AMH

Dr Deepak SanghaviHOD Clinical Chemistry

Metropolis Healthcare, Mumbai

Ovarian Reserve

Definition: The ovarian reserve is constituted by the size of the ovarian follicle pool and the quality of the oocytes.

• Ovarian reserve declines with increasing age, resulting in the decrease of a woman’s reproductive function

• There is considerable individual variation in the age of menopause and, subsequently, also in the age of subfertility Hence, chronological age is a poor indicator of reproductive aging, and thus of the ovarian reserve.

Ovarian reserve assessment Tests include :• Anti Mullerian hormone• Follicle Stimulating Hormone(FSH) (on 3rd day of the cycle)• Inhibin B (3rd day)• Oestrogen (E2)• Antral follicle count (AFC)• Ovarian volume assessment • Clomiphene citrate challenge test

However, there are potential limitations associated with each of these tests.

Follicle Stimulating Hormone (FSH)

• Usually measured Day 2 or 3 of cycle

• Women with > 10 IU/l - poor response to ART

• Variation from month to month• Lab wise variation in values due

to different techniques.• Spurious fall after hormone

therapy• Sensitivity to predict ovarian

response is poor .

Ovarian reserve markers

• Serum Oestradiol - E2 alone is of little value to asses ovarian reserve

• Progesterone -Doesn’t have any independent role in assessment of ovarian reserve. Early LH surge and elevation of P4 suggested sign of poor ovarian reserve

Inhibin B• Produced by the granulosa cells in growing follicles • A fall in day 3 inhibin-B levels may predict poor ovarian

reserve before the expected rise in day 3 FSH.• But it has high false positive rate• Levels < 45 pg/ml – poor response to inductionFactors affecting Inhibin-B measurements:• Obesity (decreases)• PCOS (increases)• Exogenous FSH administration (increases)• Oral contraceptive use (decreases).

Antral follicular count• Count of total follicles measuring 2 to 5mm in both

ovaries on Day 2/3 of periods.• So far, assessment of the number of antral follicles

by ultrasonography, the antral follicle count (AFC), best predicts the quantitative aspect of ovarian reserve (Scheffer, et al., 2003)

• Some correlation with ovarian response but only at low threshold

• If AFC < 5- significantly worse outcome.

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.

AFC can only tell of number not quality of oocytes

AMH• Alfred Jost , French

endocrinologist discovered Anti Mullerian hormone (AMH) or Müllerian inhibiting substance

• It is a dimeric glycoprotein hormone belonging to the transforming growth factor-beta family.

• It is produced by Sertoli cells of the testis in males and

• Produced by ovarian granulosa cells in females.

Patho-Physiology• Male foetal development – AMH prevents the Mullerian

ducts from developing into the uterus and other Mullerian structures, resulting in normal development of the male reproductive tract. In the absence of AMH, the Mullerian ducts and structures develop into the female reproductive tract.

• AMH serum concentrations are elevated in males under 2 years old and then progressively decrease until puberty, when there is a sharp decline.

• Females - AMH is also expressed in the follicles of females of reproductive age and inhibits the transition of follicles from primordial to primary stages.

• Follicular AMH production begins during the primary stage, peaks in the preantral and small antral stages, and then decreases to undetectable concentrations as follicles grow larger

• By contrast, AMH concentrations are low in female children until puberty. Concentrations then decline slowly over the reproductive lifespan as the size of the pool of remaining microscopic follicles decreases.

• AMH concentrations are frequently below the detection limit of current assays after natural or premature menopause

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).

Ovarian Reserve

Ovarian reserve is a complex clinical phenomenon that is influenced by age, genetics, environmental variable

Why AMH better for Ovarian reserve• - Because of Gender differences in AMH concentrations,• - its changes in circulating concentrations with sexual

development, and • - its specificity for Sertoli and granulosa cells, - Since AMH is produced continuously in the granulosa cells

of small follicles during the menstrual cycle, it is superior to the episodically released gonadotropins and ovarian steroids as a marker of ovarian reserve.

