Objective The objective of this study was to evaluate the relationship between anti-mullerian hormone (AMH), inhibin B and antral follicle count (AFC) with ovarian response. Design Retrospective study. Setting Fertility unit.Sample AFC was recorded, and a serum sample obtained on day 3 from all patients undergoing in vitro fertilisation (IVF). Patients were given 300 IU/L recombinant follicle stimulating hormone (FSH; Gonal F).The following day blood samples were collected. Methods Serum samples were assayed for FSH, AMH and inhibin B using commercial immunoassay kits and oestradiol using an in house assay. Main outcome measures Response to gonadotrophin stimulation and the number of eggs collected.Results AFC was negatively correlated to age (r ¼ À0.426, P < 0.001). Delta inhibin B (levels of inhibin B on day 4 minus day 3) had the best association to the number of eggs collected (r ¼ 0.533, P < 0.001) followed by basal AMH (r ¼ 0.51, P < 0.001) and AFC (r ¼ 0.505, P < 0.001). The number of eggs fertilised was significantly associated with basal AMH (r ¼ 0.592, P < 0.001) and inhibin B (r ¼ 0.548, P < 0.001). AMH with a cutoff of 0.2 ng/mL had the best sensitivity (87%) and specificity (64%) in predicting poor response. A cumulative score using basal FSH, basal AMH, delta E 2 (levels of oestradiol on day 4 minus day 3), delta inhibin B, AFC and age gives the best predictive statistics to identify poor responders with 87% sensitivity and 80% specificity and a positive likelihood ratio of 4.36. Conclusion Delta inhibin B had the best positive association with the number of eggs collected and basal AMH is the single best predictor of poor response. AFC has a significant association with the number of eggs collected and is predictive of clinical pregnancy. It is evident that a single parameter is of limited value in predicting ovarian response. However, we have demonstrated a cumulative score using all the above markers could be useful in predicting poor response.
The performance of existing immunoassays and bioassays for activins is compromised by the presence of activin-binding proteins such as follistatin and alpha 2 macroglobulin (alpha 2M) in biological fluids. To overcome this problem we have developed a novel two-site enzyme immunoassay procedure for activin-A which incorporates an analyte denaturation and oxidation step. The optimized assay is sensitive (detection limit approximately 10 pg/well), precise (mean within- and between-plate coefficients of variation 4.9 and 9.1% respectively) and accurate (activin-A recovery values of 102 +/- 3 and 96 +/- 5% for bovine follicular fluid (FF) and human serum respectively). In specificity tests, high concentrations of follistatin (500 ng/ml) and alpha 2M (100 microgram/ml) did not interfere with the response signal to activin-A. In addition, no significant cross-reactivity was observed with a range of related molecules including inhibin-A, inhibin-B, activin-B (all < 0.5%), bovine pro-alpha C and follistatin (both < 0.1%). Response curves parallel to the activin-A standard curve were obtained for a variety of test samples including bovine, human, ovine and porcine FF, human sera and conditioned medium from cultured bovine and human granulosa cells. Fractionation of bovine FF by SDS-PAGE confirmed assay specificity since only one peak of activin-A immunoreactivity was detected (M(r) approximately 25 k) in eluted gel slices. However, gel-permeation chromatography showed that under physiological conditions all of the detectable activin-A in bovine FF eluted with apparent M(r) values of > 700 and 60-200 k reflecting its association with binding protein(s). Analysis of bovine FF samples (n = 76) from morphologically dominant follicles during the luteal phase showed that activin-A levels were positively correlated with inhibin-A (r = +0.54; P < 0.001) and total beta subunit immunoreactivity (r = +0.32; P < 0.005) but not with total alpha subunit immunoreactivity (r = -0.09). Classification of these follicles according to oestrogenic status showed that activin-A, inhibin-A and total beta subunit levels were highest in oestrogen-inactive follicles (P < 0.01) whereas total alpha subunit levels were lowest in these follicles (P < 0.001). Activin-A levels were measurable in all human serum samples analysed, ranging from 128 pg/ml during the normal menstrual cycle, 210 pg/ml in women undergoing ovarian hyperstimulation and approximately 500 pg/ml in postmenopausal women to over 4000 pg/ml during pregnancy. In conclusion, the present assay provides a reliable method for quantitating total (i.e. bound+free) activin-A concentrations in a variety of biological samples and should prove useful for further in vivo and in vitro studies in a range of species including man.
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