. There are many other examples supporting beneficial testosterone effects on spatial ability, such as in female-male transsexuals (Van Goozen et al., 1994), older men (Janowsky et al., 1994, 2000), and hypogonadal men (Alexander et al., 1998). According to Silverman et al. (1999), mental rotation scores are positively correlated with mean testosterone levels, but not with changes in testosterone. Gonadectomized male rodents have been shown to exhibit reduced cognitive performance (object recognition, radial arm maze, testosterone-maze, inhibitory avoidance), which could be reversed by testosterone-replacement (Frye et al., 2003). There are also inconsistent reports of low salivary testosterone levels being advantageous and high salivary testosterone levels being disadvantageous for visuospatial ability (Gouchie & Kimura, 1991; Moffat & Hampson, 1996). There are also studies showing null relations (e.g.,

The possible reasons for these inconsistencies may be diverse sample characteristics such as differences in educational levels (see Kutlu et al., 2001), hand preferences, and different measurement techniques(e.g., salivary versus serum testosterone levels), which, however, may not be related to inconsistent results , according to Silverman et al. (1999). On the other hand, differences in body size may also play a role in inconsistent results (Tan et al., 2003a, 2003b). The results of the present work suggest that serum free testosterone may be beneficial for the mental rotation ability in men and women, except that when levels of testosterone are too high in men there is a detrimental effect on mental rotation ability. The relation of estradiol to mental rotation ability exhibited an inverse U-shaped relation to serum estradiol levels in right-handed women (Figure 1D), similar to that found in righthanded men as shown in Figure 1A. On the other hand, estradiol positively linearly correlated with mental rotation in right- and left-handed men (see Figures 1B and 2), like testosterone in right-handed women (Figure 1C). Inconsistent with these results, Wolf and Kirschbaum (2002) could not find a significant relation between endogenous estradiol levels and mental rotation in older men and women, suggesting no beneficial effects of estradiol in older people. There are, however, studies suggesting that high estradiol levels may be disadvantageous for spatial ability, consistent with the present results. For instance, in elderly women high estrogen levels showed negative relations to visuospatial functions (Drake et al., 2000; Barrett-Connor & Goodman-Gruen, 1999); performances on spatial tasks are hindered in the midluteal phase of the menstrual cycle as well as during pregnancy (Hampson, 1990; Hampson & Kimura, 1988; Maki et al., 2002; Silverman & Phillips, 1993). The inverse U-shaped variation of mental rotation ability is best seen during the menstrual cycle, parallel with the endogenous estradiol concentrations (see Figure 6 in Halpern & Tan, 2001). The above mentioned studies suggesting disadvantageous effects of high estradiol (E2) levels on spatial cognition in women support the present results. There are, however, advantageous effects of estradiol in relatively moderate concentrations; very low E concentrations were associated with low mental rotation scores (see Figure 1D).Beneficial effects of estrogen replacement therapy have frequently been reported, especially in post-menopausal women and in women with Alzheimer's disease (see Yaffe, Lui et al., 2000), so an optimal estradiol level would be most beneficial, but too much estradiol would be harmful for the spatial ability in women. The same was true for men in relation to

#### **3.5.2 Left-handers**

Since there were so few left-handed females, only the male left-handers were analyzed. Figure 5 illustrates the relation between the serum free testosterone and estradiol levels and the number of correct answers on the "Finding As Test". There were positive correlations between the number of correct answers and the serum testosterone and estradiol levels in these subjects. The correlation between the number of correct answers and serum free testosterone level was best described by a quadratic equation: r = .62, F(2, 15) = 12.1, p = .001; the serum estradiol level was positively linearly correlated by the equation: r = .88, F(1, 12) = 20.0, p < .001.

Fig. 5. Relations between perceptual-verbal ability (ordinate: number correct on A`s test) and serum free testosterone (A) and estradiol (B) levels in left-handed male subjects.

