Inhibin A and inhibin B reflect ovarian function in assisted reproduction but are less useful at predicting outcome

Janet E. Hall1,3, Corrine K. Weltand1 and Daniel W. Cramer2

1 Reproductive Endocrine Unit and the National Center for Infertility Research, Massachusetts General Hospital, Boston, MA 02114 and 2 Gynecologic Epidemiology Center, Department of Obstetrics and Gynecology, Brigham and Women's Hospital, Boston, MA 02115, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To test the hypothesis that dimeric inhibin A and/or inhibin B concentrations represent improved markers of in-vitro fertilization (IVF) outcome over follicle stimulating hormone (FSH), 78 women who achieved pregnancy within three assisted reproduction treatment cycles were matched to 78 women who underwent at least three assisted reproductive treatment cycles and failed to achieve pregnancy. Baseline serum inhibin B and FSH were obtained between days 1 and 4 in a cycle prior to ovarian stimulation, and inhibin A and B were measured immediately before the ovulatory stimulus and in follicular fluid from the lead follicle. Comparing pregnant and non-pregnant subjects at baseline, younger age (34.0 ± 0.5 versus 36.0 ± 0.5 years; P < 0.003) and a combination of FSH lower than the median value (11.2 IU/l) and inhibin B higher than the median value (76.5 pg/ml) were associated with pregnancy (P < 0.03), but FSH (11.7 ± 0.5 versus 12.9 ± 0.9 IU/ml) and inhibin B (89.0 ± 10.2 versus 79.7 ± 7.7 pg/ml) were not independently associated. At the time of the ovulatory stimulus, serum inhibin A (52.8 ± 3.8 versus 40.0 ± 2.7 IU/ml; P < 0.004), inhibin B (1623.8 ± 165.1 versus 859.2 ± 94.8 pg/ml; P < 0.0009) and the number of oocytes retrieved (14.6 ± 0.8 versus 10.1 ± 0.6; P < 0.0001) were predictive of pregnancy when controlled for age. Inhibin A was correlated with the number of embryos (r = 0.4; P < 0.0001). However, neither inhibin A nor inhibin B provided additional information in predicting successful outcome over age and number of oocytes. We conclude that: (i) in patients undergoing assisted reproductive technology, age and number of oocytes retrieved are the strongest predictors of success; (ii) of the parameters available prior to cycle initiation, a combination of lower FSH and higher inhibin B was associated with a greater chance for a successful outcome but an absolute cut-off could not be defined; and (iii) during ovarian stimulation, higher concentrations of inhibin A and inhibin B in serum are associated with successful IVF and mark ovarian reserve as a measure of oocyte number and quality.

Key words: FSH/inhibin/in-vitro fertilization/oestradiol/pregnancy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Assisted reproduction and, in particular, the development of in-vitro fertilization (IVF) techniques have revolutionized the treatment of infertility. Because IVF is costly and not universally successful, attempts have been made to determine the factors which predict a successful outcome in a given patient or couple. Fertility outcome with IVF is markedly decreased in women over 40 years old, presumably due to a decrease in ovarian reserve, which is a term which encompasses a decrease in both follicle number and oocyte quality. Prognostic assessment of ovarian reserve has relied upon indirect markers of ovarian function, including age (Schwartz and Mayaux, 1982Go) and follicle stimulating hormone (FSH) at baseline or in response to stimulation tests (Muasher et al., 1988Go; Scott et al., 1989Go, 1993Go; Loumaye et al., 1990Go; Tanbo et al., 1990Go; Toner et al., 1991Go; Scott and Hofman, 1995; Navot et al., 1997Go). In theory, the direct products of granulosa cells might better reflect ovarian secretory capacity and follicle number. With increased 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 turned to the possibility that this family of peptides may provide a more direct index of ovarian reserve and improved predictors of IVF outcome (McLachlan et al., 1986Go; Tsuchiya et al., 1989Go; Hughes et al., 1990Go; Matson et al., 1991Go; Haning et al., 1994Go; Pellicer et al., 1994Go; Balasch et al., 1996Go; Lockwood et al., 1996Go; Seifer et al., 1996Go, 1997Go).

