Development of a well characterized Ultra-Sensitive Human Anti-Mullerian Hormone (AMH) Chemiluminescence assay: Evaluation of Potential Clinical Applications

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 515-547-Female Reproductive Endocrinology
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-527
Jenny A. Visser*1, Joop S E Laven2, Anke McLuskey1, Yvonne V. Louwers2, Wendy van Dorp2, Axel PN Themmen1, Bhanu Kalra3, Amita Patel3 and Ajay Kumar3
1Erasmus MC, Rotterdam, Netherlands, 2Erasmus MC University Medical Center, Rotterdam, Netherlands, 3Ansh Labs, Webster, TX
Objective: Development of a sensitive and human specific AMH Chemiluminescence assay for the quantitative measurement of AMH in serum, plasma and follicular fluid.

Relevance: AMH is a homodimeric glycoprotein composed of two 55 kDa N-terminal and two 12.5 kDa C-terminal homodimers, non-covalently linked by disulfide bridges. It belongs to the transforming growth factor- family. AMH performs various physiological functions. In males, AMH is secreted by the Sertoli cells. During embryonic development, AMH is responsible for Müllerian duct regression. AMH continues to be produced by the testes until puberty and then decreases slowly to residual post-puberty values. In females, AMH is produced in small amounts by the granulosa cells of small growing follicles from the 36th week of gestation onwards until menopause when levels become undetectable.

Methodology: A two-step, sandwich-type enzymatic microplate assay has been developed to measure AMH levels in 50 µL of sample in less than 3 hours. The assay measures human AMH and uses stabilized recombinant human AMH as calibrators (0.06-14 ng/mL). The antibodies used in the assay have been selected from 74 pairs of antibodies mapped to the pro-pro, pro-mature, mature-pro and mature-mature region of AMH. This highly characterized monoclonal antibody pair in the assay measures the homodimeric form of AMH and does not detect other TGFβ family members.

Validation: AnshLabs USAMH CLIA, when compared to AMH Gen II using 94 serum samples of patients visiting the infertility clinic in the range of 0.1-46 ng/mL yielded a correlation coefficient of rs = 0.90  (p<0.0001) and a slope of 0.94 with an intercept of -0.02 ng/mL. AnshLabs US AMH CLIA when compared to the total antral follicle counts (AFC) and FSH, using 96 samples yielded a correlation coefficient (rS) = 0.64 (p<0.0001) and rs = -0.43 (p<0.0001), respectively. Total imprecision, calculated on 3 serum pools over 12 runs, 4 replicates per run, was 4.75% at 0.4 ng/mL, 3.34% at 1.06 ng/mL and 3.65% at 2.38 ng/mL. The functional sensitivity calculated at 20% CV was 0.012 ng/mL. Dilution and spiking studies showed an average recovery of 90-110%. When potential interferents (hemoglobin, triglycerides, and bilirubin) were added at twice the physiological concentrations, AMH concentrations were within ±10% of the control.

Conclusions: A highly sensitive, specific and reproducible microplate AMH assay has been developed that measures human AMH and correlates well with antral follicle counts. The performance of the AMH assay is ideal for research investigation into infertility testing, polycystic ovary syndrome, menopause screening, ovarian reserve and granulosa cell tumor monitoring after therapy.

Disclosure: JSEL: Researcher, Merck BV, Founder, Genovum, Researcher, Ferring Pharmaceuticals. Nothing to Disclose: JAV, AM, YVL, WV, APT, BK, AP, AK

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