#### **4. Discussion**

#### **4.1 Mental rotation ability**

The results suggest that testosterone may be beneficial for mental rotation ability in women (see Figure 1C). Moffat and Hampson (1996) also found a positive correlation between visuospatial performance and salivary testosterone level in right-handed females, but there was no significant correlation in left-handed females. The sample size for the left-handed women was not large enough in the present work to make any statistical inferences.

The beneficial effects of testosterone on spatial ability has also been experimentally verified in humans and animals. Aleman et al (2004) found that a single testosterone administration improved mental rotation ability in young women; Roof and Havens (1992) found in rats that males outperformed females in the Morris water maze (spatial ability), and females given testosterone performed better than females without testosterone.

In right- and left-handed men, the mental rotation ability increased with testosterone for a large testosterone spectrum, but tended to decrease with very high testosterone levels, which was especially visible in right-handers (see Figures 1A and 2). In contrast to women, there are inconsistencies in the scientific literature with regard to testosterone effects on spatial ability in men. Some authors found a direct correlation, while others found an inverse relation, or an inverse U-shaped relation. The present work suggests relatively high testosterone levels would be advantageous, but very low and very high testosterone levels would be disadvantageous for the mental rotation ability in right- and left-handed men.

The present results favor the hypothesis that serum free testosterone is beneficial for visuospatial ability for a large part of the testosterone spectrum regardless of hand

Since there were so few left-handed females, only the male left-handers were analyzed. Figure 5 illustrates the relation between the serum free testosterone and estradiol levels and the number of correct answers on the "Finding As Test". There were positive correlations between the number of correct answers and the serum testosterone and estradiol levels in these subjects. The correlation between the number of correct answers and serum free testosterone level was best described by a quadratic equation: r = .62, F(2, 15) = 12.1, p = .001; the serum estradiol level was positively linearly correlated by the equation: r = .88, F(1,

**B. LH MEN**

**SERUM ESTRADIOL (pg/mL)** 0 10 20 30 40 50 60

The results suggest that testosterone may be beneficial for mental rotation ability in women (see Figure 1C). Moffat and Hampson (1996) also found a positive correlation between visuospatial performance and salivary testosterone level in right-handed females, but there was no significant correlation in left-handed females. The sample size for the left-handed

The beneficial effects of testosterone on spatial ability has also been experimentally verified in humans and animals. Aleman et al (2004) found that a single testosterone administration improved mental rotation ability in young women; Roof and Havens (1992) found in rats that males outperformed females in the Morris water maze (spatial ability), and females

In right- and left-handed men, the mental rotation ability increased with testosterone for a large testosterone spectrum, but tended to decrease with very high testosterone levels, which was especially visible in right-handers (see Figures 1A and 2). In contrast to women, there are inconsistencies in the scientific literature with regard to testosterone effects on spatial ability in men. Some authors found a direct correlation, while others found an inverse relation, or an inverse U-shaped relation. The present work suggests relatively high testosterone levels would be advantageous, but very low and very high testosterone levels would be disadvantageous for the mental rotation ability in right- and left-handed men. The present results favor the hypothesis that serum free testosterone is beneficial for visuospatial ability for a large part of the testosterone spectrum regardless of hand

women was not large enough in the present work to make any statistical inferences.

given testosterone performed better than females without testosterone.

Fig. 5. Relations between perceptual-verbal ability (ordinate: number correct on A`s test) and serum free testosterone (A) and estradiol (B) levels in left-handed male subjects.

**NUMBER CORRECT ON A's**

30

35

40

45

50

55

**3.5.2 Left-handers** 

12) = 20.0, p < .001.