The inhibins are dimeric peptides composed of an {alpha}-subunit along with a ßA-subunit, inhibin A, or a ßB-subunit, inhibin B (Vale et al., 1988Go). Inhibin B concentrations rise across the luteal–follicular transition and peak in the mid-follicular phase, suggesting secretion by the developing cohort of follicles in a cycle (Groome et al., 1996Go; Welt et al., 1997Go). In contrast, inhibin A does not begin to increase until just after the increase in oestradiol in the late follicular phase, suggesting secretion by the dominant follicle (Groome et al., 1994Go; Lambert-Messerlian et al., 1994Go). The pattern of secretion of the inhibins suggests that early follicular phase inhibin B may mark the number or quality of developing follicles at baseline, while inhibin A may indicate follicle maturity. To test the hypothesis that baseline inhibin B concentrations or gonadotrophin-stimulated inhibin A and/or inhibin B concentrations would serve as improved markers of IVF outcome, we examined baseline inhibin B between days 1 and 4, prior to ovarian stimulation and gonadotrophin-stimulated inhibin A and B in serum drawn immediately before the ovulatory stimulus, and compared them with standard markers of IVF outcome using a case-control design. Granulosa cell secretory dynamics were further assessed by measurement of follicular fluid inhibin A and inhibin B from the largest follicle.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subject population
The study was approved by the Institutional Review Boards of the Brigham and Women's Hospital and the Massachusetts General Hospital and all subjects gave their written informed consent. All subjects were enrolled between 1994 and 1997, as part of a larger study of epidemiological and biological predictors of IVF success in couples seeking their first IVF or gamete intra-Fallopian transfer (GIFT) treatment at one of the three major reproductive clinics in greater Boston [Boston IVF, Brigham and Women's IVF Clinic, and Reproductive Sciences Center (formerly IVF America)]. All subjects were deemed acceptable for IVF therapy and therefore represent a population prescreened prior to acceptance into the IVF programmes according to clinical criteria including age <43–45 years and FSH cut-offs equivalent to those of Scott et al. (1989) or based on clinical information available at the centres. Subjects were ineligible if they required donor eggs or semen. In the larger study, the average eligibility and participation was 65% of couples approached and approximately 1500 couples have been enrolled to date.

The study reported herein is a nested case-control design in which cases (pregnant) are defined as women who became pregnant within the first three cycles of IVF or GIFT and were discharged from the study with a continuing pregnancy as documented by a viable fetus on ultrasound (n = 78). An equal number of controls (non-pregnant) were randomly selected from women who had undergone at least three cycles of IVF within a similar time frame and at the same IVF centre and had failed to become pregnant (n = 78). Additional criteria included the availability of banked serum from the serial bloods used to monitor gonadotrophin stimulation, including the specimen obtained immediately prior to human chorionic gonadotrophin (HCG) stimulation and follicular fluid from the first follicle aspirated on the day of egg retrieval. Age was not matched to allow it to be tested as a variable in subjects preselected for IVF. This study included ovulatory patients with tubal disease and/or endometriosis, patients with unexplained infertility and couples with mild male factor infertility in whom donor spermatozoa and intracytoplasmic sperm injection were not recommended. The vast majority of patients had IVF performed, while there were six GIFT cycles in each of the case and control groups.

Protocol
In patients admitted to the study, an additional baseline blood sample was collected between days 1 and 4 of the menstrual cycle prior to their first treatment cycle (available in 68 cases and 44 controls) and assayed for FSH, inhibin B and oestradiol. The longest time frame between baseline sampling and the cycle used for analysis was 3 months. Patients generally underwent 10 days of gonadotrophin releasing hormone agonist down-regulation followed by exogenous gonadotrophin stimulation (Humegon, Organon, Mt. Pleasant, NJ, USA). Blood samples were collected for the purpose of this study on the day of HCG administration, and assayed for inhibin A, inhibin B and oestradiol. For cases (pregnant), the specimen from the successful cycle was used and, for controls (non-pregnant), the most recent available specimen from a failed cycle was chosen. Follicular fluid was obtained from the first, and generally the largest, follicle aspirated and was assayed for inhibin A and inhibin B. The aspirate was not diluted with a saline wash. The follicular fluid aspirate was matched to the pre-HCG serum specimen by treatment cycle.

