**2.1 Follicular phase**

The follicular phase starts from the first day of menstrual cycle D1 until ovulation. Basal body temperature chart normally shows lower values during this phase. Development growth trajectories of the ovarian follicles characterize this first phase of the cycle. Folliculogenesis starts during the last few days of the preceding menstrual cycle and continues till the release of the mature follicle at the time of ovulation. The primary development of the follicle to the preantral follicle is not gonadotropin dependent, and further follicular growth beyond this point requires gonadotropin action.

The secretion of gonadotropins from anterior pituitary is regulated by gonadotropin releasing hormone (GnRH), steroid hormones, and various peptides released by the dominant follicle. The growth of follicular size and number of granulosa cells in each follicle leads to an increase in estradiol serum concentrations in the early follicular phase. FSH receptors exist only on the granulosa cell membrane. There is increase in the number of FSH receptors with the gradual rise in serum FSH levels during the late follicular phase. The rise in serum FSH along with the rise in FSH

### **Figure 2.**

*Theca and granulosa cells (two cells) respond to luteinizing hormone and follicle stimulating hormone (two gonadotropins) to produce 17-OH progesterone and estradiol (two hormones).*

receptors leads to an increase in estradiol secretion by granulosa cells. It has to be noted at this point clearly that the increase in FSH receptor numbers is due to an increase in the population of granulosa cells and not an increase in the concentration of FSH receptors per granulosa cell. FSH has a positive stimulating effect for the formation of LH receptors on granulosa cells thus allowing the production of small quantities of progesterone and 17-hydroxyprogesterone (17-OHP) that exert a positive feedback on the estrogen-primed pituitary to augment luteinizing hormone (LH) release. Without LH, the increased progesterone synthesis from granulosa cells under the influence of FSH advances the endometrium and the resulting asynchronous development reduces the chances of embryo implantation (**Figure 2**). This phenomenon is also called as ENLOP or Elevated Non Luteinised Origin of Progesterone leading to displaced window of implantation. The addition of LH in follicular phase reduces premature progesterone increase and improves the likelihood of implantation and clinical pregnancy.

FSH is elevated during the early follicular phase, declines as estradiol secretion increases from the granulosa cells, has a second rise before ovulation and then begins to decline until ovulation. FSH also stimulates several steroidogenic enzymes including aromatase, and 3β-hydroxysteroid dehydrogenase (3β-HSD). The secondary rise of FSH is important for inducing LH receptors on theca cells and granulosa cells and thereby preparing for the action of LH surge.

### **2.2 Luteal phase**

The positive feedback from the rising estrogen levels during the follicular phase results in gradual increase in LH level by mid follicular phase that is low to start with during the early follicular phase. Estradiol levels must be greater than 200 pg/mL for approximately 50 hours in duration for the positive feedback effect of LH release to occur. During the early follicular phase, LH secretion occurs at a pulse frequency of

**65**

**3. Ovarian reserve**

*Ovarian Reserve*

**Figure 3.**

*DOI: http://dx.doi.org/10.5772/intechopen.89772*

60 to 90 minutes with relatively constant pulse amplitude. During the late follicular phase prior to ovulation, the pulse frequency and may be amplitude of LH secretion increases. LH pulse amplitude increase results in ovulation [1, 2]. After ovulation

*The cyclical harmonious balance of gonadotropin secretion from pituitary and estrogen and progesterone* 

The reduction of steroid production by the corpus luteum and the sudden fall of inhibin A allow the follicle stimulating hormone (FSH) to increase during the last few days of the menstrual cycle. In the late luteal phase, as corpus luteum lso degenerates there is no the estrogen and progesterone secretion from the ovary resulting in FSH rise because of increased GnRH pulsatile secretion. This elevation in FSH is very crucial for the recruitment of a cohort of ovarian follicles in each ovary out of that one follicle will be destined to ovulate during the next menstrual cycle. As menstruation starts the FSH levels begin to decline due to the negative feedback of estrogen and the negative effects of inhibin B produced by the developing follicle

