Development of a sensitive enzyme immunoassay for anti-Müllerian hormone and the evaluation of potential clinical applications in males and females.
OBJECTIVE
Recent studies have found anti-Müllerian hormone (AMH) to be a potentially important marker for the assessment of ovarian reserve and prediction of the success of in vitro fertilization (IVF) treatment. The objectives of this study were to develop a sensitive and specific assay for AMH and to evaluate the potential application of the assay. This assay will be then available to our collaborators in the UK and overseas.


METHODS
Samples obtained as part of another prospective cross-sectional study from infertility patients and another prospective longitudinal study from pregnant women were used in this study to measure AMH using a new double-antibody enzyme-linked immunosorbent assay (ELISA).


METHODS
AMH levels were evaluated in (i) serum and seminal fluid from males (normal and male factor infertility males), (ii) serum and follicular fluid from females (normal and female with unexplained infertility) and (iii) serum, amniotic fluid (AF) and coelomic fluid (CF) from pregnant women. AMH levels in the samples were measured by a newly developed ELISA.


RESULTS
The assay had a detection limit of<0.078 ng/ml. High recoveries of spiked recombinant protein were observed from male and female sera and also from follicular, seminal, coelomic and amniotic fluids. The intra- and interassay coefficients of variation (CVs) were 3.6% and 4.0%, respectively. Serially diluted human samples gave dose-response curves parallel to the standard curve. Immunoreactivity was stable to sample storage at room temperature for several days and to multiple cycles of freezing and thawing. In seminal fluid, the AMH concentrations in a group of men with male factor infertility were insignificantly different from those in fertile men. By contrast, serum AMH concentrations were lower in the male factor infertility group than the normal group of patients. Women with unexplained infertility had similar concentrations of AMH in serum and follicular fluid compared to controls. Pregnant women had higher concentrations of AMH in the circulation in early pregnancy compared with nonpregnant women, suggesting a foeto-placental contribution and a possible biological role for this molecule in early pregnancy.


CONCLUSIONS
We have developed a sensitive and specific assay for AMH. Serum AMH in men with male factor infertility is lower than in normal men. Levels of AMH in pregnancy are higher than normal menstrual cycle levels suggesting a foeto-placental contribution.
Al-Qahtani
A
A
School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford, UK.
Muttukrishna
S
S
Appasamy
M
M
Johns
J
J
Cranfield
M
M
Visser
J A
JA
Themmen
A P N
AP
Groome
N P
NP
D000653:Amniotic Fluid / Q000737:chemistry; D000818:Animals; D054304:Anti-Mullerian Hormone; D015415:Biomarkers / Q000032:analysis / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004812:Epidemiologic Methods; D005260:Female; D005300:Fertility Agents, Female / Q000494:pharmacology; D015571:Follicular Fluid / Q000737:chemistry; D006023:Glycoproteins / Q000032:analysis* / Q000097:blood / Q000494:pharmacology; D006064:Gonadotropins, Equine / Q000494:pharmacology; D006801:Humans; D007247:Infertility, Female / Q000378:metabolism*; D007248:Infertility, Male / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010062:Ovulation Induction; D011247:Pregnancy; D011261:Pregnancy Trimester, First; D012661:Semen / Q000737:chemistry; D013735:Testicular Hormones / Q000032:analysis* / Q000097:blood / Q000494:pharmacology
D015415:Biomarkers; D005300:Fertility Agents, Female; D006023:Glycoproteins; D006064:Gonadotropins, Equine; D013735:Testicular Hormones; D054304:Anti-Mullerian Hormone