**A. LH MEN**

**NUMBER CORRECT ON As**

30

**4. Discussion** 

**4.1 Mental rotation ability** 

35

40

45

50

55

**SERUM TESTOSTERONE (ng/dL)** 2 4 6 8 10 12 14 preference. Consistent with a curvilinear relation, Beech (2001) found that moderate baldness reflecting high levels of blood dihydrotestosterone is related to better performance in the mental rotation test in males. Supporting the dominant beneficial effect of testosterone on spatial ability in men, Christiansen and Knussman (1987) found a positive linear correlation between testosterone (salivary and serum) and spatial performance. There are many other examples supporting beneficial testosterone effects on spatial ability, such as in female-male transsexuals (Van Goozen et al., 1994), older men (Janowsky et al., 1994, 2000), and hypogonadal men (Alexander et al., 1998). According to Silverman et al. (1999), mental rotation scores are positively correlated with mean testosterone levels, but not with changes in testosterone. Gonadectomized male rodents have been shown to exhibit reduced cognitive performance (object recognition, radial arm maze, testosterone-maze, inhibitory avoidance), which could be reversed by testosterone-replacement (Frye et al., 2003). There are also inconsistent reports of low salivary testosterone levels being advantageous and high salivary testosterone levels being disadvantageous for visuospatial ability (Gouchie & Kimura, 1991; Moffat & Hampson, 1996). There are also studies showing null relations (e.g., McKeever et al., 1987; McKeever & Deyo, 1990).

The possible reasons for these inconsistencies may be diverse sample characteristics such as differences in educational levels (see Kutlu et al., 2001), hand preferences, and different measurement techniques(e.g., salivary versus serum testosterone levels), which, however, may not be related to inconsistent results , according to Silverman et al. (1999). On the other hand, differences in body size may also play a role in inconsistent results (Tan et al., 2003a, 2003b). The results of the present work suggest that serum free testosterone may be beneficial for the mental rotation ability in men and women, except that when levels of testosterone are too high in men there is a detrimental effect on mental rotation ability.

The relation of estradiol to mental rotation ability exhibited an inverse U-shaped relation to serum estradiol levels in right-handed women (Figure 1D), similar to that found in righthanded men as shown in Figure 1A. On the other hand, estradiol positively linearly correlated with mental rotation in right- and left-handed men (see Figures 1B and 2), like testosterone in right-handed women (Figure 1C). Inconsistent with these results, Wolf and Kirschbaum (2002) could not find a significant relation between endogenous estradiol levels and mental rotation in older men and women, suggesting no beneficial effects of estradiol in older people. There are, however, studies suggesting that high estradiol levels may be disadvantageous for spatial ability, consistent with the present results. For instance, in elderly women high estrogen levels showed negative relations to visuospatial functions (Drake et al., 2000; Barrett-Connor & Goodman-Gruen, 1999); performances on spatial tasks are hindered in the midluteal phase of the menstrual cycle as well as during pregnancy (Hampson, 1990; Hampson & Kimura, 1988; Maki et al., 2002; Silverman & Phillips, 1993). The inverse U-shaped variation of mental rotation ability is best seen during the menstrual cycle, parallel with the endogenous estradiol concentrations (see Figure 6 in Halpern & Tan, 2001). The above mentioned studies suggesting disadvantageous effects of high estradiol (E2) levels on spatial cognition in women support the present results. There are, however, advantageous effects of estradiol in relatively moderate concentrations; very low E concentrations were associated with low mental rotation scores (see Figure 1D).Beneficial effects of estrogen replacement therapy have frequently been reported, especially in post-menopausal women and in women with Alzheimer's disease (see Yaffe, Lui et al., 2000), so an optimal estradiol level would be most beneficial, but too much estradiol would be harmful for the spatial ability in women. The same was true for men in relation to serum testosterone levels, as seen Figure 1A.

Serum Free Testosterone and Estradiol Levels in

**4.3 Perceptual-verbal ability ("Finding As Test")** 

eugonadal and hypogonadal men.

relations to cognitive abilities.

detrimental; rather it seemed to be beneficial for this ability.

effects of estradiol are both recorded in the scientific literature.