Historical information collected for each subject included the age at enrolment, the number of prior pregnancies, the smoking history and primary infertility diagnosis. Information related to the stimulation cycle included the number of eggs retrieved at the time of aspiration, the number of fertilized eggs (eggs that exhibit two pronuclei and have undergone at least one normal division determined at 24 h) and the pregnancy status.

Assays
Plasma FSH was measured by radioimmunoassay as previously described (Crowley et al., 1980Go; Filicori et al., 1984Go). All samples were analysed in duplicate. FSH concentrations are expressed in IU per litre, as equivalents of the Second International Reference Preparation of human menopausal gonadotrophins. The inter- and intra-assay coefficients of variation were similar to those previously described (Hall et al., 1994Go). Oestradiol was measured at the time of the HCG administration using a microparticle enzyme immunoassay (Abbott Laboratories, Abbott Park, IL, USA) as previously described (Khoury et al., 1993Go). The intra-assay coefficient of variation was <6% and the inter-assay coefficient of variation was 6% for 500 and 1125 pg/ml standards and 10% for a 144 pg/ml standard. The functional sensitivity of the assay was 50 pg/ml. To attain the greater sensitivity required for baseline measurements, oestradiol was measured at baseline using a radioimmunoassay (Coat-a-Count, Diagnostic Products Corporation, Los Angeles, CA, USA). The intra-assay coefficient of variation was 6% and the inter-assay coefficient of variation was 7%. The sensitivity of the assay was 8 pg/ml.

Matched samples for dimeric inhibin A and inhibin B in the pre-HCG sample and the follicular fluid were measured in the same assay and samples from pregnant and non-pregnant subjects were combined in each assay plate in random order. Baseline samples for inhibin B were assayed separately with randomization of pregnant and non-pregnant samples on each plate. Inhibin A was measured in duplicate by enzyme-linked immunosorbent assay (ELISA) (Serotec, Oxford, UK) as previously described (Lambert-Messerlian et al., 1994Go). The assay utilizes a lyophilized human follicular fluid calibrator standardized as equivalents of the World Health Organization recombinant human inhibin A preparation 91/624, and values are reported as IU per ml. The intra-assay coefficient of variation for the dimeric inhibin A assay was 3.9%, the inter-assay coefficient of variation was 6.8%, and the assay sensitivity was 1 IU/ml.

Inhibin B was also measured by ELISA (Serotec) as previously described (Seminara et al., 1996Go). The lowest measurable concentration of inhibin B (mean ± 2 SD of multiple zero standard measurements) was 15 pg/ml. However, dilution of human serum samples revealed a clinical detection limit of 50 pg/ml, with less than 20% within plate variation. Thus, 50 pg/ml was used as the lower limit of detection of the assay. The working range for the assay was 50–500 pg/ml and all samples with concentrations in excess of 500 were appropriately diluted to read within this range. The intra-assay coefficient of variation was 4–6% in samples pretreated separately, however, intra-assay variability was close to zero for duplicate samples pretreated together. Therefore, samples were measured individually after the pretreatment step. The inter-assay coefficient of variation was 15–18% using 120, 250 and 720 pg/ml standards.

