1 Institut Clínic of Gynecology, Obstetrics and Neonatology and 2 Hormonal Laboratory, Faculty of Medicine-University of Barcelona, Hospital Clínic-Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
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Abstract |
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Key words: assisted reproduction treatment/FSH/inhibin B/low responders/ovarian reserve
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Introduction |
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Although ovarian reserve declines with age, it is a biological and not just a chronological function. In fact, the most important aspect of diminished ovarian reserve is that the timing of its onset is highly variable (Scott and Hofmann, 1995). Thus, a useful biomarker of ovarian response to ovulation induction is needed. The ideal parameter to estimate ovarian reserve would be easily measurable, minimally invasive, inexpensive, and have good predictive value (Sharara and Scott, 1997). Basal FSH concentrations on cycle day 3 have been described by some investigators as meeting these criteria (Scott and Hofmann, 1995; Sharara and Scott, 1997
).
Other authors, however, have recently stressed that FSH concentrations alone may be an unreliable indicator of reproductive potential in older women because FSH measurement represents an indirect assessment of ovarian reserve (Seifer et al., 1997, 1999
). On the contrary, the inhibin B concentration would offer a more direct assessment because inhibin B is produced by granulosa cells (Seifer et al., 1997
, 1999
). In addition, the decrease in inhibin B would be the earliest marker of the decline in follicle number across reproductive ageing (Welt et al., 1999
). Thus, Seifer et al. (1997, 1999) reported that women with declining ovarian responsiveness and clinical outcomes in assisted reproduction treatment consistent with declining ovarian reserve had decreased day 3 serum inhibin B concentrations despite having non-elevated day 3 serum FSH concentrations. However, other recent reports disagree (Corson et al., 1999
; Hall et al., 1999
). Because studies are discordant, the need for further investigation has been stressed (Corson et al., 1999
).
The present study is an attempt to assess further the usefulness of day 3 inhibin B concentration as a predictor of response to ovarian stimulation and pregnancy in an assisted reproduction treatment programme.
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Materials and methods |
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Stimulation regimen
Ovarian stimulation was carried out with FSH under pituitary suppression with gonadotrophin-releasing hormone (GnRH) agonist according to a protocol previously reported (Balasch et al., 1996). In all women, pituitary desensitization was achieved by s.c. administration of leuprolide acetate (Procrin; Abbott Laboratories, Madrid, Spain) (1 mg daily, which was reduced to 0.5 mg after ovarian arrest was confirmed) started in the mid-luteal phase of the previous cycle. Gonadotrophin stimulation of the ovaries was started when serum oestradiol concentrations declined to <50 pg/ml and a vaginal ultrasonographic scan showed an absence of follicles >10 mm diameter. On days 1 and 2 of ovarian stimulation, six ampoules/day of highly purified FSH (75 IU per ampoule) (Neo-Fertinorm, Serono SA, Madrid, Spain) were administered s.c. On days 3-7 of ovarian stimulation, two ampoules per day of FSH were administered to each patient. From day 8 onward, FSH was administered on an individual basis according to the ovarian response. The criteria for human chorionic gonadotrophin (HCG) administration were the presence of two or more follicles >18 mm in diameter with
5 follicles measuring
14 mm in association with a consistent rise in serum oestradiol concentration. Oocyte aspiration was performed with vaginal ultrasonography 3536 h after HCG administration. Up to four embryos per patient were replaced and the luteal phase was supported with additional doses of HCG. The cycle was cancelled when there were <3 follicles with diameter
14 mm after 89 days of gonadotrophin therapy or when requirements for HCG injection were not accomplished following 4 or 5 additional treatment days.
Hormone analyses and ultrasonography
Hormones were measured using commercially available kits. FSH serum concentrations were measured by an immunoenzymatic assay with two monoclonal antibodies (Immuno 1, Technicon; Bayer, Tarrytown, NY, USA) and data are expressed in terms of International Reference Preparation (IRP) 78/549. The sensitivity of the assay was 0.1 IU/l and the inter-assay coefficient of variation (CV) was 2.7%. oestradiol concentrations in serum were estimated by a competitive immunoenzymatic assay (Immuno 1). The sensitivity was 10 pg/ml and the interassay CV was 5%. Dimeric inhibin B was measured by a solid-phase sandwich enzyme-linked immunosorbent assay which uses two monoclonal antibodies (Serotec, Oxford, UK). The first monoclonal antibody is specific for the ßB-subunit of inhibin; the second one is directed to the -subunit and coupled to alkaline phosphatase. The assay utilizes an immunopurified mixture of inhibin forms from human follicular fluid calibrated against recombinant 32 kDa inhibin B. The sensitivity of the assay was 15 pg/ml and the intra-assay and inter-assay CV were <11 and 15% respectively. Ultrasonic scans were performed using a 5 mHz vaginal transducer attached to an Aloka sector scanner (Model SSD-620; Aloka Co. Ltd, Tokyo, Japan). Pregnancy was diagnosed by increasing serum concentrations of ß-HCG after embryo transfer, and the subsequent demonstration of an intrauterine sac by ultrasonography.
