IT IS NOW recognized that inhibin plays an important

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1 X/00/$03.00/0 Vol. 85, No. 2 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright 2000 by The Endocrine Society Relationship between Serum Inhibin A and B and Ovarian Follicle Development after a Daily Fixed Dose Administration of Recombinant Follicle-Stimulating Hormone* T. ELDAR-GEVA, D. M. ROBERTSON, N. CAHIR, N. GROOME, M. P. GABBE, V. MACLACHLAN, AND D. L. HEALY Monash IVF (T.E.-G., M.P.G., V.M., D.L.H) and the Department of Obstetrics and Gynecology, Monash University (T.E.-G., D.L.H), Prince Henry s Institute of Medical Research (D.M.R., N.C.), Clayton, Victoria 3168, Australia; and Oxford Brookes University (N.G.), Oxford, United Kingdom ABSTRACT The aim of this study was to investigate the relationship of serum inhibin A and inhibin B to ovarian follicular development in women undergoing pituitary down-regulation and ovarian stimulation with a fixed daily dose of recombinant human FSH in an in vitro fertilization program. Thirty-eight patients were treated randomly with either 100 or 200 IU/day recombinant human FSH (Puregon) for a period of 9 14 days. Serum FSH, inhibin A, inhibin B, 17 -estradiol, and follicular size and number were determined before FSH treatment and every second day from days 4 6 throughout FSH treatment. Serum FSH increased in a dose-related manner to reach a maximum by days 4 6 and remained unchanged over the duration of treatment. Serum inhibin A and 17 -estradiol also increased with increasing FSH dose and continued to rise throughout the FSH treatment period. By contrast, serum inhibin B was increased by days 4 6 at both doses of FSH to reach a Received November 3, Revision received July 30, Accepted October 25, Address all correspondence and requests for reprints to: Dr. T. Eldar- Geva, Monash IVF and the Department of Obstetrics and Gynecology, Monash University, Clayton, Victoria 3168, Australia. * This study was supported in part by Program Grants and from the National Health and Medical Research Council of Australia and by Organon. maximum by days 7 8, remaining unchanged thereafter. Serum inhibin B and, to a lesser extent, inhibin A correlated significantly with the number of oocytes retrieved even when assessed early (days 4 6) in the treatment period (inhibin B vs. number of oocytes: r 0.89; P 0.001; inhibin A vs. number of oocytes: r 0.61; P 0.05). Serum inhibin A, inhibin B, and 17 -estradiol were weakly correlated with the number of follicles less than 11 mm when assessed on a daily basis; stronger correlations were observed with the greater than 11-mm follicles during the late stages of treatment. It is concluded that serum inhibin B levels determined during the early stages (e.g. days 4 6) of fixed dose FSH treatment provide an early indicator of the number of recruited follicles that are destined to form mature oocytes. In this context, serum inhibin B may be of predictive value in monitoring ovarian hyperstimulation treatment for in vitro fertilization. (J Clin Endocrinol Metab 85: , 2000) IT IS NOW recognized that inhibin plays an important role in regulating FSH secretion (1 3). Two forms of biologically active inhibin (inhibin A and B) have been identified. Based on their tissue localization, as assessed by in situ hybridization (4, 5) and immunocytochemical methods (6, 7), and their serum patterns during the menstrual cycle (8 10), it is believed that inhibin B is produced by a cohort of recruited follicles, whereas inhibin A is primarily a product of the dominant follicle. These data suggested that inhibin B may be a suitable marker of ovarian follicle reserve in the assessment of in vitro fertilization (IVF) outcome in women (11, 12), whereas inhibin A may provide an alternative index to serum 17 estradiol (E 2 ) of dominant follicular development (13). However, studies exploring the relationship between controlled FSH stimulation, inhibin A and B, and ovarian follicular development are limited. The opportunity to explore in detail the relationship between serum inhibin A and B and ovarian follicular development arose as a side study to a multicenter trial aimed at the investigation of the efficacy of a fixed daily dose regimen of 100 or 