Data analysis
Chi-square analysis was used for comparison of categorical variables between pregnant and non-pregnant subjects. Unpaired t-testing was used for comparison of continuous variables between pregnant and non-pregnant subjects and generalized linear modelling was used to confirm that the same relationships persisted after adjusting for age. Spearman correlations were used to examine the associations between continuous variables. Multiple logistic regression analysis was then used to determine the effect of categorical or continuous variables as determinants of successful pregnancy, adjusting for the presence of other variables. Separate models were used for baseline and treatment characteristics so that the smaller number of subjects with baseline data would not decrease the power of the model. Results are expressed as mean ± SE unless otherwise indicated. A P-value of <0.05 was considered significant for all analyses except correlations in which multiple variables were compared and a P-value of <0.005 was considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Baseline characteristics
In an analysis of variables reduced to categories, age differed significantly between pregnant and non-pregnant subjects (34.0 ± 0.5 versus 36.0 ± 0.5 years) but parity, smoking history, and fertility diagnosis did not (Table IGo). Although baseline FSH was slightly lower (11.7 ± 0.5 versus 12.9 ± 0.9 IU/l) and baseline oestradiol (48.5 ± 6.5 versus 38.4 ± 3.0 pg/ml) and inhibin B (89.0 ± 10.2 versus 79.7 ± 7.7 pg/ml) slightly higher in pregnant than in non-pregnant subjects respectively, there was no significant difference between the two groups (Figure 1Go). Baseline FSH, oestradiol and inhibin B were also not significantly different between pregnant and non-pregnant subjects when analysed as categorical variables expressed in quartiles based on the distribution of data in all subjects (Table IGo). However, a combination variable which separated subjects based on the FSH and inhibin B medians for the whole group demonstrated a significant difference between pregnant and non-pregnant subjects such that the fewest pregnancies were seen in the high FSH, low inhibin B group and the greatest number of pregnancies in the low FSH, high inhibin B group (Table IGo). Women with FSH < 11.22 IU/l and inhibin B >= 76.5 pg/ml had a threefold greater chance of success compared with those with FSH >= 11.22 IU/l and inhibin B < 76.5 pg/ml in a multivariate model controlling for age. A combination variable based on the median baseline oestradiol and inhibin B or oestradiol and FSH was not different between pregnant and non-pregnant subjects.


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Table I. Distribution of baseline characteristics between subjects succeeding or failing to become pregnant after assisted reproductive techniques
 


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Figure 1. Baseline FSH and inhibin B, drawn between days 1 and 4 of the menstrual cycle prior to treatment. Each {square} represents one pregnant (case) and each {circ} one non-pregnant (control) subject. The horizontal line represents the mean level for each hormone. The dotted lines encompass two standard deviations around the mean for follicle stimulating hormone (FSH) and inhibin B in normal cycling women, and the solid line represents the lower limit of detection for inhibin B. There was no significant difference between the mean levels for FSH or inhibin B between pregnant and non-pregnant subjects.

 
Treatment cycle characteristics
There was no difference in the number of pregnant compared with non-pregnant subjects in either the GIFT or IVF groups (Table IGo). The number of oocytes retrieved was significantly higher in pregnant than in non-pregnant subjects, independent of the age of the patient (Table IIGo). The number of embryos transferred was not different between pregnant and non-pregnant subjects but became significant when controlled for age (Table IIGo). On the day of HCG administration, both serum inhibin A and inhibin B concentrations were significantly higher in pregnant than in non-pregnant subjects (Table IIGo; Figure 2Go), while oestradiol was not (range 447.2 to 4907.8 versus 472.3 to 4325.4 pg/ml for cases and controls respectively). Follicular fluid inhibin B was also higher in cases than in controls, but follicular fluid inhibin A was not (Table IIGo; Figure 2Go). Age adjustment did not change the significance of these variables.


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Table II. Comparison of treatment characteristics as continuous variables between pregnant and non-pregnant subjects
 


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Figure 2. Gonadotrophin-stimulated serum inhibin B and inhibin A concentrations at the time of human chorionic gonadotrophin administration (upper panels), and follicular fluid (FF) inhibin B and inhibin A levels in the first, and largest follicle aspirated 36 h later (lower panels). Each {square} represents one pregnant (PR; case) and each {circ} one non-pregnant (Not PR; control) subject. The horizontal line represents the mean level for each hormone. Mean serum inhibin B (P < 0.0001) and inhibin A (P < 0.007) and follicular fluid inhibin B (P < 0.006) were significantly higher in pregnant than in non-pregnant subjects.

 
The number of oocytes retrieved was positively correlated with stimulated inhibin A (r = 0.54; P < 0.0001), inhibin B (r = 0.69; P < 0.0001) and oestradiol (r = 0.58; P < 0.0001). Inhibin A (r = 0.40, P < 0.0001) was correlated with the total number of embryos. Use of a multivariate logistic regression indicated that age, serum inhibin A and inhibin B on the day of HCG administration and oocyte number were predictive of pregnancy (Table IIIGo). However, controlling for age and number of oocytes in the model indicated that inhibin A and inhibin B provided no additional information in predicting a successful outcome (Table IIIGo).