Statistics and probability testing
For statistical analysis Student's t-test and 2-test were used as appropriate. Mean and SD were calculated for variables in each group of patients (cancelled and punctured cycles). A multivariate logistic regression model was performed to assess the joint effect of variables and to estimate the sensitivity and specificity values for all possible combinations of the study variables. The best joint model was obtained by a stepwise procedure considering P < 0.05 as the P-value associated with a variable to enter in the model. The discrimination attained between the study groups (cancelled versus punctured assisted reproduction treatment cycles, and pregnancy versus non-pregnancy cycles) was evaluated with receiver operating characteristic (ROC) analysis (Hanley and McNeil, 1982
; Zweig and Campbell, 1993
). Sensitivity, specificity, and the area under the ROC curve (AUCROC) which is a quantitative measure of accuracy, were obtained for each model. 95% confidence intervals (CI) were calculated for each of the estimates. The models' AUCROC were compared using the method of Hanley and McNeil (1982). Areas of 1.0 and 0.5 denote no overlapping and no discrimination, respectively, between groups. Data were analysed by Statistics Package for Social Sciences (SPSS, Chicago, IL USA) statistical software and MedCalc Software (Mariakarke, Belgium).
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Results |
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Discussion |
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Because pituitary FSH secretion increases with declining ovarian reserve, day 3 serum FSH concentrations are being routinely used in most assisted reproduction treatment programmes and they have been found to be a better predictor of assisted reproduction treatment outcome than age (Scott and Hofmann, 1995; Barnhart and Osheroff, 1998). Both age and FSH concentrations, however, are indirect markers of ovarian function and this has been the reason to explain the low sensitivity of day 3 serum FSH concentrations as an indicator of reproductive potential in women undergoing assisted reproduction treatment (Barnhart and Osheroff, 1998
). Theoretically, the direct products of granulosa cells might better reflect ovarian secretory capacity and follicle number. Inhibin is a heterodimeric glycoprotein produced by granulosa cells and consisting of
- and ß-subunits. There are two distinct molecular forms of the ß-subunit that exist (ßA and ßB) and, when combined with an
-subunit, form inhibin A and inhibin B, respectively. With increasing understanding of the control of synthesis and secretion of the inhibins and their potential endocrine role in the regulation of FSH in the human, attention has focused to the possibility that this family of peptides may provide a more direct index of ovarian reserve and improved predictors of assisted reproduction treatment outcome.
Earlier studies using immunoreactive -inhibin measurements found that ovarian inhibin secretion decreases with declining ovarian function with advancing age (Hughes et al., 1990
; Buckler et al., 1991
; Pellicer et al., 1994
) as a result of a reduced cellular function rather than a decrease in the follicular population (Pellicer et al., 1994
). Accordingly, in a previous report where the same study design as in the present investigation was used, it was shown that basal FSH and immunoreactive
-inhibin had similar predictive properties in IVF cycles stimulated with gonadotrophins under pituitary suppression (Balasch et al., 1996
). The cross-reactivity of inhibin assays, however, has been a source of confusion for many years (Hayes et al., 1998
). Recently two-site directed assays have been developed capable of reliably and specifically measuring the inhibin dimers, inhibin A and inhibin B (Groome et al., 1994
, 1996
). Thus, it has been claimed that these new assays will become widespread in their use, enabling the identification of situations in which measurements of inhibin A and B are of clinical value (Hayes et al., 1998
).
In the female, the pattern of secretion of the inhibins suggests that early follicular phase inhibin B may reflect the number of follicles present at baseline, whereas inhibin A may indicate follicle maturity (Groome et al., 1994, 1996
; Hayes et al., 1998
). On this basis, recent reports have focused on the potential usefulness of basal serum inhibin B measurement in predicting successful outcome in assisted reproduction treatment. Thus, Seifer et al. (1997) reported significantly higher cancellation rates and lower pregnancy rates in women with `low' (<45 pg/ml) day 3 serum inhibin concentrations. Recent studies, however, have shown that neither 45 pg/ml (Corson et al., 1999
) nor any other inhibin B threshold value (Hall et al., 1999
) were good predictors of assisted reproduction treatment outcome. In fact, as much as 118 of 156 patients (76%) studied by Seifer et al. (1997) had `normal' basal serum inhibin B concentrations despite a mean age of 35 years. As recently stressed (Hall et al., 1999
), considering that the lowest measurable concentration of inhibin B by enzyme-linked immunosorbent assay (Serotec) is 15 pg/ml and inter-assay CV 15%, a considerable overlap can be expected when measuring concentrations of ~50 pg/ml. Also, in the study by Seifer et al. (1997), basal FSH concentrations were significantly higher in the group of patients having `low' inhibin B but both hormones were not compared with respect to their predictive value. Finally, in that study,
95% of patients investigated had basal FSH concentrations
9.5 IU/l, a value considered by those authors as the upper limit associated with a normal ovarian reserve. In contrast, basal FSH concentrations in patients included in the present study ranged between 3.92 and 19.45 IU/l (35% of women having FSH >9.45 IU/l).
