Ovarian reserve and infertility Dr. Sundus Yousif Kellow C.A.B.O.G

Definition Ovarian reserve is a term used to describe the functional potential of the ovary and reflects the number and quality of oocytes within it. A good test of ovarian reserve should be predictive of conception (with or without treatment) and should indicate how long current levels of ovarian activity can be maintained before ovarian ageing sets in.

Ovarian Reserve

Ovarian reserve is a complex clinical phenomenon that is influenced by age, genetics, environmental variable For the general practitioner performing an infertility evaluation, we recommend focusing on the following groups of women for ovarian reserve testing: • women over 30 years of age • women with a history of exposure to a confirmed gonadotoxin, i.e., tobacco smoke, chemotherapy, radiation therapy. • women with a strong family history of early menopause or premature ovarian failure. • women who have had extensive ovarian surgery, i.e., cystectomy and unilateral oophorectomy.

In a subfertile population attending for fertility treatment, a test of ovarian reserve should guide us in prognosticating outcome in individual cases by (i) predicting the chances of pregnancy and live birth with or without treatment and ; (ii) selecting an optimal dose of ovarian stimulation where treatment using ovarian stimulation is planned.

Markers of ovarian reserve A/Static tests      1 - Age      2 - Basal serum FSH      3 - Basal serum estradiol      4 - Basal LH/FSH ratio      5 - Basal serum inhibin-B level      6 - Basal serum anti-Müllerian hormone level      7 - Basal ovarian volume      8 - Basal antral follicle count      9 - Ovarian stromal blood flow     

B/Dynamic tests 1 - Clomiphene citrate challenge test (CCCT) 2 - GnRH agonist stimulation test (GAST) 3 - Exogenous FSH ovarian reserve test (EFORT) The ideal ORT that may be usefule clinically has yet to be ascertained. Age and fertility: In many cases, a woman’s age is the single most important indicator of fertility potential. A woman’s fertility starts decreasing in her late twenties, and decreases further after age 35. While a 20 year old woman and a 40 year old woman ovulate the approximate same number of times each year, their monthly pregnancy rate, or fecundity, is much different.

Natural Decline of Oocytes with Age

FSH FSH is the hormone released by the pituitary gland in the brain to stimulate the ovaries to produce a dominant follicle (which contains an egg). A “good quality” egg releases certain substances (e.g. inhibin-B, estrogen) that suppress the FSH level (negative feedback). When the egg quality is compromised, these negative feedback signals are weak and there is a resultant increase in FSH levels. Day 3 FSH is an indirect measure of the size of the follicle cohort and is regulated by various factors, including inhibins, activins, estradiol and follistatins .

It is important to realize that FSH levels have low sensitivity, meaning that not everyone with a diminished ovarian reserve will have an abnormally elevated FSH level. Large inter-cycle variations in basal FSH remain a frequent problem. An elevated FSH does mean that achieving a successful pregnancy with any type of fertility treatment, including with IVF, will be compromised. At the same time, a mild elevation in the FSH level may be a reason to pursue infertility treatment more aggressively and proceed directly to IVF.

Antral follicle count measurements *A normal ovary should have a volume of at least 3 cc with at least 6 – 15 antral follicles. *Antral follicles are small, fluid filled cysts that are normally found in the ovaries. The higher the antral follicle count, the better the fertility potential. *Small ovaries may indicate compromised fertility potential, as there may be less follicles - and therefore less eggs - available within the ovaries. *The performance of AFC for predicting failure to achieve pregnancy is poor. This is because while AFC determines the number of oocytes, a clinically relevant outcome (pregnancy or live birth) depends on oocyte quality as well as quantity.

5-10 per side

Low A F Count <6 in total

PCO

Factors affecting AFC measurements: • Oral contraceptive use (decreases) • Polycystic ovary syndrome (PCOS) (increases). Drawbacks of AFC: • Accurate assessment of AFC requires an experienced sonographer and can be limited in patients who have had pelvic surgery or uterine fibroids and in those who are obese • Moderate interobserver and intercycle variability of AFC determinations limits its reproducibility. • As with basal FSH measurement, the intercycle variability of AFC does not correlate well with IVF outcome in individual patients.

Serum estradiol Elevated basal estradiol may predict the poor response even when basal FSH is normal. The value of cycle day 3 estradiol levels in the prediction of ovarian reserve is still debatable. Inhibin-B Inhibin-B is mainly produced by the granulosa cells in growing follicles and offers a more immediate assessment of ovarian activity than other serum tests. A fall in day 3 inhibin-B levels may predict poor ovarian reserve before the expected rise in day 3 FSH.