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Table III. Parameters and P-values for selected treatment characteristics as predictors of successful in-vitro fertilization
 
Inhibin dynamics
At baseline, there was no correlation between age, FSH or inhibin B. However, baseline inhibin B was positively correlated with the number of oocytes retrieved after gonadotrophin stimulation (r = 0.30; P < 0.002).

Gonadotrophin-stimulated inhibin B was positively correlated with stimulated inhibin A and oestradiol on the day of HCG administration, and with the follicular fluid concentration of inhibin B in the first follicle aspirated 36 h later (Figure 3Go). Gonadotrophin-stimulated inhibin A was positively correlated with oestradiol on the day of HCG administration (r = 0.65; P < 0.0001), but not with follicular fluid inhibin A.



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Figure 3. Correlation of gonadotrophin-stimulated inhibin B with stimulated inhibin A, oestradiol and follicular fluid inhibin B from the first and largest follicle aspirated. Each {square} represents one pregnant (case) and each {circ} one non-pregnant (control) subject. Correlation coefficients and P-values are indicated.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the current study, dimeric inhibin B at baseline and inhibin A and inhibin B after gonadotrophin stimulation have been examined as markers of IVF outcome and compared with age and day 3 FSH using a nested case-control design. We have shown that gonadotrophin-stimulated inhibin A and inhibin B concentrations correlate with the number of oocytes retrieved and are associated with an increased chance for a successful outcome even after adjustment for age. However, from a clinical perspective, the most useful markers are those that can be assessed before a patient begins treatment. In this population of subjects preselected for IVF by age and FSH concentration, age remained a significant factor predicting pregnancy. FSH and inhibin B did not independently predict pregnancy, but baseline inhibin B may enhance the predictive value of FSH.

Lower gonadotrophin-stimulated oestradiol concentrations and follicle number were shown to correlate with the age-related decline in ovarian reserve (Rosenwaks et al., 1986). Previous studies have found a positive correlation between the number of oocytes retrieved and inhibin as measured by the Monash assay (inhibin A, inhibin B and {alpha}-inhibin) on the day of HCG administration after gonadotrophin stimulation (McLachlan et al., 1986Go; Hughes et al., 1990Go; Matson et al., 1991Go; Haning et al., 1994Go), but did not find an association between stimulated inhibin and successful IVF outcome (Hughes et al., 1990Go). A positive correlation between gonadotrophin-stimulated dimeric inhibin A and follicle number has also been described in a small series in which pregnancy outcome was not examined (Lockwood et al., 1996Go). In the current study, a positive correlation was seen between inhibin A and oocyte number and between inhibin B and oocyte number, and higher concentrations of inhibin A and inhibin B were present in patients who conceived. Thus, higher stimulated inhibin A and inhibin B concentrations appear to measure ovarian reserve as a reflection of oocyte number and are positively associated with a successful pregnancy outcome.

Earlier studies suggest that inhibin concentrations reflect follicle quality but these studies did not distinguish between inhibin A and inhibin B. In-vitro studies of granulosa cells from older infertile women (Pellicer et al., 1994Go) and women with high FSH concentrations and poor IVF outcome (Seifer et al., 1996Go) demonstrate decreased inhibin secretion in culture (Pellicer et al., 1994Go; Seifer et al., 1996Go). Consistent with in-vitro findings, follicular fluid inhibin concentrations were lower in older subjects with poor pregnancy outcome (Dionyssiou-Asteriou et al., 1993Go). In the current study, follicular fluid inhibin B in the first follicle aspirated was 1.5-fold higher in subjects who became pregnant, consistent with studies in the mouse in which inhibin B identified the best quality follicles (Smitz and Cortvrindt, 1998Go). Further, inhibin A was correlated with the number of oocytes and with the number of embryos, also consistent with mouse studies in which inhibin A marked follicular maturation (Smitz and Cortvrindt, 1998Go). Taken together, these studies suggest that inhibin B reflects granulosa cell secretory capacity and therefore follicle quality, while inhibin A may be a better marker of follicular maturation and consequently oocyte quality.