- Furthermore, AMH concentrations are unaffected by pregnancy or use of oral or vaginal estrogen- or progestin-based contraceptives.

AMH – Normal range

High (often PCOS) - Over 3.0 ng/ml

Normal Over 1.0 ng/ml

Low normal range 0.7 - 0.9 ng/ml

Low 0.3 – 0.6 ng/ml

Very Low Less than 0.3 ng/ml

Uses of AMH• Assessment of menopausal status, including premature

ovarian failure • Assessing ovarian status, including follicle development,

ovarian reserve, and ovarian responsiveness, as part of an evaluation for infertility and assisted reproduction protocols such as in vitro fertilization 

• Assessing ovarian function in patients with polycystic ovarian syndrome 

• Evaluation of infants with ambiguous genitalia and other intersex conditions 

• Evaluating testicular function in infants and children • Diagnosing and monitoring patients with antimullerian

hormone-secreting ovarian granulosa cell tumors

Anti-Mullerian Hormone (AMH)• AMH levels are indicative for the size of the growing follicle

pool and considered as marker of ovarian aging.• AMH levels decline with age hence decline in AMH levels

may be the earliest marker of ovarian aging. • Changes in serum AMH levels occur relatively early in the

sequence of events associated with ovarian aging.• Changes in serum levels of FSH, Inhibin B and E2 occur

relatively late in the reproductive aging process. • There is strong correlation of serum AMH levels with AFC

(Antral follicle count)• AMH is marker of ovarian responsiveness in women

undergoing treatment for infertility. It helps to predict outcome of IVF procedure

• Antral follicle count (AFC) by USG during the early follicular phase best predicts the quantitative aspect of ovarian reserve.

• In women undergoing treatment for infertility, ovarian aging is characterized by decreased ovarian responsiveness to exogenous gonadotropin administration and poor pregnancy outcome.

• On the one hand, correct identification of poor responders by assessment of their ovarian reserve before entering an in vitro fertilization (IVF) program is important.

• AMH serum levels show highly correlation with the number of antral follicles before treatment and number of oocytes retrieved upon ovarian stimulation. Several studies show that AMH is an excellent marker of ovarian responsiveness in IVF treatment

• Lower AMH levels (<0.3 ng/ml) predict poor ovarian response with sensitivity of 85 % and specificity of 82.3 %

Invitro fertilization

• AMH levels > 6.79 ng/ml are associated with increased incidence of ovarian hyper stimulation syndrome.

• PCOS is one of the most common endocrine disorders in women of reproductive age. It is characterized by an-ovulation manifested as oligo- or amenorrhea. Elevated levels of circulating androgens, and polycystic ovaries as visualized by ultrasound.

• Polycystic ovarian syndrome can elevate serum AMH concentrations because it is associated with the presence of large numbers of small follicles.

AMH in PCOS

Factors that may influence serum AMH A) Factors that decrease MIS / AMH Increasing age Increasing body mass index Administration of gonadotropins Administration of Chemotherapy or Radiation OophorectomyB) Factors that increase MIS / AMH PCOSC) Factors that do not influence MIS / AMH Day of menstrual cycle GNRH agonists Oral contraceptives Pregnancy

REPORT FORMAT

CONCLUSION• Serum AMH levels decrease with age in pre-menopausal

women and hence marker for ovarian aging.• Serum levels of AMH correlate strongly with the number of

antral follicles, suggesting that AMH levels reflect the size of the primordial follicle pool.

• Assessment of the ovarian reserve is particularly important in the IVF clinic, where AMH may be useful as a predictor of poor response.

• Since a considerable proportion of sub-fertility is due to postponement of childbearing, measurement of AMH levels to assess the ovarian reserve may also be of interest in women in general.