Perceptual-Verbal and Spatial Abilities; Differences in Sex and Hand Preference 75

curvilinear correlations (close to inverse U) between testosterone and Cattell IQ, especially in young women. The present results showed the relations of serum free testosterone vs Cattell IQ in men, and serum estradiol vs Cattel IQ in women were insignificant, but the relations of testosterone to Cattell IQ in women, and estradiol to Cattell IQ in men showed inverse-U shaped relations (see Figure 3). So, only optimal estradiol concentrations in men and optimal

The finding As test is a perceptual-verbal test, usually yielding higher scores for females than males (e.g., Kimura & Hampson, 1994; Halpern & Tan, 2001). In the current study this test also exhibited sex-related differential correlations with testosterone and estradiol (see Figure 4). In left-handed male subjects, testosterone and estradiol levels showed positive correlations with the score on the test, but the relations were different in right-handers. The results were inconsistent with Gouchie and Kimura (1991) who reported that sex hormones may not influence mental ability tests that favor women or do not typically show a sex difference. Interestingly, Tan et al. (2003a) have reported the sex difference on the Finding As Test increased using testosterone as covariate; but disappeared with covariates of estradiol and

testosterone concentrations in women are beneficial for this visuospatial ability.

progesterone, suggesting the remarkable dependence of this test on hormonal milieu.

Serum free testosterone level was inversely related to perceptual-verbal ability in righthanded men: increasing testosterone levels seemed to be detrimental (Figure 4A). Van Goozen et al. (1994) have also found that verbal fluency deteriorated after testosterone levels were increased into the physiological range for men. Alexander et al. (1998) could not find any significant association between testosterone and perceptual speed in this test in

In women, there was a positive relation between testosterone and perceptual-verbal ability: the score on the test increased with serum free testosterone levels (Figure 4C). In support, Drake et al. (2000) reported that higher testosterone levels were associated with superior verbal fluency in older women. A positive correlation was also found between testosterone and verbal memory in healthy elderly women (Wolf & Kirschbaum, 2002). It was argued that high levels of male sex steroids may impair performance on tests in which women outperform men (see Almeida, 1999; Wolf et al., 2000). In the present work, women outperformed men in the Finding As Test, but, despite that, testosterone was not

Interestingly, there was an inverse U-shaped relation between estradiol and the Finding As Test score in right-handed males and females. This means there is an optimum estradiol level for this perceptual-verbal ability, as also shown for the pre-ovulatory phase of the menstrual cycle, where serum estradiol level first increases and then decreases towards the ovulation (see Figure 5 in Halpern & Tan, 2001). So, advantageous and disadvantageous

The serum testosterone and estradiol levels were found to be positively correlated with the perceptual-verbal ability in the left-handed male subjects (Figure 5). There is no report from the current literature to compare the present results on this topic. The results suggest opposite relations of serum testosterone and estradiol levels to the perceptual-verbal ability in right- and left-handers, accentuating the importance of cerebral laterality in sex-hormone

The scientific literature abounds with examples of hormonal effects on various abilities. For example, estrogen was found to have a possible positive effects on oral reading in postmenopausal women (Shaywitz et al., 2003), but an association between estrogen level and

These two relations partly support Nyborg's (1983) optimal gonadal hormone theory, which argues that there is an inverted U-shaped relationship between brain estrogen and spatial ability, with males tending more than females to occupy the peak of the curve. Nyborg assumed, however, that testosterone does not affect the brain directly, but exerts its effects by aromatization to estrogen in the brain.

