PCOCheck AMH ELISA: A Clinical Case Report to Resolve Miss-Matched Antral Follicle Counts (AFC) to Serum AMH

Abstract

Introduction: At times clinicians wonder why a subject’s Anti-Mullerian Hormone (AMH) does not correlate to the antral follicle count (AFC). AMH is secreted as a full-length protein and undergoes proteolytic cleavage at amino acid 451 to become biologically active. Additional proteolytic processing takes place at aa229. This processing, which may differ between individuals with different clinical conditions, exposes new antigenic sites which affect AMH measurements. Moreover, AMH epitopes might be masked by protein interaction in the circulation. Clinical Case: A 24-year-old woman gravida 0 presented with metrorrhagia for several months. The bleeding pattern was unresponsive to a trial of oral contraceptive pills, and they were discontinued. The patient’s history is remarkable for a microprolactinoma, managed with dopamine agonist. She has facial acne, but no hirsutism. Her BMI is 24 kg/m2. Laboratory studies revealed normal testosterone (27 ng/mL) and DHEAS (64 mcg/dL). Her prolactin was 1.1 ng/mL Thyroid function tests were in the normal range. hCG was negative. A random progesterone was 12 ng/mL, consistent with an ovulatory cycle. Her ovarian assessment showed FSH of 6.6 IU/L, LH of 6.7 IU/L, estradiol of 51 pg/mL, Inhibin B of 139 pg/mL, all in the normal range. Transvaginal pelvic ultrasound determined the ovarian volumes to be 8 and 6 mL, respectively, with a basal antral follicle count of 15 per side. This would have predicted a normal AMH level in this patient. However, AMH levels were repeatedly found to be low (0.05 to 0.1 ng/mL) using the pro-Mature AMH assay. As this patient was interested in future fertility, additional studies to further investigate her AMH production were carried out. AMH Measurement: Novel AMH ELISAs with antibody epitopes specific to Pro-Mature, Mature-Mature and Pro-Pro were used to analyze the sample. The observed AMH concentration by Pro-AMH ELISA, Mature-Mature ELISA and PCOCheck ELISAs were 0.1 ng/mL, 0.14 ng/mL, and 4.99 ng/mL, respectively. AMH Pro-Pro assay (PCOCheck) uses a linear epitope two-sided antibody that is not impacted by post-translational modifications, known AMH mutations or conformational changes due to thermal instability. Conclusions: The AMH measurements indicate that the patient’s serum may not contain pro-mature associated form of AMH or the C-fragmented AMH in circulation. This may be due to mutated AMH in the C-terminus and can result in C-truncation. This subject’s AMH is mostly fragmented and contains enriched pro-region AMH, which was measured by PCOCheck ELISA. The Teaching: Tests such as PCOCheck ELISA, targeted to specific regions of AMH will provide clinicians better tools for patient management. Such tests will also help resolve the observed AMH discrepancies with AFC using legacy AMH tests. PCOCheck ELISA can be a valuable tool for assessment of ovarian reserve or polycystic ovary syndrome (PCOS).