During the normal menstrual cycle, inhibin B concentrations are highest in the early to mid-follicular phase and decrease in the late follicular phase (Groome et al., 1996Go; Welt et al., 1999Go), suggesting secretion by developing follicles. Inhibin A rises in the late follicular phase of the normal menstrual cycle as a product of the pre-ovulatory follicle (Groome et al., 1994Go; Lambert-Messerlian et al., 1994Go; Welt et al., 1999Go). It is unclear, however, whether inhibin B is also produced by the pre-ovulatory follicle. While one in-vitro study found inhibin B protein in a pre-ovulatory follicle (Yamoto et al., 1992Go), a second was unable to demonstrate ß-subunit mRNA or inhibin B protein (Roberts et al., 1993Go). Data from the present study and others (Lockwood et al., 1996Go) demonstrate a progressive rise in inhibin B in association with gonadotrophin stimulation, which stands in contrast to the pattern of inhibin B in the normal menstrual cycle. The positive correlation between serum inhibin B and inhibin A and between serum inhibin B and follicular fluid inhibin B in this study suggests that inhibin B can be made by pre-ovulatory follicles during exogenous gonadotrophin stimulation.

Previous studies have demonstrated that inhibin is stimulated by luteinizing hormone in vitro in human granulosa cells (Hillier et al., 1991Go). Thus, the absence of a correlation between serum inhibin A drawn prior to HCG administration and follicular fluid inhibin A from the lead follicle at the time of oocyte retrieval may represent the difference in the timing of sample collection, with serum collected before and follicle fluid collected after the HCG stimulus. We have shown that inhibin B does not increase in response to HCG in normal women or in women with polycystic ovarian syndrome (Hall et al., 1996Go), perhaps accounting for the preserved relationship between stimulated inhibin B in serum and follicular fluid.

The most useful predictive information for an infertile couple is that which can be obtained before beginning assisted reproductive techniques. Basal inhibin concentrations have been evaluated previously as predictive markers for pregnancy in IVF cycles. In two (Tsuchiya et al., 1989Go; Balasch et al., 1996Go) of three (Hughes et al., 1990Go) previous studies, higher inhibin on day 2–3 was associated with a greater number of oocytes retrieved in IVF cycles (Balasch et al., 1996Go) and with subsequent pregnancy (Tsuchiya et al., 1989Go) and was determined to be a better predictor of the response to exogenous gonadotrophins than age (Balasch et al., 1996Go), and equivalent to day 3 FSH. These previous studies used assays which utilized antibodies directed at the {alpha}-subunit of inhibin, therefore measuring inhibin A, inhibin B and the non-biologically active {alpha}-subunit (Robertson et al., 1989Go, 1997Go; Schneyer et al., 1990Go). It is likely, however, that {alpha}-inhibin and inhibin B were the inhibin species measured in the early follicular phase in these earlier studies, since they are the only species measured in significant quantities at this time in the menstrual cycle (Groome et al., 1996Go; Welt et al., 1997Go). Using a specific assay for inhibin B (Groome et al., 1996Go), the association between high follicular phase FSH concentrations in older ovulatory women and low concentrations of inhibin B has suggested that inhibin B concentrations may be both a biochemical index of a decreased pool of pre-antral follicles and an endocrine negative feedback regulator of FSH secretion (Klein et al., 1996Go). Seifer et al. (1997) demonstrated that, after controlling for age, day 3 FSH and day 3 oestradiol, pregnancy rates were higher in patients with day 3 inhibin B concentrations >=45 pg/ml than in patients with lower values.

Results of the present study do not support the use of day 3 inhibin B as a predictive marker of IVF outcome. There was tremendous overlap in baseline inhibin B concentrations between pregnant and non-pregnant subjects, and inhibin B alone failed to predict pregnancy. The difference in study design or selection of patients may explain the difference in the current results and those of Seifer et al. (1997). Specifically, the preselection of subjects for entrance into IVF in this study may have decreased the ability to detect differences in inhibin B at baseline. However, if inhibin B were a better marker of outcome than FSH alone, its predictive value should have been detected since the current study had sufficient power to detect a 40% difference in inhibin B concentrations in pregnant compared with those of non-pregnant subjects, well under the difference described previously (Seifer et al., 1997Go). Further, it must be noted that the normal range for inhibin B has changed over the course of assay development, presumably because of changes in the inhibin standards. Whereas Seifer et al. (1997) cite 45 pg/ml as the upper end of the 95% confidence limits for day 3 inhibin B, day 3 inhibin B is 86 ± 38 pg/ml (mean ± 1 SD) in our population of 46 normal women (Welt et al., 1999Go) measured concurrently with samples from the present study. Thus, the inhibin B value of 45 pg/ml has little meaning in the present inhibin B assay (Serotec).