There was a positive correlation between serum estradiol level and mental rotation ability in the right-handed male subjects (see Figure 1B) and this suggests that estradiol is beneficial for the mental rotation ability in men. The free testosterone level was also found to be beneficial for this ability for a large spectrum of testosterone levels. Consequently, the positive relation of serum estradiol level to mental rotation ability is to be expected, since testosterone is a circulating pro-hormone, which, through aromatization, is converted to E2, the principal ligand for estrogen receptors (see Simpson, et al., 2002). The male estradiol should be very important, since men maintain a high circulating level of the active precursor testosterone, which is available for conversion to estradiol (estradiol, or active estrogen) throughout life in extragonadal sites.

The activating effects of testosterone and estradiol on the mental rotation ability was also observed in left-handed male subjects (see Figure 2), in contrast to the results of Moffat and Hampson (1996), who could not find any relation of testosterone to spatial ability either in left-handed males or in left-handed females.

Jordan et al. (2002) have reported that women and men exhibited activations in different cortical areas during mental rotation even when performances were similar. This suggests the sex-related differences in the relations between sex hormones and mental rotation ability may be accounted for by different cerebral origins of the visuospatial abilities.

#### **4.2 Cattell's culture fair intelligence test**

There is no significant sex difference in Cattell IQ (see also Tan et al., 2003 a, b). Despite that, there were some significant relations between sex hormones and IQs. The results did not support the notion that there is no relation between testosterone and performance on some cognitive abilities at which men are not usually better (Gouchie & Kimura, 1991). The fluid intelligence, as measured by Cattell's Culture Fair Intelligence Test, has frequently been reported to be related to serum total testosterone levels in men and women (Tan, 1990 a, b; Tan & Akgun, 1992; Tan et al., 1993).

Athough Cattell's Culture Fair Intelligence Test also measures the visuospatial ability, which is similar to mental rotation ability, the relations between testosterone and estradiol to Cattell IQs were quite different from those for the mental rotation test (see Figure 3). So, different visuospatial tests may produce different results. Here again, the correlations exhibited sex-dependent results: there was no significant relation between testosterone and IQ in males, and no significant relation between estradiol and IQ in females. testosterone in women and estradiol in men exhibited curvilinear correlations with Cattell IQ. These results suggest the possible effects of testosterone and estradiol on Cattell IQ may reflect the independent, direct effects of these sex hormones on nonverbal intelligence. The differential sex-related correlations may be due to the sex-related cortical activation patterns as for the mental rotation task (see Jordan et al., 2002).

The results are good examples for the optimal hormone levels for the highest cognitive abilities. Halpern and Tan (2001) showed fluctuations in Cattell IQ with menstrual cycle, and found an inverse U-shaped relation between serum estradiol level and Cattell IQ using a larger sample size. Using the total serum testosterone level, Tan and Tan (1998) reported there were curvilinear correlations (close to inverse U) between testosterone and Cattell IQ, especially in young women. The present results showed the relations of serum free testosterone vs Cattell IQ in men, and serum estradiol vs Cattel IQ in women were insignificant, but the relations of testosterone to Cattell IQ in women, and estradiol to Cattell IQ in men showed inverse-U shaped relations (see Figure 3). So, only optimal estradiol concentrations in men and optimal testosterone concentrations in women are beneficial for this visuospatial ability.

#### **4.3 Perceptual-verbal ability ("Finding As Test")**

74 Sex Hormones

These two relations partly support Nyborg's (1983) optimal gonadal hormone theory, which argues that there is an inverted U-shaped relationship between brain estrogen and spatial ability, with males tending more than females to occupy the peak of the curve. Nyborg assumed, however, that testosterone does not affect the brain directly, but exerts its effects

There was a positive correlation between serum estradiol level and mental rotation ability in the right-handed male subjects (see Figure 1B) and this suggests that estradiol is beneficial for the mental rotation ability in men. The free testosterone level was also found to be beneficial for this ability for a large spectrum of testosterone levels. Consequently, the positive relation of serum estradiol level to mental rotation ability is to be expected, since testosterone is a circulating pro-hormone, which, through aromatization, is converted to E2, the principal ligand for estrogen receptors (see Simpson, et al., 2002). The male estradiol should be very important, since men maintain a high circulating level of the active precursor testosterone, which is available for conversion to estradiol (estradiol, or active estrogen)