**Factors affecting Inhibin-B measurements: • Obesity (decreases) • PCOS (increases) • Exogenous FSH administration (increases) • Oral contraceptive use (decreases). Anti-Müllerian hormone Antimüllerian hormone (AMH) also known as Müllerian Inhibiting Substance (MIS) is a new diagnostic marker of ovarian function. The existence of AMH was first proposed in 1947 by Professor Alfred Jost. This hormone is made in the testes of men. It was thought not to exist in women. In recent years, it has been found in women starting at puberty.

Anti-Müllerian hormone (AMH) is produced by the granulosa cells of the recruited follicles until they become sensitive to FSH . AMH has been identified as a regulator of the recruitment, preventing the depletion of all primordial follicle pool at once. AMH is a glycoprotein growth factor and a member of the transforming growth factor superfamily(TGF-B)with a molecular weight of 140kDa. It is primarily produced by the pool of early-growing follicles, which are believed to serve as a proxy for the number of primordial follicles in the ovary

AMH expression in mouse ovaries AMH expression in mouse ovaries. (A) AMH is expressed in granulosa cells of primary (P), preantral (PA) and small antral (SA) follicles. (B) AMH expression disappears in antral (A) and atretic (At) follicles. Expression is lost last in the granulosa cells surrounding the oocytes. AMH expression was detected using a monoclonal antibody that recognises rat, mouse and human AMH. Reproduction. 2006 Jan;131(1):1-9 Copyright ©2010 Society for Reproduction and Fertility

Model of AMH action in the ovary Model of AMH action in the ovary. Progressing stages of folliculogenesis are depicted. AMH is produced by the small growing (primary and preantral) follicles in the postnatal ovary and has two sites of action. It inhibits initial follicle recruitment (1) and inhibits FSH-dependent growth and selection of preantral and small antral follicles (2).

What is the role of AMH in assessing ovarian aging and ovarian reserve? • AMH levels decrease over time even in “fertile” women who have regular menstrual cycles. • AMH levels correlate well with the ovarian antral follicle count and were the only levels that decreased longitudinally over time compared with FSH, estradiol, and inhibin-B levels. With ovarian aging, the first change is a decrease in AMH levels, followed by a decline in inhibin-B and finally by an increase in FSH levels.

• AMH levels do not vary significantly during the menstrual cycle and can therefore be drawn on any day of the cycle! • Women who are overweight have 65% lower AMH levels than thin women, indicating that obesity may be associated with decreased ovarian reserve and/or with ovarian dysfunction.  

What are the factors that influence AMH levels? Factors that decrease MIS/AMH • Increasing age • Obesity • Administration of gonadotropins • Administration of chemotherapy or radiation • Surgical removal of one or both ovaries B. Factors that increase MIS/AMH • Polycystic Ovarian Syndrome

C. Factors that do not influence MIS/AMH • Day of menstrual cycle • GnRH agonists • Birth Control Pills • Pregnancy What are “normal” and “abnormal” levels of AMH? • AMH levels less than 0.2 - 0.5 ng/mL are associated with increased IVF cycle cancellation rates and fewer eggs retrieved from the ovaries. • AMH levels greater than 2.5 ng/mL are associated with greater number of eggs retrieved and a better fertility potential.

• Recent data suggest that AMH levels may reflect fertility potential more accurately than conventional markers like FSH, inhibin-B or estradiol levels. • AMH levels may be better indicators of the ultimate chance that a woman will achieve a pregnancy than FSH levels. • A high AMH level (greater than 3.6 ng/mL) may predict that a woman is at increased risk for ovarian hyperstimulation syndrome. In such women, the dose of medications with IVF can be reduced to avoid this side effect of fertility treatments.

AMH levels have been found to be two to three times higher in PCOS women , making it difficult to find a threshold value for poor ovarian reserve without a significant overlap with normal values. Dynamic tests Another approach towards identifying ovarian reserve involves dynamic testing. This involves taking a baseline serum sample, stimulating the ovaries (FSH/Clomiphene/GnRH agonist) and then retesting the serum level again for the same marker. All the dynamic tests are more expensive, invasive and associated with the side effects of administered stimulation regimens.

Combination of tests Combinations of various markers (AFC, AMH and inhibin-B) have been tried, and a joint scoring system has been developed which predicts a poor response to gonadotrophin stimulation at best with 87% sensitivity and 80% specificity and a positive likelihood ratio of 4.36%. However, they have not been tested for prediction of pregnancy .

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