**5.1 Antral follicle count**

*Innovations in Assisted Reproduction Technology*

"Insipient Ovarian Failure."

ovarian failure.

*3.1.4 Inhibin B*

is not recommended [19].

thus resulting in misinterpretation of the test.

**4. Clomiphene citrate challenge test**

*3.1.5 Basal estradiol*

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

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

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

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

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

**68**

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

### **Figure 4.**

*(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.*

in the early menstrual cycle, the AFC is believed to correlate strongly with the number of primordial follicles present in the ovary and, thus, the ovarian reserve. Antral Follicles are routinely measured by 2 D transvaginal ultrasonography in the early follicular phase, by taking the mean of two perpendicular measurements. Inversion made is useful for counting multiple follicles. The numbers of follicles in both ovaries are added for the total Antral Follicle count. (AFC). AFC has been predominantly used as a marker of ovarian reserve over a period of time. A count of 8–10 is taken as a normal response of ovaries. Different diameters are used to define antral follicles of varying sizes as those measuring 2–6 and 7–10 mm. There is no clear consensus regarding the size of antral follicles, which truly represent ovarian reserve. The number of small antral follicles (2–6 mm) is significantly related to age and also to all endocrine ORTs tested, suggesting the number of small antral follicles represents the functional ovarian reserve. It is seen that the number of antral follicles of 2–6 mm in size decreases with age and correlates with other markers such as serum basal FSH and CCCT whereas follicles of size 7–10 mm remains constant and thus, the former appears to be a more reliable marker of ovarian reserve. Measurements taken repeatedly of the antral follicles have shown that there is only a limited intercycle variability. 3D ultrasound imaging also does not carry any better advantage in comparison to 2D ultrasound for the detection of functional ovarian reserve [22–25]. Meta-analyses showed that women with AFC less than four were 8.7 times more likely not to get pregnant after IVF (two studies; 95% CI,) than women with AFC four or more. The sensitivity and specificity of AFC to predict cycle cancelation was 66.7 and 94.7%, respectively [21].