The activating effects of testosterone and estradiol on the mental rotation ability was also observed in left-handed male subjects (see Figure 2), in contrast to the results of Moffat and Hampson (1996), who could not find any relation of testosterone to spatial ability either in

Jordan et al. (2002) have reported that women and men exhibited activations in different cortical areas during mental rotation even when performances were similar. This suggests the sex-related differences in the relations between sex hormones and mental rotation ability

There is no significant sex difference in Cattell IQ (see also Tan et al., 2003 a, b). Despite that, there were some significant relations between sex hormones and IQs. The results did not support the notion that there is no relation between testosterone and performance on some cognitive abilities at which men are not usually better (Gouchie & Kimura, 1991). The fluid intelligence, as measured by Cattell's Culture Fair Intelligence Test, has frequently been reported to be related to serum total testosterone levels in men and women (Tan, 1990 a, b;

Athough Cattell's Culture Fair Intelligence Test also measures the visuospatial ability, which is similar to mental rotation ability, the relations between testosterone and estradiol to Cattell IQs were quite different from those for the mental rotation test (see Figure 3). So, different visuospatial tests may produce different results. Here again, the correlations exhibited sex-dependent results: there was no significant relation between testosterone and IQ in males, and no significant relation between estradiol and IQ in females. testosterone in women and estradiol in men exhibited curvilinear correlations with Cattell IQ. These results suggest the possible effects of testosterone and estradiol on Cattell IQ may reflect the independent, direct effects of these sex hormones on nonverbal intelligence. The differential sex-related correlations may be due to the sex-related cortical activation patterns as for the

The results are good examples for the optimal hormone levels for the highest cognitive abilities. Halpern and Tan (2001) showed fluctuations in Cattell IQ with menstrual cycle, and found an inverse U-shaped relation between serum estradiol level and Cattell IQ using a larger sample size. Using the total serum testosterone level, Tan and Tan (1998) reported there were

may be accounted for by different cerebral origins of the visuospatial abilities.

by aromatization to estrogen in the brain.

throughout life in extragonadal sites.

left-handed males or in left-handed females.

**4.2 Cattell's culture fair intelligence test** 

Tan & Akgun, 1992; Tan et al., 1993).

mental rotation task (see Jordan et al., 2002).

The finding As test is a perceptual-verbal test, usually yielding higher scores for females than males (e.g., Kimura & Hampson, 1994; Halpern & Tan, 2001). In the current study this test also exhibited sex-related differential correlations with testosterone and estradiol (see Figure 4). In left-handed male subjects, testosterone and estradiol levels showed positive correlations with the score on the test, but the relations were different in right-handers. The results were inconsistent with Gouchie and Kimura (1991) who reported that sex hormones may not influence mental ability tests that favor women or do not typically show a sex difference. Interestingly, Tan et al. (2003a) have reported the sex difference on the Finding As Test increased using testosterone as covariate; but disappeared with covariates of estradiol and progesterone, suggesting the remarkable dependence of this test on hormonal milieu.

Serum free testosterone level was inversely related to perceptual-verbal ability in righthanded men: increasing testosterone levels seemed to be detrimental (Figure 4A). Van Goozen et al. (1994) have also found that verbal fluency deteriorated after testosterone levels were increased into the physiological range for men. Alexander et al. (1998) could not find any significant association between testosterone and perceptual speed in this test in eugonadal and hypogonadal men.

In women, there was a positive relation between testosterone and perceptual-verbal ability: the score on the test increased with serum free testosterone levels (Figure 4C). In support, Drake et al. (2000) reported that higher testosterone levels were associated with superior verbal fluency in older women. A positive correlation was also found between testosterone and verbal memory in healthy elderly women (Wolf & Kirschbaum, 2002). It was argued that high levels of male sex steroids may impair performance on tests in which women outperform men (see Almeida, 1999; Wolf et al., 2000). In the present work, women outperformed men in the Finding As Test, but, despite that, testosterone was not detrimental; rather it seemed to be beneficial for this ability.