• The elevated levels of AMH in these women strongly suggest that serum AMH levels may also be used in the diagnosis of PCOS.•AMH levels are strongly correlated with the size of the follicle pool, and because of the lack of cycle variations, serum levels of AMH are a good candidate for inclusion in standard diagnostic procedures to assess other ovarian dysfunctions, such as premature ovarian failure

References1) Jenny A visser, et al. Antimullerian hormone: a new marker for

ovarian function. 2006 Society for Reproduction and Fertility. ISSN 1470-1626

2) Kit insert- Beckman coulter (generation II) 3) La Marca A, Broekmans FJ, Volpe A, et al: ESHRE Special

Interest Group for Reproductive Endocrinology-AMH Round Table. Anti-Mullerian hormone (AMH): what do we still need to know? Hum Reprod 2009 Sep;24(9):2264-2275

4) Rey R: Anti-Mullerian hormone in disorders of sex determination and differentiation. Arq Bras Endocrinol Metabol 2005 Feb;49(1):26-36

5) La Marca A, Volpe A: The Anti-Mullerian hormone and ovarian cancer. Hum Reprod Update 2007 May-Jun;13(3):265-273

Thank you

Materials & Method• A retrospective study of AMH test human serum samples

was done for 14080 samples over a period of 6 months (June – Nov 2013).

• AMH test was done by ELISA method by Beckman coulter kit - 2nd generation

• It is a two step sandwich type immunoassay. • In the first step the AMH is captured by a monoclonal

antibody bound to the wells of a micro-titer plate. • In the second step a bio-tinylated monoclonal antibody is

added together with streptavidin- peroxidase. This bio-tinylated antibody binds to the solid phase antibody-antigen complex and in turn binds with the conjugate.

• After incubation the wells are washed and the antigen complex bound to the well are detected by addition of a chromo-genic substrate.

• The intensity of the color developed is proportional to the AMH concentration in the sample or calibrator.

A pilot study done in house on normal population of 105 samples for validating the reference ranges used for stratifying female infertile patients.

AGE GROUP

AMH Values (ng / ml) FSH values

mIU/mL

Optimal Satisfactory Low fertility

Very low

High Average

Years  4.0 - 6.79 2.19 - 4.0 0.3-1.0 1.0-1.5 1.5-2.19 < 0.3 > 6.79

Upto 20 (8) 4 3 0 0 0 0 1 4.57

20 - 40 (89) 18 25 13 1 10 7 15 7.92

Above 40 (8) 0 0 0 0 0 8 0 21.34

Data analyzed gender wise

AMH Levels FEMALE

MALE

CHILD

<0.3 ng/mL 2064 6 2

0.3-2.19 ng/mL 5615 13 3

2.19-4.0 ng/mL 2559 11 4

4.0-6.79 ng/mL 1688 13 0

>6.79 ng/mL 2085 10 7

Total 14011 53 16

Data analyzed age wiseTotal No. Patient AGE <0.3 ng/mL 0.3 - 2.19

ng/mL2.19- 4.0

ng/mL4.0-6.79 ng/mL >6.79 ng/mL

2045 < 20 yrs 310(15.16%) 776(37.95%) 337(16.48%) 257(12.57%) 365(17.84%)

10900 20-40 yrs 1125(10.30%) 4422(40.57%) 2190(20.10%) 1434(13.16%) 1729(15.87%)

1135 > 40 yrs 637(56.10%) 433(38.15%) 47(4.15%) 10(0.89%) 8(0.71%)

Impression : For Female Age 20-40 yrs group (10900 samples), it was found that 1125 (10.30%) <0.3 ng/mL, 4422 (40.57%) 0.3 to 2.19 ng/mL , 2190 (20.10%) 2.19 to 4 ng/mL, 1434 (13.16%) 4 to 6.69 ng/mL & 1729 (15.87%) > 6.79 ng/mL. Age alone cannot always predict ovarian reserve as shown in above data that the relationship between them is skewed.