Age was the most significant predictor of pregnancy in the current study. However, in contrast to earlier studies (Muasher et al., 1988Go; Scott et al., 1989Go Scott et al., 1995; Toner et al., 1991Go), baseline FSH did not predict pregnancy outcome. This difference may be due, at least in part, to clinical preselection criteria which excluded women with high FSH concentrations from IVF therapy. It has previously been shown that maximum FSH is not correlated with parameters of ageing in women under the age of 35 years whose FSH concentrations are in the normal range (Schipper et al., 1998Go). Although in our preselected population the combination of FSH greater than the median and inhibin B less than the median may strengthen the information derived from FSH and age, the combination is not absolutely predictive of failure to become pregnant. Baseline oestradiol alone or in combination with FSH was also not predictive of pregnancy in this study, although others have found that it enhanced the predictive value of FSH (Licciardi et al., 1995Go; Smotrich et al., 1995Go).

In conclusion, during assisted reproductive cycles, stimulated serum inhibin A and inhibin B concentrations are associated with pregnancy and mark ovarian reserve as a measure of oocyte number and follicle quality. The correlation between serum and follicular fluid inhibin B concentrations and their association with pregnancy suggests that inhibin B may reflect granulosa cell secretory capacity. In patients undergoing assisted reproductive technology, the strongest predictors of success are age and number of oocytes retrieved, and neither FSH nor inhibin B at baseline predicts pregnancy in a population preselected clinically for IVF.


    Acknowledgments
 
We would like to thank Drs. Mark Hornstein, Patricia McShane and Doug Powers, directors of the participating IVF Centers, for their critical contributions to this study. In addition, we would like to acknowledge the support of the Reproductive Endocrine Sciences Center Assay Core at Massachusetts General Hospital and its director Dr. Patrick Sluss who has provided critical input. We would like to thank Rebecca F.Liberman, MPH, Evelyn Y.Li, and Mary F.DePari for assistance with data analysis, Cam Fraer, BSN, for database support and Drs. R.Barbieri, W.F.Crowley Jr, and A.L.Schneyer for their helpful reviews of the manuscript.

This work was supported by NIH U54 HD29164, R01 HD32153, M01RR01066 and P30 HD28138.


    Notes
 
3 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Balasch, J., Creus, M., Fabregues, F. et al. (1996) Inhibin, follicle-stimulating hormone and age as predictors of ovarian response in in vitro fertilization cycles stimulated with gonadotropin-releasing hormone agonist-gonadotropin treatment. Am. J. Obstet. Gynecol., 127, 1226–1230.

Crowley, W.F., Beitins, I.Z., Vale, W. et al. (1980) The biologic activity of a potent analogue of gonadotropin releasing hormone in normal and hypogonadotropic men. N. Engl. J. Med., 302, 1052–1057.[Abstract]

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Groome, N.P., Illingworth, P.J., O'Brien, M. et al. (1994) Detection of dimeric inhibin throughout the human menstrual cycle by two-site enzyme immunoassay. Clin. Endocrinol., 40, 717–723.[ISI][Medline]

Groome, N.P., Illingworth, P.J., O'Brien, M. et al. (1996) Measurement of dimeric inhibin B throughout the human menstrual cycle. J. Clin. Endocrinol. Metab., 81, 1401–1405.[Abstract]

Hall, J.E., Taylor, A.E., Martin, K. et al. (1994) Decreased release of gonadotropin-releasing hormone during the preovulatory midcycle luteinizing hormone surge in normal women. Proc. Natl Acad. Sci. USA, 91, 6894–6898.[Abstract/Free Full Text]

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Submitted on July 28, 1998; accepted on November 9, 1998.