Ovarian reserve plays an important role in achieving pregnancy following any treatment in infertile and sub fertile women. The main function of the ovary in a woman is the production of a mature and viable oocyte that is capable of fertilization and subsequently leads to an embryo development and implantation. At birth, each ovary has a fixed number of oocytes available for folliculogenesis during the later life. This fixed number of available oocytes is termed as "the ovarian reserve" of the woman. Delayed childbearing, voluntary or involuntary, is a common feature in couples visiting fertility clinics nowadays as they are career oriented and mostly working. The estimation of ovarian reserve is routinely performed prior to interventions through various ovarian reserve tests (ORTs) in an effort to predict the response and outcome in couples in vitro fertilization techniques and to even counsel them. The ovarian reserve estimation has to be routinely performed prior

corpus luteum is formed and theca cells continue to secrete progesterone.

*secretion from ovary results in LH surge resulting in final maturation of oocyte and ovulation.*

[1, 2]. The cyclical hormone changes are depicted in **Figure 3**.

### **Figure 3.**

*Innovations in Assisted Reproduction Technology*

hood of implantation and clinical pregnancy.

cells and thereby preparing for the action of LH surge.

receptors leads to an increase in estradiol secretion by granulosa cells. It has to be noted at this point clearly that the increase in FSH receptor numbers is due to an increase in the population of granulosa cells and not an increase in the concentration of FSH receptors per granulosa cell. FSH has a positive stimulating effect for the formation of LH receptors on granulosa cells thus allowing the production of small quantities of progesterone and 17-hydroxyprogesterone (17-OHP) that exert a positive feedback on the estrogen-primed pituitary to augment luteinizing hormone (LH) release. Without LH, the increased progesterone synthesis from granulosa cells under the influence of FSH advances the endometrium and the resulting asynchronous development reduces the chances of embryo implantation (**Figure 2**). This phenomenon is also called as ENLOP or Elevated Non Luteinised Origin of Progesterone leading to displaced window of implantation. The addition of LH in follicular phase reduces premature progesterone increase and improves the likeli-

*Theca and granulosa cells (two cells) respond to luteinizing hormone and follicle stimulating hormone (two* 

*gonadotropins) to produce 17-OH progesterone and estradiol (two hormones).*

FSH is elevated during the early follicular phase, declines as estradiol secretion increases from the granulosa cells, has a second rise before ovulation and then begins to decline until ovulation. FSH also stimulates several steroidogenic enzymes including aromatase, and 3β-hydroxysteroid dehydrogenase (3β-HSD). The secondary rise of FSH is important for inducing LH receptors on theca cells and granulosa

The positive feedback from the rising estrogen levels during the follicular phase results in gradual increase in LH level by mid follicular phase that is low to start with during the early follicular phase. Estradiol levels must be greater than 200 pg/mL for approximately 50 hours in duration for the positive feedback effect of LH release to occur. During the early follicular phase, LH secretion occurs at a pulse frequency of

**64**

**2.2 Luteal phase**

**Figure 2.**

*The cyclical harmonious balance of gonadotropin secretion from pituitary and estrogen and progesterone secretion from ovary results in LH surge resulting in final maturation of oocyte and ovulation.*

60 to 90 minutes with relatively constant pulse amplitude. During the late follicular phase prior to ovulation, the pulse frequency and may be amplitude of LH secretion increases. LH pulse amplitude increase results in ovulation [1, 2]. After ovulation corpus luteum is formed and theca cells continue to secrete progesterone.