The hypothesis that women with declining ovarian reserve may have decreased day 3 serum inhibin B concentrations despite having non-elevated day 3 serum FSH concentrations was further tested and expanded in a later study by the same team of authors (Seifer et al., 1999). In this latter study, however, 47 women who had previously proven declining ovarian reserve on the basis of a poor response to exogenous gonadotrophin stimulation in a previous assisted reproduction treatment cycle were compared with 109 women having tubal factor or male factor. The mean age of patients in the study group (40 years) was significantly higher than that of control patients (34 years) and study patients received a different stimulation protocol with leuprolide acetate and gonadotrophins than controls in order to improve their poor response to stimulation. Thus, inclusion criteria and study design in that report (Seifer et al., 1999
) were different from those used in the present investigation where only women undergoing the first cycle of assisted reproduction treatment were included. The results of the current study, as well as those reported by others (Corson et al., 1999
; Hall et al., 1999
), do not support the use of day 3 inhibin B as a predictive marker of ovarian response and pregnancy in an assisted reproduction treatment general programme. The current study has demonstrated that: (i) basal FSH is a better marker of ovarian response than inhibin B and age; (ii) age, but not basal FSH or inhibin B, was independently associated with pregnancy rate. The combination of any two or all three of these variables studied did not improve the predictive value of FSH or age alone.
The current results indicate that as a single predictor of ovarian response to gonadotrophin stimulation basal FSH is not surpassed by inhibin B. This seems in contrast with studies indicating that inhibin B determination as a direct measure of ovarian function may be a more sensitive indicator of ovarian reserve than indirect indicators such as pituitary FSH secretion (Danforth et al., 1998). However, to presume that a singular deficiency in inhibin activity could account for age-related elevations in FSH fails to consider the extensive structural and functional overlap that exists among the other component FSH regulatory proteins known to mediate FSH regulation. On the contrary, the monotropic rise in FSH is more likely to be dictated by the sum effect of inhibitory inputs stemming from inhibins, follistatins, as well as oestradiol, and the stimulatory inputs from GnRH and activin (Reame et al., 1998
).
A remarkable feature of the present study is that it provides evidence indicating that, unexpectedly, the predictive ability of basal inhibin B is not better than that afforded by immunoreactive -inhibin measurement in previous studies (Balasch et al., 1996
). In our previous report an immunoenzymatic method was used which utilized antibodies directed at the
-subunit of inhibin, thus measuring inhibin A, inhibin B and the non-biologically active
-subunit (Balasch et al., 1996
). Recent studies (Burger et al., 1998
; Hayes et al., 1998
), however, have shown that only
-inhibin and inhibin B are measured in significant quantities in the early follicular phase of the menstrual cycle, and thus they were probably the only inhibin species measured at this time in the menstrual cycle in those earlier studies. In fact, when immunoreactive inhibin concentrations across the menstrual cycle have been compared with those obtained using the two-site directed assays, the overall pattern of inhibin secretion was similar except that the magnitude of the changes observed was greater with the new assays (Hayes et al., 1998
).
In conclusion, in patients undergoing their first treatment cycle in an assisted reproduction treatment general programme, the stronger predictors of success were age and basal FSH rather than inhibin B. Basal FSH concentration was a better predictor of cancellation rate than age, but age was a stronger predictor of pregnancy rate. However, several facts should be considered before the results of the present study are applied in clinical practice. First, the predictive value of FSH in predicting the ovarian response of 79% may be flattered because of the study design which ensured a high prevalence of cancellation (33%). In a non-selected IVF/ICSI population the prevalence of cancellation is generally much lower. Since the predictive value of a diagnostic test is dependent on the prevalence of outcome, one can assume that the accuracy of basal FSH in predicting ovarian response in clinical practice will be lower. Thus, the predictive performance of FSH for cancellation and of age in discriminating between pregnant and not pregnant using the best discriminating cut-off value should be determined in an unselected assisted reproduction treatment general population. On the other hand, it is possible that a greater number of women with decreased ovarian reserve could have been identified using the clomiphene citrate challenge test which may uncover an abnormality that would be missed by obtaining a basal day 3 FSH value alone (Barnhart and Osheroff, 1998). Finally, further work is warranted before widespread usage of the inhibin B assay which is technically challenging and not readily available. Thus, there is no international assay standard for inhibin B yet and both follicular fluid and recombinant standards are being used. This makes comparison of results between laboratories difficult and may explain discrepancy between different studies.
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Notes |
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References |
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Submitted on April 6, 2000; accepted on July 27, 2000.