1 Women's Institute and Thomas Jefferson University, Department of Obstetrics and Gynecology, Philadelphia, PA and 2 Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Washington, Seattle, WA, USA
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Abstract |
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Key words: inhibin-B/ovarian reserve testing
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Introduction |
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Seifer et al. studied 156 women who underwent 178 cycles of assisted reproductive technology and measured day 3 inhibin-B concentrations in an effort to determine if this hormone was predictive of a subsequent poor response to ovulation induction (Seifer et al., 1997). Follicle stimulating hormone (FSH) and oestradiol concentrations were also measured in their study. Using a threshold value for inhibin-B of >45 pg/ml (conversion factor to SI units: 1), women whose serum concentration was less than this value demonstrated a decreased oestrogen response to stimulation, a reduced number of oocytes retrieved, three times the cancellation rate and only a 28% clinical pregnancy rate per initiated cycle compared with women having a `normal' value.
The current study was designed to examine further a possible role for inhibin-B as a predictor of the ability to achieve pregnancy in a group of infertile patients, who also had a standard day 3 and day 10 clomiphene citrate challenge test.
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Materials and methods |
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Laboratory determinations
Oestradiol determinations were done with an automated immunomagnetic separation assay (Bayer, Tarrytown, N.Y., USA) with intra- and inter-assay coefficients of variation (CV) between 2 and 8% depending on concentration. FSH determinations in Philadelphia were performed with a similar automated technique with intra-assay and inter-assay CV of 4.0 and 4.6%, respectively. FSH and oestradiol determinations were done in batches. FSH determinations in Seattle were performed with a solid phase, two-site fluoroimmunometric (Delfia, Wallace, Inc., Gaithersburg, MA, USA) assay in batches with CV of 2.3 and 4.6% (Wallace Inc.). The inhibin-B assay was an ELISA with a monoclonal antibody directed against the Beta B subunit. Ninety-six well plates coated with antibody were kindly provided by Nigel Groome (Oxford Brookes University, Oxford, UK) and the human recombinant inhibin-B standard was graciously donated by Teresa Woodruff (Northwestern University, Chicago, IL, USA). The assay detection limit was <15 pg/ml; activin A and B, follistatin, and purified human pro-C have <0.1% cross-reactivity, and recombinant inhibin-A has a 0.05% cross-reactivity in this assay system. The intra- and inter-assay CV were <10%. All samples were assayed in duplicate in serial assays using a polynomial least squares analysis programme. The lower limit of detectability was 15.6 pg/ml for each of three assays. No samples were measured on different assays and then analysed. Figure 1
shows the standard curve of the assay.
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Results |
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Day 3 oestradiol concentrations >80 pg/ml (SI Unit conversion factor = 3.67) were found in four patients with FSH >10 mIU/ml on either or both day 3 and day 10, but this number was too small to permit analysis of the possible inverse relationship to pregnancy. Day 3 FSH concentrations were not related to oestradiol concentrations.
Table III shows the correlation and P values between inhibin-B, oestradiol and FSH on cycle days 3 and 10. No significant correlations were found.
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Discussion |
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The study by Hofmann et al. correlated inhibin-B concentrations with results of CCCT in 19 patients with normal ovarian reserve testing and in 15 whose CCCT was abnormal (Hofmann et al., 1998). Inhibin-B concentrations were higher on day 10 than day 3 for all patients, and women with normal CCCT results had higher inhibin-B concentrations on both days than those with diminished ovarian reserve. There was a negative correlation between FSH and inhibin-B concentrations on day 3 (r = 0.37) and on day 10 (r = 0.41). A positive correlation was found between day 10 oestradiol and inhibin-B (r = 0.67). As documented in Table III
, no such correlations were found in our sample of 106 women.
Klein et al. found that women aged 4045 compared with younger women had lower serum inhibin-B concentrations both in the early follicular phase and on the day of oocyte retrieval in a natural cycle, but no differences in follicular fluid concentrations could be found when the older patients were compared with women aged 2025 years (Klein et al., 1996).
Although not the major focus of this study, it was found that the CCCT was once again validated as having prognostic significance in evaluation of women aged >35 years and in those with a poor stimulation history. The correlation between laboratories for FSH results was surprisingly good, considering that different methods of analysis were employed. The differences were well within the limits described for aliquot sampling by multiple laboratories (Hershlag et al., 1992).
It was not possible to find any value in inhibin-B testing either on day 3 or day 10 at the usual normal concentration threshold value of 45 pg/ml. Because these results are discordant with those reported by Seifer et al., corroborating studies are necessary (Seifer et al., 1997). A plausible explanation for the discordance between this study and prior studies in relation to inhibin-B as an indicator of advanced reproductive age is assay methodology. There is no international assay standard for inhibin-B yet, so comparisons of results between laboratories remains a problem. The studies by Seifer et al. (1997) and Hofmann et al. (1998) used as their assay standard an immunopurified preparation of human follicular fluid. The standard contains at least four forms of inhibin (33, 36, 55 and 66 kDa) that are biologically and immunologically active (Robertson et al., 1996
). This study used a recombinant inhibin-B standard as a single molecular form that represents the single most common and biologically active form (33 kDa) of inhibin-B. There are two schools of thought as to which standard is more relevant and there is no consensus yet. Furthermore, it should be recognized that the inhibin-B assay as performed with either standard is technically challenging and not readily available.
A recent study (Hall et al., 1999) dealt with ihibin B serum measurements in 78 patients achieving pregnancy in assisted reproduction treatment in up to three cycles matched to 78 patients who were unsuccessful. It was concluded that the results did not support the use of day 3 inhibin-B as a predictive marker of IVF outcome. There was extensive overlap in baseline inhibin-B concentrations between pregnant and non-pregnant subjects, and inhibin-B alone failed to predict pregnancy. Additionally, another study (Schipper et al., 1998
) found a lack of correlation between maximum early follicular phase serum FSH concentrations and inhibin-B concentrations when measurements were performed on samples drawn daily and assayed with the Serotec follicular fluid standard.
One could argue that the group tested represented a heterogeneous population with infertility based on varied pathologies; but this is exactly what a screening test is all aboutsomething applicable to the general population. FSH concentrations, whether solely as day 3 determinations or with a full clomiphene citrate challenge, have clinical relevance, whereas inhibin-B may or may not. Pregnancy as an endpoint is associated with many confounding variablessemen quality, tubal function and others besides ovarian function. Therefore, any screening test for ovarian function or oocyte quality has more value as a negative predictor than as a positive one, but establishment of an absolute threshold value above or below which pregnancy will not occur is probably not a useful clinical exercise. Instead, quoting pregnancy rates with any specific therapy according to (in this case) FSH concentrations seems to be a more useful approach. For the same reason calculations have not been included of predictive value or receiver operating curves since patients with FSH values of 15 mIU/ml were excluded from the data set, and the inhibin-B data showed no statistical correlation with pregnancy initiation. Until the inhibin-B assay is standardized and more readily available it would be prudent for clinicians to continue to assess ovarian reserve with measurements of FSH and oestradiol before and after clomiphene citrate stimulation.
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Acknowledgments |
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Notes |
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References |
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Submitted on January 6, 1999; accepted on July 13, 1999.