The reduction of steroid production by the corpus luteum and the sudden fall of inhibin A allow the follicle stimulating hormone (FSH) to increase during the last few days of the menstrual cycle. In the late luteal phase, as corpus luteum lso degenerates there is no the estrogen and progesterone secretion from the ovary resulting in FSH rise because of increased GnRH pulsatile secretion. This elevation in FSH is very crucial for the recruitment of a cohort of ovarian follicles in each ovary out of that one follicle will be destined to ovulate during the next menstrual cycle. As menstruation starts the FSH levels begin to decline due to the negative feedback of estrogen and the negative effects of inhibin B produced by the developing follicle [1, 2]. The cyclical hormone changes are depicted in **Figure 3**.

### **3. Ovarian reserve**

Ovarian reserve plays an important role in achieving pregnancy following any treatment in infertile and sub fertile women. The main function of the ovary in a woman is the production of a mature and viable oocyte that is capable of fertilization and subsequently leads to an embryo development and implantation. At birth, each ovary has a fixed number of oocytes available for folliculogenesis during the later life. This fixed number of available oocytes is termed as "the ovarian reserve" of the woman. Delayed childbearing, voluntary or involuntary, is a common feature in couples visiting fertility clinics nowadays as they are career oriented and mostly working. The estimation of ovarian reserve is routinely performed prior to interventions through various ovarian reserve tests (ORTs) in an effort to predict the response and outcome in couples in vitro fertilization techniques and to even counsel them. The ovarian reserve estimation has to be routinely performed prior

to any interventions for infertility through various ovarian reserve tests (ORTs) in an effort to predict the response and outcome in couples seeking help for infertility treatment such. The widely used tests are basal follicle stimulating hormone, Anti-Mullerian Hormone (AMH) and Antral Follicle Count (AFC). Ovarian reserve reduction is a physiological phenomenon characterized by declining follicular pool and oocyte quality. The reduction of ovarian reserve starts at about 30 years in south Asian population and at 35 years in Caucasians [3].This rate of age-related reduction of follicle count in the human ovary is more than doubles when numbers fall below a critical figure of 25,000 at ~37.5 years of age [4].

### **3.1 Ovarian reserve testing**

Ovarian reserve tests (ORT) serve as an indirect measures of a woman's remaining follicular pool when she presents herself for infertility treatment. ORT should be easy to perform, should be sensitive, specific, valid, and help to individualize the starting dose of gonadotropins for multifollicular development. The ovarian reserve testing helps to differentiate normoresponders, hyporesponders, and hyperresponders. Ovarian reserve is deciphered through a number of markers. These markers also help to prognosticate poor responders. Ovarian reserve is predicted clinically using a combination of clinical, biochemical and biophysical tests.

The tests are being used all over the world but the sensitivity and specificity of these test to detect the oocyte number, quality, and fecundity has to be still ascertained with further research [5]. More recently, their value in predicting hyperresponse and hypo response and thus using safe stimulation regimes to prevent OHSS is also explored [2]. The interpretation of the results of the ovarian reserve test is complicated by the lack of uniform definitions for hypo or hyper-responders and uniform threshold values to identify abnormal results. Several static and dynamic ovarian functional markers like biological (age), biochemical, biophysical, and histological tests have been used to identify ovarian reserve [1, 2].

In most cases of decreased ovarian reserve, the cause remains undetected. In specific cases like exposure to chemotherapy, pelvic irradiation, and genetic abnormalities there is a premature decrease in ovarian pool of oocytes. Cigarette smoking has been associated with a decrease in ovarian reserve. With diminished ovarian reserve a reproductive age woman has regular periods with normal or shortened duration of menstrual cycles but there is a decrease in response to ovarian stimulation and fecundity. Thus women of same age can differ in their response to ovarian stimulation and thus the fecundity can vary.

