*3.1.3 Anti-Mullerian hormone*

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 used for counseling couples wishing to delay childbirth. Still the available evidence is not sufficient enough to suggest that serum AMH can be used as a single marker to predict pregnancy. Furthermore, studies of the levels of follicular fluid AMH has shown that oocytes obtained from follicles with higher levels of AMH have a better fertility potential compared to those with lower AMH levels [14–16]. Serum AMH estimations have also been useful to diagnose "Transitional Ovarian Failure" and "Insipient Ovarian Failure."

Studies conducted longitudinally in fertile women have clearly shown a decline in serum AMH levels with progressing age. AMH is one of the earliest markers to show a decline progressively in young women with aging thus offering the probability of a screening test for women to counsel against delay in childbirth. Levels of 0.5–1.26 ng/ml of AMH suggests impending menopause in next 3–5 years [17, 18]. AMH is also a most promising marker for predicting age of natural menopause too. The serum levels of AMH are not controlled by hypothalamus pituitary axis that makes it important marker in diagnosing conditions such as PCOS and premature ovarian failure.

### *3.1.4 Inhibin B*

Inhibin B is a glycoprotein hormone produced by small ovarian follicular granulosa cells and thereby it is an indirect indicator of the follicular pool. Inhibin B is not a reliable parameter for measuring ovarian reserve though serum levels <45 pg/ml have been associated with poor response to controlled ovarian stimulation since it is not a reliable predictor of pregnancy. Inhibin B levels are lower in poor responders than in women with normal ovarian reserve. Inhibin B levels if exaggerated in stimulated cycle is an indicator of hyper response thus it can be used to monitor the response to exogenous FSH. Use of Inhibin B as a sole predictor of ovarian response is not recommended [19].

### *3.1.5 Basal estradiol*

Estradiol is a steroid hormone secreted by the granulosa cells of the growing ovarian follicles. Day 2 or Day 3 basal estradiol is commonly assessed for observing the early oocyte development. Estradiol also exerts a negative feedback on the secretion of FSH from the pituitary thus high basal estradiol can reduce the FSH levels. Thus it is a helpful parameter in combination with FSH to establish the baseline ovarian reserve. Elevated basal estradiol has been associated with a poor response to ovarian stimulation. An early rise in serum estradiol is a characteristic sign of reproductive aging and can lower the elevated basal FSH into normal range thus resulting in misinterpretation of the test.

### **4. Clomiphene citrate challenge test**

This is a dynamic test of ovarian function. This basically involves the Day 3 testing of basal FSH levels and serum estradiol. Administering 100 mg of Clomiphene citrate tablets per day for 5 days from Day 5 to Day 9 of the cycle follows this. The FSH level is measured on Day 10 of the cycle. In cases of low reserve FSH is elevated on Day 10. FSH is the primary stimulus for final follicular maturation. It is under negative feedback from estradiol and inhibin B. Basal FSH levels are elevated it indicates a diminished follicular pool. Clomiphene citrate challenge test is a good predictor of ovarian reserve but not an absolute indicator of ovarian hypofunction. Thus the clinical value of CCCT is not clearly better than basal FSH and AFC in

**69**

**Figure 4.**

*Ovarian Reserve*

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

**5. Ultrasound parameters**

**5.1 Antral follicle count**

combination. The Day 3 or Day10 FSH levels more than 10 mIU/ml is considered abnormal by most studies. Though there are different opinions still CCCT remains a gold standard in testing ovarian reserve. The justification of this test is that as the ovarian follicle develops in patients with normal ovarian function and will produce levels of inhibin and estradiol sufficient enough to suppress FSH production by Day 10 of the cycle concerned [20].Other dynamic tests used are GAST (GnRH Analogue Stimulation Test) and EFFORT (Exogenous FSH Ovarian Reserve Test).

After menarche, gradually a regular bi-fortnight ovulation is established. The immature oocyte covered with granulosa and theca cells rests in a small fluid filled cavity called as the antral follicle. These small fluid filled cavities are visualized sonologically in early follicular phase. These reflect follicles that were selected from the primordial follicle in this wave (wave theory of folliculogenensis) and so the antral follicle counts may vary in various mentstral cycles. There is no clearcut consensus on the criteria to identify antral follicles. Various litreture reviews suggest that the follicles with a diameter of 2 to 10 mm can be considered as AFa [5, 21]. Thus, the antral follicle count (AFC) is the number of follicles with cavity less than 10 mm in diameter with Transvaginal Ultrasound (TVUS) imaging in the early follicular phase of the cycle (**Figure 4a–c**). Antral follicle count is a quantitative aspect of ovarian aging. As a direct marker of the cohort of growing follicles

*(a) Ovarian antral follicular count in hypo responders; (b) ovarian antral follicular count in normal* 

*responders; and (c) ovarian antral follicular count in hyper responders.*

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

combination. The Day 3 or Day10 FSH levels more than 10 mIU/ml is considered abnormal by most studies. Though there are different opinions still CCCT remains a gold standard in testing ovarian reserve. The justification of this test is that as the ovarian follicle develops in patients with normal ovarian function and will produce levels of inhibin and estradiol sufficient enough to suppress FSH production by Day 10 of the cycle concerned [20].Other dynamic tests used are GAST (GnRH Analogue Stimulation Test) and EFFORT (Exogenous FSH Ovarian Reserve Test).