200 IU recombinant human FSH (Puregon, Organon, Lane Cove, Australia) in promoting ovarian hyperstimulation in pituitary-suppressed infertile women undergoing IVF. In the work reported here, the relationships among circulating levels of inhibin A, inhibin B, FSH, and E 2 at different times during FSH treatment; the number and size of ovarian follicles; and the number of oocytes retrieved after treatment were examined. The results suggest that serum inhibin B determined early during FSH treatment provides an index of the number of recruited follicles that will ultimately reach maturity and may provide a useful early marker in the clinical management of controlled ovarian hyperstimulation. Subjects Subjects and Methods Thirty-eight women, aged yr (mean, 32.7 yr), undergoing IVF treatment were included in the study. The inclusion criteria used were 1) subjects should have normal ovulatory cycles with a mean length of between days, a body mass index between kg/m 2, and be in good physical and mental health; and 2) the cause of infertility must be treatable by IVF or intracytoplasmic sperm injection (ICSI) procedures. 607

2 608 ELDAR-GEVA ET AL. JCE&M 2000 Vol 85 No 2 The exclusion criteria used were 1) infertility attributed to endocrine abnormalities such as hyperprolactinemia, polycystic ovarian syndrome, and absence of ovarian function; 2) previous IVF, gamete intrafallopian transfer or zygote intrafallopian transfer attempts in which less than three oocytes were retrieved; 3) chronic cardiovascular, hepatic, renal, or pulmonary disease; 4) history of current abuse or abuse within the last 12 months of alcohol or drugs; 5) treatment with investigational drugs within 3 months before screening; and 6) presence of ovarian cysts more than 2 cm. The study protocol was approved by the Epworth Hospital research and ethics committee (Melbourne, Australia). Informed consent was obtained from all participants. Treatment protocol This study consists of a subset of a larger, randomized, double-blind clinical trial (14) examining the efficacy of a daily fixed dose regimen of 100 or 200 IU recombinant human FSH in pituitary-suppressed infertile women undergoing controlled ovarian hyperstimulation for IVF. After taking a blood sample for determination of basal hormone levels, patients were initially treated with a GnRH agonist, nafarelin (0.5 mg/day; Synarel, Searle, Sydney, Australia), by intranasal administration for 14 days, starting in the midluteal phase of the ovarian cycle. When E 2 levels were below 150 pmol/l and any ovarian cysts were excluded by vaginal ultrasound, 100 or 200 IU recombinant human FSH (Puregon, Organon) were administered daily sc. The ovarian response was monitored by serum E 2 and transvaginal ultrasonography every 1 3 days from the fourth day of FSH treatment. The ultrasonography detection limit was 1 mm, and its reproducibility in detecting follicles, as assessed from the coefficient of variation of repeated measurements, was 6%. The number and size of follicles with diameter greater than 2 mm were recorded. Serum samples were collected before FSH administration and every 1 3 days from days 4 to days 9 14 of treatment. Serum was stored at 20 C until assayed for progesterone, FSH, LH, inhibin A, and inhibin B. Treatment was continued for a maximum of 3 weeks until at least three follicles larger than 17 mm developed. hcg (5000 IU, im; Profasi, Serono, Melbourne, Australia) was given to promote ovulation. Oocytes were retrieved transvaginally 36 h later under general anesthesia. Routine IVF or ICSI and embryo culture were used as indicated, and a maximum of three embryos were transferred 2 6 days after oocyte retrieval. Eighteen patients were randomized to receive 100 IU FSH/day, and 20 patients received 200 IU FSH/day. Both groups were comparable in demographic and infertility characteristics (Table 1). TABLE 1. Demographic and clinical data in patients receiving a fixed daily dose of FSH Characteristic 100 IU/day (n 18) 200 IU/day (n 20) Age (yr) BMI (kg/m 2 ) Wt (kg) No. of patients with cause of infertility Tubal/endometriosis 1 3 Male factor 8 9 Multiple causes 4 3 Idiopathic 5 5 Duration of infertility (yr) Treatment cycle no No. of patients with primary 10 (56) 14 (70) infertility (%) Duration of FSH treatment a (days) Serum LH (IU/L) Serum progesterone (nmol/ L; h before hcg treatment) Values are the mean SD. a P 0.05, 100 vs. 200 IU/L. Oocyte maturation assessment In patients with severe male factor infertility, the ICSI procedure was used (11 cases each were treated with 100 and 200 IU/day FSH). After hyaluronidase treatment, oocyte maturation were assessed according to the criteria described previously (15). Hormone assays Serum concentrations of E 2, progesterone, LH, FSH, and PRL were measured using a chemiluminescent immunoassay (Diagnostic Products, Los Angeles, CA). The inter- and intraassay coefficients of variation were, respectively, 9.2% and 4.9% for E 2, 12.3% and 9.2% for progesterone, 7.8% and 1.9% for LH, 4.3% and 3.6% for FSH, and 3.7% and 1.7% for PRL. Serum inhibin A was measured using an / A -subunit dimeric enzyme-linked immunosorbent assay (16), with the WHO inhibin A preparation (91/624) used as standard. The assay sensitivity was 2 pg/ml, and the between-assay variation was 18%. Serum inhibin B concentrations were measured by the method of Groome et al. (10), using a human inhibin B preparation isolated from human follicular fluid by N. Groome. The assay sensitivity was 15 pg/ml, and the between-assay variation was 18%. Statistical analyses Samples with hormone values below the assay detection limit were assigned values equal to the detection limit of that assay. Testing for differences between treatment groups was performed using both the Mann-Whitney test (for differences between medians) and the twosample t test (for differences between means). Similar statistical results were found with the use of both tests in this study. The 2 test was used to test for differences between infertility groups. One-way ANOVA was used to analyze serial changes in serum hormones concentrations and number of follicles. Results are expressed as the mean sd unless otherwise indicated. Differences were considered statistically significant at P Results Serum hormone levels Daily administration of either 100 or 200 IU FSH produced a significant (P 0.05) dose-related increase in serum FSH, E 2, and inhibin A (Fig. 1). By contrast, serum inhibin B showed a maximum response with either FSH dose. The time course of response to FSH treatment differed among the various hormones. After 4 6 days of treatment, serum FSH values showed stable levels for the duration of FSH administration at both FSH doses, whereas inhibin A and E 2 showed a continuous significant (P 0.05) increase from days 4 6 to 9 10 days of FSH treatment. By contrast, inhibin B was significantly (P 0.05) increased by 4 6 days of treatment to reach a maximum by days 7 8 and remained unchanged thereafter for the duration of FSH treatment. Serum LH levels remained unchanged in patients treated with either 100 or 200 IU FSH/day (Table 1). Serum progesterone levels measured h before hcg administration were comparable in the two groups(table 1). Follicle number and size The follicles were divided into three groups according to diameter: less than 11, 11 14, and 15 or more mm, respectively. The number of 11- to 14-mm and 15-mm or more follicles significantly (P 0.05) increased with FSH treatment, although no change was observed in the number of less than 11-mm follicles (Fig. 2). A significant FSH dose-related increase was observed in the numbers of oocytes and mature

3 SERUM INHIBIN A AND B IN IVF 609 oocytes collected and the total number of embryos per patient transferred and frozen (Fig. 2). As the duration of FSH treatment was significantly longer for the 100 IU FSH/day group (10.8 days) compared to the 200 IU FSH/day group (9.3 days; Table 1), the serum hormone levels and follicle count were also expressed in terms of the last day of treatment (day of hcg administration). As shown in Figs. 1 and 2 and Table 2, a similar FSH doseresponse effect with these parameters was observed compared to FSH responses during the latter days of treatment. FIG. 1. Time course of effect of daily FSH treatment on serum hormone levels. The pretreatment sample was obtained during the luteal phase of a normal cycle before GnRH agonist treatment. The range in number of subjects is presented in brackets; for days 11 12, the first number presented