### *3.1.1 Age*

It is long established that ovarian reserve reduces progressively with age. This is due to a combination of two factors the body 'spending' the eggs through routine ovulation and the ovaries aging and preparing for menopause. Individual variation of the ovarian reserve can be explained by the two instances given as- a young woman with certain reproductive health problems may start out with smaller than the normal reserve of healthy eggs, and some women's reserves decrease more quickly than others with age. Fecundity in both natural and stimulated ovarian cycles declines with maternal age, beginning in the late 20s and becoming more abrupt in the late 30s. The fall in ovarian reserve with age in is a universal phenomenon in all ethinic groups. The initiation and rate of this decline varies considerably with ethinicity. Calender Age *per se* cannot determine ovarian responses. Ovarian reserve can also be traced indirectly by other biochemical and biophysical markers of ovarian function [6–11].

**67**

*Ovarian Reserve*

*DOI: http://dx.doi.org/10.5772/intechopen.89772*

One of the most classically used biochemical levels to measure ovarian reserve is the Basal follicle stimulating hormone (FSH) levels measured on day 3 of the menstrual cycle. An increase in FSH levels occurs due to follicle depletion as the age of the woman progresses [9, 10]. The measurement of FSH is easy, and inexpensive reproducible and its specific. FSH levels are known to have diurnal, intraand intercycle variations that have to be kept in mind. There is definite precise parameter value to detect a woman with poor ovarian reserve. A vague demarking values more than 25 IU/L was used arbitrarily in some studies to detect high basal FSH. Several subsequent reviews did not identify values to satisfy the specificity and sensitivity for basal FSH as a test for poor ovarian response to stimulation or prediction of non-pregnancy. In women with regular menstrual cycles, FSH can predict a poor response adequately only at very high levels, and hence will be helpful only to a small number of women as a screening test for ovarian reserve testing and further counseling. It is thus clear that the ovarian aging begins several years prior to any elevation in FSH levels is noted and thus a normal test cannot rule out a poor ovarian response in some women. When FSH level is combined with other markers it can be used to counsel couples and planning treatment option regarding a poor response but it should not be used to exclude regularly cycling women from ART. The specificity of basal FSH testing in a general sub-fertile population or elevated levels in young, regularly cycling women is thus unclear and needs further studies [11–13]. Additionally the reliability of FSH is challengeable because of its pulsatile and circadian release and its isoforms. There are no cut off values available

Anti-Mullerian hormone (AMH) is a dimeric glycoprotein exclusively produced by granulosa cells of preantral (primary and secondary) and very small and small antral follicles (2–6 mm) in the ovary. The serum levels of AMH reflect the number of follicles that have made the transition from primordial pool into the growing pool but still it is not under gonadotrophin control. The secretion of AMH starts once there is a follicular transition from the primordial to the primary stage, and it continues until the follicles reach till the follicles attain antral stages of diameters 2–6 mm. The number of the small Antral Follicles indirectly reflects size of the primordial follicle pool. With the decrease in the number of the antral follicles with age, AMH production seems to reduce and become undetectable at and after menopause. The physiological function of AMH is to modulate primordial follicle recruitment. It inhibits the action of FSH on follicular growth and selection. AMH is considered to be reflective of FSH independent follicular growth, so it is a direct measure of ovarian reserve. AMH reflects qualitative and quantitative assessment of ovarian reserve. AMH levels also strongly correlate with basal antral follicle count (AFC) measured by transvaginal ultrasonography. Serum AMH levels correlate inversely with age from 25 years onwards and reaches undetectable levels after menopause, thus AMH levels is an important ovarian reserve marker. Serum AMH levels can be measured on any day of the cycle and does not exhibit inter-cycle variability unlike other biochemical markers. Threshold values of 0.2–1.26 ng/ml, have been used to identify poor responders with 80–87% sensitivity and 64–93% specificity. Thus by understanding of its clinical implications, AMH too has the potential to predict a hyper-response during treatment as well. The nomograms of the values of AMH can predict and identify the age-related physiological decline in the AMH levels and thus ovarian reserve, and abnormal deviation in the levels of AMH can be

*3.1.2 Basal follicle stimulating hormone*

to predict poor responders.

*3.1.3 Anti-Mullerian hormone*
