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
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Abstract |
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Key words: FSH/inhibin/in-vitro fertilization/oestradiol/pregnancy
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Introduction |
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The inhibins are dimeric peptides composed of an -subunit along with a ßA-subunit, inhibin A, or a ßB-subunit, inhibin B (Vale et al., 1988
). Inhibin B concentrations rise across the lutealfollicular transition and peak in the mid-follicular phase, suggesting secretion by the developing cohort of follicles in a cycle (Groome et al., 1996
; Welt et al., 1997
). 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., 1994
; Lambert-Messerlian et al., 1994
). 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.
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Materials and methods |
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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., 1980; Filicori et al., 1984
). 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., 1994
). 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., 1993
). 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., 1994). 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., 1996). 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 50500 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 46% 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 1518% 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.
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Results |
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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 3). 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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Discussion |
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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 -inhibin) on the day of HCG administration after gonadotrophin stimulation (McLachlan et al., 1986
; Hughes et al., 1990
; Matson et al., 1991
; Haning et al., 1994
), but did not find an association between stimulated inhibin and successful IVF outcome (Hughes et al., 1990
). 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., 1996
). 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., 1994) and women with high FSH concentrations and poor IVF outcome (Seifer et al., 1996
) demonstrate decreased inhibin secretion in culture (Pellicer et al., 1994
; Seifer et al., 1996
). Consistent with in-vitro findings, follicular fluid inhibin concentrations were lower in older subjects with poor pregnancy outcome (Dionyssiou-Asteriou et al., 1993
). 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, 1998
). 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, 1998
). 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., 1996; Welt et al., 1999
), 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., 1994
; Lambert-Messerlian et al., 1994
; Welt et al., 1999
). 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., 1992
), a second was unable to demonstrate ß-subunit mRNA or inhibin B protein (Roberts et al., 1993
). Data from the present study and others (Lockwood et al., 1996
) 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., 1991). 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., 1996
), 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., 1989; Balasch et al., 1996
) of three (Hughes et al., 1990
) previous studies, higher inhibin on day 23 was associated with a greater number of oocytes retrieved in IVF cycles (Balasch et al., 1996
) and with subsequent pregnancy (Tsuchiya et al., 1989
) and was determined to be a better predictor of the response to exogenous gonadotrophins than age (Balasch et al., 1996
), and equivalent to day 3 FSH. These previous studies used assays which utilized antibodies directed at the
-subunit of inhibin, therefore measuring inhibin A, inhibin B and the non-biologically active
-subunit (Robertson et al., 1989
, 1997
; Schneyer et al., 1990
). It is likely, however, that
-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., 1996
; Welt et al., 1997
). Using a specific assay for inhibin B (Groome et al., 1996
), 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., 1996
). 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., 1997). 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., 1999
) 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., 1988; Scott et al., 1989
Scott et al., 1995; Toner et al., 1991
), 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., 1998
). 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., 1995
; Smotrich et al., 1995
).
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.
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Acknowledgments |
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This work was supported by NIH U54 HD29164, R01 HD32153, M01RR01066 and P30 HD28138.
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Notes |
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References |
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Submitted on July 28, 1998; accepted on November 9, 1998.