refers to the 200-IU dose group. *, P 0.05; **, P 0.01; ***, P Relationship between serum hormone levels and number of oocytes retrieved No significant correlations were observed between serum inhibin A (r 0.16; n 19) and inhibin B (r 0.21; n 19) on day 21 (midluteal phase) of the previous menstrual cycle and the number of eggs collected during the subsequent treatment cycle. To investigate the ability of serum hormone levels or follicular number during early FSH treatment to predict treatment outcome, correlation coefficients were determined between the number of oocytes retrieved and serum inhibin A, inhibin B, and E 2 determined on days 4 6, 7 8, and 9 10 and the last day (12 36 h before hcg administration; Table 2 and Fig. 3). Highly significant correlations (r ) were found between serum inhibin B levels and the number of oocytes on all days of treatment. Serum inhibin A and E 2 showed significant, but lower, correlations (r 0.78) with oocyte number later in the treatment period. The number of 11- to 14-mm and more than 15-mm follicles correlated highly (r 0.63) with oocyte number in the middle to late stages of treatment (Table 2). Relationship between serum hormone levels and follicular number The relationship between serum ovarian hormone levels, and follicle number and size at various stages of FSH treatment was examined (Table 3). Correlations of limited (P 0.05) or no significance were observed on any day of treatment between the number of less than 11-mm follicles and serum FSH, E 2, and inhibin A and B. Significant correlations (P ) were observed between the number of 11- to 14-mm follicles and serum E 2 and inhibin B levels during days 7 12 of FSH treatment. The highest correlations (r ) were observed between the more than 15-mm follicles and both serum E 2 and inhibin A on days 9 10 of treatment. Interrelationships among the various hormones As shown in Table 4, a highly significant correlation (P ) was observed between serum inhibin A and B on days 4 8 of treatment, with a less significant correlation (P NS to P 0.05) observed toward the end of the treatment period (days 9 10, day of hcg administration). Serum E 2 correlated closely with inhibin A (P ) during the early stages (days 4 10) and with inhibin B (P 0.001) during the later stages (days 9 10, day of hcg administration) of the treatment cycle. The correlations between the number of less than 11-mm follicles on day 0 and the number of oocytes retrieved were r 0.46 and P 0.01 (n 31). Discussion This study has provided insights into the relationship between FSH, inhibin A, and inhibin B and follicle development. The study protocol consisted of the daily administra-

4 610 ELDAR-GEVA ET AL. JCE&M 2000 Vol 85 No 2 FIG. 2. Time course of effect of daily FSH treatment on ovarian follicles as assessed by ultrasonography. The bottom panel also shows the total number of oocytes retrieved, the number of metaphase II mature oocytes collected, and the number of resultant embryos available for transfer. The range in number of subjects is presented in brackets; for days 11 12, the first number presented refers to the 200-IU dose group. **, P 0.01; ***, P TABLE 2. Correlations expressed as Pearson s correlation coefficients between the number of oocytes retrieved and serum hormones levels and follicle numbers determined on days 4 6, 7 8, and 9 10 of FSH treatment and h before hcg administration (last day) No. of oocytes retrieved vs. Days 4 6 Days 7 8 Days 9 10 Last day FSH 0.08 (17) 0.45 (25) a 0.44 (30) a 0.50 (35) b E (17) 0.57 (25) b 0.78 (30) c 0.73 (35) c Inhibin A 0.61 (14) a 0.70 (25) c 0.68 (28) c 0.50 (32) b Inhibin B 0.89 (14) c 0.79 (25) c 0.79 (28) c 0.72 (32) c No. of 11-mm follicles 0.33 (17) 0.49 (24) a 0.14 (28) 0.25 (34) No. of 11- to 14-mm follicles 0.33 (14) 0.75 (24) c 0.73 (29) c 0.69 (33) c No. of 15-mm follicles 0.28 (6) (14) 0.69 (27) c 0.63 (35) c The number of subjects is presented in parentheses. The difference in subject numbers between days of treatment relates to which days the subjects gave blood. a P b P c P