Interestingly, there was an inverse U-shaped relation between estradiol and the Finding As Test score in right-handed males and females. This means there is an optimum estradiol level for this perceptual-verbal ability, as also shown for the pre-ovulatory phase of the menstrual cycle, where serum estradiol level first increases and then decreases towards the ovulation (see Figure 5 in Halpern & Tan, 2001). So, advantageous and disadvantageous effects of estradiol are both recorded in the scientific literature.

The serum testosterone and estradiol levels were found to be positively correlated with the perceptual-verbal ability in the left-handed male subjects (Figure 5). There is no report from the current literature to compare the present results on this topic. The results suggest opposite relations of serum testosterone and estradiol levels to the perceptual-verbal ability in right- and left-handers, accentuating the importance of cerebral laterality in sex-hormone relations to cognitive abilities.

The scientific literature abounds with examples of hormonal effects on various abilities. For example, estrogen was found to have a possible positive effects on oral reading in postmenopausal women (Shaywitz et al., 2003), but an association between estrogen level and

Serum Free Testosterone and Estradiol Levels in

Vol.33, pp. 85-94.

187-195.

1289-1293.

10, pp. 3681-3685.

No.2, pp. 59-69.

No. 3, pp. 599-603.

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verbal memory has not been generally supported (see Green et al., 2000). Wolf & Kirschbaum, (2002) have indeed shown that in older women higher estradiol levels were associated with better verbal memory (paired associates). Anti-androgen therapy in combination with estrogen treatment was shown to have no enhancing effect on verbal fluency in adult men (Slabbekoorn et al., 1999). Women were shown to have enhanced verbal articulation during the pre-ovulatory phase of the menstrual cycle when only estrogren levels were high (Hampson, 1990).Manipulations of sex hormone levels appeared to be unrelated to verbal skills and perceptual speed in transsexuals (Slabbekoorn et al., 1999), while in menopausal women estrogen replacement therapy have been shown to improve cognition, especially verbal memory (Wolf, 2003). Serum free and bioavailable estradiol may have beneficial effects for cognitive decline in older women (Yaffe, Haan et al., 2000), but this benefit may be obtained with a low dose of estrogen (Tierney, 2000).

#### **5. Conclusions and perspectives**

Since the most prominent sex difference in mental rotation ability—being better in men than women— reversed under covariation of bodily measures such as height and weight, to be better in women than men, and similar changes were observed in other cognitive tests, the sex differences in perceptual-verbal and spatial abilities were re-studied in the present work, which recruited men and women with similar height and weight and with known right- or left-hand preference.

The mental rotation ability predominantly increased with serum free-testosterone levels in right- and left-handed men and women, but there were detrimental effects of very large testosterone concentrations in the same individuals. The serum estradiol concentrations showed a direct relationship to mental rotation test scores in right- and left-handed men, but an inverse U-shaped relation in right-handed women. The relation of perceptual-verbal ability *vs* testosterone exhibited a different pattern: in right-handers the correlation was negative, while in left-handers it was positive. This suggests an optimum estradiol level could be beneficial for this ability in men and women.

The sex-neutral Cattell IQ did not show any significant relationship either to the serum testosterone level in right-handed men or to the estradiol level in right-handed women. Optimal testosterone and estradiol levels (not too high and not too low) were needed for the highest Cattell IQs in right-handed men and women, but if levels of these hormones were too high or too low this was detrimental for the sex-neutral fluid intelligence.

