Anti mullerian hormone Lab investigations

1. AMH
Dr Deepak Sanghavi
HOD Clinical Chemistry
Metropolis Healthcare, Mumbai

2. 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.

3. 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.

4. 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 .

5. 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

6. 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 induction
Factors affecting Inhibin-B measurements:
• Obesity (decreases)
• PCOS (increases)
• Exogenous FSH administration (increases)
• Oral contraceptive use (decreases).

7. 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.

8. 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

9. 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.

10. 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.

11. • 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

12. 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).

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

14. 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.

15. 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

16. 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

17. 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

19. • 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

20. • 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

21. 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
Oophorectomy
B) Factors that increase MIS / AMH
PCOS
C) Factors that do not influence MIS / AMH
Day of menstrual cycle
GNRH agonists
Oral contraceptives
Pregnancy

22. REPORT FORMAT

23. 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.

24. • 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

25. References
1) 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

26. Thank you

27. 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.

28. 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

29. 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

30. Data analyzed age wise
Total No.
Patient
AGE <0.3 ng/mL
0.3 - 2.19
ng/mL
2.19- 4.0
ng/mL
4.0-6.79
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.