5 SERUM INHIBIN A AND B IN IVF 611 FIG. 3. Correlation between the number of oocytes retrieved and serum inhibin A and inhibin B measured on days 4 6 of FSH treatment. tion of FSH at either of two fixed doses (100 or 200 IU) that resulted in stable plasma FSH concentrations from days 4 6 to days 9 14 of FSH treatment. Under this hormonal regimen the number of small follicles remain relatively unchanged, whereas the number of medium and large follicles increased during the treatment period. Serum inhibin B increased early during treatment, whereas serum E 2 and inhibin A increased late during treatment. The temporal relationship between follicle development and serum hormone levels after FSH treatment provides a means to delineate the contribution of each follicle group to the overall serum hormone levels. On days 4 6 of FSH treatment, when the small ( 11 mm) follicles dominate, inhibin B provides the largest response, suggesting that these small follicles are largely producing inhibin B. A similar finding has been observed previously by Anderson and colleagues (17) after FSH treatment of women with polycystic ovarian syndrome. On days 7 8 of treatment, although the numbers of medium-sized (11 14 cm) follicles are increasing, the numbers of large ( 15 mm) follicles are still low. Serum inhibin A and B and E 2 correlate strongly with the number of medium-sized follicles, suggesting that these follicles readily produce these hormones, with inhibin B showing the most marked response. The late rise (days 9 12) in serum E 2 and inhibin A correlates closely with the number of 15-mm or more follicles, whereas serum inhibin B, which does not show a late rise, is less strongly correlated. These data support the proposition that large follicles produce large amounts of both E 2 and inhibin A compared with earlier time intervals. Although these findings are consistent with earlier studies, the origin of inhibin B and its responsivity to FSH is still unclear. The high correlation between inhibin A and B during the early and middle stages of FSH treatment suggests that both small and medium sized follicles are producing these inhibins (8, 13, 18). However, during the late stages of treatment, serum inhibin A and B correlate significantly with E 2, but less so with each other. On the other hand, it has been shown previously, based on immunocytochemical (6, 7) and in situ hybridization (4, 5) studies, that the A -subunit, but not the B -subunit protein and ribonucleic acid message are localized to the dominant human follicle. It is not clear how to reconcile these different observations. One explanation may relate in part to the continued production of inhibin A, inhibin B, and E 2 by the medium-sized follicles developing in parallel with the large follicles and by the changing patterns of inhibin A, inhibin B, and E 2 production as the large follicle develops. A highly significant correlation was observed between the serum levels of inhibin B during FSH treatment and the number of oocytes collected up to 8 days later (Table 2). Based on the observation that inhibin B is produced by immature follicles (4 7), these data suggest that inhibin B is being produced by immature follicles receptive to FSH stimulation and destined for subsequent maturation. The size of this FSH-responsive follicle pool probably represents a small proportion of the total follicle number, as no or limited correlations were observed between serum inhibin B levels and the number of small ( 11-mm) follicles at any stage of treatment. It is presumed that the remainder is already programmed for apoptotic death. This high correlation (r 0.79) between serum inhibin B levels observed early in FSH treatment and subsequent number of oocytes collected suggests that the measurement of inhibin B may be useful as a marker of ovarian response during early FSH treatment. In fact, the correlation coefficient is higher than that noted between the number of less than 11-cm follicles detected on day 0 (r 0.46) and the subsequent number of oocytes collected. As serum inhibin B levels at the 200-IU FSH dose are already elevated by days 4 6 of treatment, examination of inhibin B levels before day 4 may provide an even earlier marker of treatment outcome. Although previous studies have proposed that inhibin B may be a good marker of ovarian reserve (10 13), there have been few reported studies that have explored the relationship of serum inhibins, ovarian follicle number, and treatment outcome early in the FSH treatment period. Burger et al. (19) reported that serum inhibin B levels showed a larger incremental increase compared to inhibin A after a single injection of FSH early in the follicular phase. Lockwood and co-work-