The results of the present work suggest that the sex-related differences in cognitive abilities may depend upon the hand preference and the cognitive tests used to measure the cognitive abilities. Sex-hormones may exert beneficial or detrimental effects on the cognitive abilities, depending upon their serum concentrations and cerebral laterality differences. The results also suggest that bodily measures, hand preference, and sex as basic variables should be considered in basic research and clinical evaluations, as well as in future treatment for cognitive impairments and related cognitive disorders such as dementia in men and women. The results may also be enlightening for future research on the physiological mechanisms of cognitive abilities.

#### **6. Acknowledgments**

This study was partly supported by the Turkish Academy of Sciences.

#### **7. References**

76 Sex Hormones

verbal memory has not been generally supported (see Green et al., 2000). Wolf & Kirschbaum, (2002) have indeed shown that in older women higher estradiol levels were associated with better verbal memory (paired associates). Anti-androgen therapy in combination with estrogen treatment was shown to have no enhancing effect on verbal fluency in adult men (Slabbekoorn et al., 1999). Women were shown to have enhanced verbal articulation during the pre-ovulatory phase of the menstrual cycle when only estrogren levels were high (Hampson, 1990).Manipulations of sex hormone levels appeared to be unrelated to verbal skills and perceptual speed in transsexuals (Slabbekoorn et al., 1999), while in menopausal women estrogen replacement therapy have been shown to improve cognition, especially verbal memory (Wolf, 2003). Serum free and bioavailable estradiol may have beneficial effects for cognitive decline in older women (Yaffe, Haan et al., 2000), but this benefit may be obtained with a low dose of estrogen (Tierney, 2000).

Since the most prominent sex difference in mental rotation ability—being better in men than women— reversed under covariation of bodily measures such as height and weight, to be better in women than men, and similar changes were observed in other cognitive tests, the sex differences in perceptual-verbal and spatial abilities were re-studied in the present work, which recruited men and women with similar height and weight and with known right- or

The mental rotation ability predominantly increased with serum free-testosterone levels in right- and left-handed men and women, but there were detrimental effects of very large testosterone concentrations in the same individuals. The serum estradiol concentrations showed a direct relationship to mental rotation test scores in right- and left-handed men, but an inverse U-shaped relation in right-handed women. The relation of perceptual-verbal ability *vs* testosterone exhibited a different pattern: in right-handers the correlation was negative, while in left-handers it was positive. This suggests an optimum estradiol level

The sex-neutral Cattell IQ did not show any significant relationship either to the serum testosterone level in right-handed men or to the estradiol level in right-handed women. Optimal testosterone and estradiol levels (not too high and not too low) were needed for the highest Cattell IQs in right-handed men and women, but if levels of these hormones were

The results of the present work suggest that the sex-related differences in cognitive abilities may depend upon the hand preference and the cognitive tests used to measure the cognitive abilities. Sex-hormones may exert beneficial or detrimental effects on the cognitive abilities, depending upon their serum concentrations and cerebral laterality differences. The results also suggest that bodily measures, hand preference, and sex as basic variables should be considered in basic research and clinical evaluations, as well as in future treatment for cognitive impairments and related cognitive disorders such as dementia in men and women. The results may also be enlightening for future research on the physiological

too high or too low this was detrimental for the sex-neutral fluid intelligence.

This study was partly supported by the Turkish Academy of Sciences.

**5. Conclusions and perspectives** 

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mechanisms of cognitive abilities.

**6. Acknowledgments** 

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lordosis of rats through effects on midbrain 3 alpha,5 alpha-THP concentrations.


**1. Introduction** 

**4** 

*Turkey* 

**Sex Hormones and Infertility** 

*Erciyes University / Department of Obstetric and Gynecology* 

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

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

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

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

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

progesteron, and a functional uterus that can be responded by these hormones.

extracellular signals to the nucleus to affect a physiologic response.

lengthening of the interval between pulses with higher amplitude (5).

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

minutes as a result of rapid proteolytic cleavage.

Iptisam Ipek Muderris and Gokalp Oner