6 612 ELDAR-GEVA ET AL. JCE&M 2000 Vol 85 No 2 TABLE 3. Correlation between the number of ovarian follicles of different sizes and serum endocrine markers during FSH treatment Follicles Days FSH E 2 Inhibin A Inhibin B 11 mm (18) 0.20 (18) 0.01 (15) 0.37 (15) (23) 0.22 (23) 0.22 (23) 0.56 (23) a (30) b 0.14 (30) 0.13 (27) 0.13 (26) (17) 0.32 (17) 0.14 (17) 0.14 (17) mm (15) 0.46 (15) 0.14 (12) 0.19 (12) (24) 0.62 (24) a 0.65 (24) c 0.69 (24) c (31) 0.47 (31) a 0.35 (28) 0.70 (27) c (17) 0.55 (17) b 0.28 (17) 0.52 (17) b 15 mm (6) 0.49 (6) 0.03 (5) 0.34 (5) (13) 0.42 (13) 0.11 (13) 0.04 (13) (31) b 0.80 (31) c 0.76 (28) c 0.52 (25) a (18) 0.67 (18) a 0.47 (18) b 0.46 (18) The number of subjects is presented in parentheses. a P b P c P TABLE 4. Correlation coefficients between inhibin A, inhibin B, FSH, and E 2 on different days of treatment Day 0 (n 35) Days 4 6 (n 17 19) ers (13) found that the levels of inhibin A and B rose markedly after 8 days of FSH stimulation, with serum inhibin B increasing more than serum inhibin A in women undergoing ovarian stimulation. However, in contrast to the present study, they did not determine serial changes in serum inhibins earlier during treatment. Siefer and colleagues (12) also proposed that inhibin B was a good marker of treatment outcome based on serum inhibin B levels determined in the early follicular phase of the previous spontaneous menstrual cycle. Earlier studies (20 25) using the Monash inhibin RIA (20) to determine serum inhibin levels in FSH hyperstimulation cycles, showed that E 2 and inhibin levels increased in parallel during ovarian hyperstimulation. However, this RIA detected both free -subunit and inhibin dimers and thus was unable to identify changes in the specific dimeric forms. Porchet et al. (18), using an assay recognizing both heterodimeric inhibin and free -subunit together, found that serum inhibin levels were the first pharmacodynamic markers to increase after FSH treatment in pituitary down-regulated women. Results from this study would suggest that the primary inhibin form responsible is inhibin B. In conclusion, these studies have further clarified the relationship between ovarian hormone production and follicle number and development after FSH stimulation. During the early stages of treatment, the elevated serum inhibin B levels are attributed to production by small follicles. In contrast, the late increase in serum E 2 and inhibin A is associated with production by large follicles. Medium-sized follicles appear Days 7 8 (n 30) Days 9 10 (n 31 34) to be capable of producing inhibin A, inhibin B, and E 2. Serum inhibin B levels determined during the early stages (e.g. days 4 6) of fixed dose FSH treatment provide an early indicator of the number of recruited follicles that are destined to form mature oocytes. The marked FSH stimulation of inhibin B early in treatment suggests that inhibin B may be a useful predictor in monitoring ovarian hyperstimulation treatment for IVF. Acknowledgments We thank all the clinical, nursing, embryology, and administration staff of Monash IVF and Monash US for Women for their invaluable help. The authors acknowledge Organon for supplying the recombinant FSH. References Last day (n 35 38) Inhibin A vs. inhibin B 0.44 a 0.81 b 0.66 b 0.45 c 0.31 Inhibin A vs. E b 0.58 a 0.57 b 0.39 c Inhibin A vs. FSH a 0.16 Inhibin B vs. E c 0.56 a 0.70 b 0.63 b Inhibin B vs. FSH E 2 vs. FSH c n, Number of subjects. P P P Burger HG Inhibin. Reprod Med Rev. 1: Baird DT, Smith KB Inhibin and related peptides in the regulation of reproduction. Oxf Rev Reprod Biol. 15: Vale WW, Hseuh A, Rivier C, Yu J The inhibin/activin family of hormones and growth factors. In Peptide growth factors and their receptors. In: Sporn MA, Roberts AB, eds. Handbook of experimental physiology. Berlin: Springer-Verlag; vol 95: Roberts VJ, Barth S, El-Roeiy A, Yen SSC Expression of inhibin/activin subunits and follistatin messenger ribonucleic acids and proteins in ovarian follicles and corpus luteum during the human menstrual cycle. J Clin Endocrinol Metab. 77: Jaatinen TA, Penttila TL, Kaipia A, Ekfors T, Parvinen M, Toppari J Expression of inhibin, A and B messenger ribonucleic acids in the normal human ovary and in polycystic ovarian syndrome. J Endocrinol. 143: Rabinovici J, Spencer SJ, Doldi N, Goldsmith PC, Schawall R Jaffe RB Activin-A as an intraovarian modulator: actions, localization, and regulation of the intact dimer in human ovarian cells. J Clin Invest. 89:

7 SERUM INHIBIN A AND B IN IVF Yamoto M, Minami S, Nakano R Immunohistochemichal localization of inhibin/activin subunits in human ovarian follicles during the menstrual cycle. J Clin Endocrinol Metab. 77: Muttukrishna S, Fowler PA, Groome NP, Mitchell GG, Robertson WR, Knight PG Serum concentrations of dimeric inhibin during the spontaneous human menstrual cycle and after treatment with exogenous gonadotrophin. Hum Reprod. 9: Groome NP, Illingworth PJ, O Brien M, Pal R, Rodger FE, Mather JP Mc- Neilly AS Measurement of dimeric inhibin B throughout the human menstrual cycle. J Clin Endocrinol Metab. 81: Welt CK, Martin KA, Taylor AE, et al Frequency modulation of folliclestimulating hormone (FSH) during the luteal-follicluar transition: evidence for FSH control of inhibin B in normal women. J Clin Endocrinol Metab. 82: Klein NA, Illingworth PJ, Groome NP, McNeilly AS, Battaglia MR Soules MR Decrease inhibin B secretion is associated with the menotrophic FSH rise in older, ovulatory women: a study of serum and follicular levels of dimeric A and B in spontaneous menstrual cycles. J Clin Endocrinol Metab. 81: Seifer DB, Lambert-Messerlian G, Gardiner AC, Blazer AS, Berk CA Day 3 serum inhibin B is predictive of assisted reproductive technologies outcome. Fertil Steril. 67: Lockwood GM, Muttukrshna S, Groome NP, Knight PG, Ledger WL Circulating inhibins and activin A during GNRH-analogue down-regulation and ovarian hyperstimulation with recombinant FSH for in vitro fertilisationembryo transfer. Clin Endocrinol (Oxf). 45: Out HJ, Lindenberg S, Mikkelsen AL, et al A prospective, randomized, double-blind clinical trial to study the efficacy and efficiency of a fixed dose of recombinant follicle stimulating hormone (Puregon) in women undergoing ovarian stimulation. Hum Reprod. 14: Veek L Oocyte assessment and biological performance. Ann NY Acad Sci. 541: Groome NP, O Brien M Two-site immunoassays for inhbin and its subunits. Further applications of the synthetic peptide approach. J Immunol Methods. 165: Anderson RA, Groome NP, Baird DT Inhibin A and inhibin B in women with polycystic ovarian syndrome during treatment with FSH to induce monoovulation. Clin Endocrinol (Oxf). 48: Porchet HCP, le Cotonnec J-Y, Loumaye E Clinical pharmacology of recombinant human follicle-stimulating hormone. III. Pharmacokinetic-pharmacodynamic modeling after repeated subcutaneous administration. Fertil Steril. 61: Burger HG, Groome NP, Robertson DM Both inhibin A and B respond to exogenous FSH in the follicular phase of the human menstrual cycle. J Clin Endocrinol Metab. 83: McLachlan RI, Robertson DM, Healy DL, de Kretser DM, Burger HG Plasma inhibin levels during gonadotrophin induced ovarian hyperstimulation for IVF: a new index of follicular function? Lancet. 1: Buckler HM, Healy DL, Burger HG Purified FSH stimulates production of inhibin by the human ovary. J Endocrinol. 122: Hughes EG, Robertson DM, Handesman DJ, Hayward S, Healy DL, de Kretser DM Inhibin and estradiol responses to ovarian hyperstimulation: effects of age and predictive values. J Clin Endocrinol Metab. 70: Matson PL, Mpriss ID, Sun JG, Ibrahim ZHZ Lieberman BA Serum inhibin as an index of ovarian function in women undergoing pituitary suppression and ovarian stimulation in an in vitro fertilization program. Horm Res. 35: Hee JP, MacNaughton J, Bangah M, Zissimos M, McCloud PI, Healy DL, Burger HG Follicle-stimulating hormone induced dose-dependent stimulation of immunoreactive inhibin secretion during the follicular phase of the human menstrual cycle. J Clin Endocrinol Metab. 76: Mitchell R, Buckler HM, Matson P, et al Oestradiol and immunoreactive inhibin-like secretory patterns following controlled ovarian hyperstimulation with urinary (Metrodin) or recombinant FSH (Puregon). Hum Reprod. 11: