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Both Inhibin A and B Respond to Exogenous Follicle-Stimulating Hormone in the Follicular Phase of the Human Menstrual Cycle¹

Prince Henry’s Institute of Medical Research, Monash Medical Center (H.G.B., D.M.R.), Clayton, Victoria 3168, Australia; and Oxford Brookes University (N.P.G.), Oxford, OX3 O8P United Kingdom

Address all correspondence and requests for reprints to: Dr. Henry G. Burger, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Monash Medical Center, Level 4, Block 4, 246 Clayton Road, Clayton, Victoria 3168, Australia. E-mail: henry.burger{at}med.monash.edu.au

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
To ascertain whether changes in the concentrations of the dimeric inhibins A and/or B (INH-A and INH-B) contributed to the previously described dose-dependent increase in immunoreactive inhibin (INH) in response to FSH during the follicular phase of the human menstrual cycle, both dimers were measured by specific two-site assays in stored serum samples from regularly cycling normal volunteers who had received saline as a control (n = 5) or FSH [100 IU (n = 6) or 200 IU (n = 5)] between days 3–5 of the menstrual cycle. Both INH-A and INH-B showed a dose-dependent increase in response to administered FSH; INH-A rose from 13.5 to 35.9 ng/L (P < 0.01), and INH-B rose from 77.8 to 205 ng/L (P < 0.05) at 36 h after 200 IU FSH. Highly significant correlations were observed between INH and each of the specific inhibin dimers (A: r = 0.79, P < 0.001; B: r = 0.76, P < 0.001), and the responses of the two dimers were also highly correlated (r = 0.59, P < 0.001). The response of each inhibin was also highly correlated with the response of serum estradiol (A: r = 0.45, P < 0.001; B: r = 0.40, P < 0.001). When analyzed by ANOVA, the INH response of INH-B was significantly above the control value at 36 h after treatment with both 100 and 200 IU FSH, whereas the response of INH-A was significant only at 200 IU. It is concluded that the concentrations of both dimeric INH-A and INH-B are stimulated by increases in FSH within the physiological range in the follicular phase of the human menstrual cycle and that both contribute to the previously observed rise in INH.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
MANY of the early observations concerning the physiology of inhibin were based on the relatively nonspecific Monash RIA, which detects both of the dimeric inhibins, A and B (INH-A and INH-B), as well as the inhibin -subunit (1). Among those early studies, it was reported that purified FSH, administered in the early part of the follicular phase of the human menstrual cycle, stimulated immunoreactive inhibin (INH) levels in a dose-dependent fashion; 200 IU FSH led to a 107% increase in INH, consistent with a physiological role for FSH in the regulation of granulosa cell production of INH (2).

The recent availability of specific two-site assays for dimeric INH-A and INH-B prompted us to assess whether the previously reported increase in INH represented an increase in INH-A, INH-B, or a combination of the two.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Details of this study have been reported previously (2). In brief, serum samples were available from 16 normal volunteers, aged 21–37 yr, who had participated as unpaid volunteers. All had regular menstrual cycles (26–30 days), were of normal body mass index, and were not taking oral contraceptives. They were randomly allocated to receive a single im injection of normal saline (150 mmol/L NaCl; n = 5) or 100 IU (n = 6) or 200 IU (n = 5) purified FSH (Metrodin, Serono, Milan, Italy) in the early follicular phase of the menstrual cycle, between days 3–5. All but one individual commenced the study at 1400 ± 1 h. Venous blood samples were collected just before the injection (0 h), then at 9, 18, 26, 36, 45, 54, 66, and 78 h thereafter. Serum was separated and frozen at -20 C until assayed recently for INH-A and INH-B.

Hormone assays

Dimeric INH-A and INH-B were measured by enzyme-linked immunosorbent assays (ELISA) as specified by the authors (3, 4). INH-A was measured in terms of the First International Standard for INH-A (human recombinant, 91/624 30–34K, National Institute of Biological Standards and Control, Potters Bar, UK) in terms of its nominal vial content (5 µg). INH-B standard was provided by Dr. Groome. The sensitivities for INH-A and INH-B ELISAs, as determined by 2 SD above the mean blank value, were 10 and 25 ng/L, respectively. The between-plate within-assay variations for INH-A and INH-B ELISAs were 6.4% and 7.9% (n = 6), and between-assay variations from eight assays were 12% and 19%, respectively. INH-B showed less than 0.1% cross-reaction in the INH-A assay, whereas INH-A showed less than 0.5% cross-reaction in the INH-B assay.

Statistical analysis

The data were log transformed because of the heterogeneity of variances when comparing control and treatment groups. Differences between groups were assessed by ANOVA of logged data, followed by the least significant difference statistic, as described previously (2).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Figure 1 depicts the results obtained in control subjects and in volunteers given 100 and 200 IU FSH. The peak responses of both dimeric inhibins were observed 36 h after FSH injection. There was no significant response of either inhibin to the injection of saline. INH-A rose from 9.9 to 20.6 ng/L after 100 IU FSH (P < 0.05) and from 13.5 to 35.9 ng/L in response to 200 IU (P < 0.01) at 36 h. INH-B rose from 36.7 to 97.2 ng/L in response to 100 IU FSH (P < 0.05) and from 77.8 to 205 ng/L (P < 0.05) in response to 200 IU at 36 h. By ANOVA comparing treatment with control groups at 36 h, INH-A increased 271% compared with the control value at 200 IU FSH (P < 0.01), but the response at 100 IU was not significant. In contrast, INH-B increased 216% at 100 IU FSH (P < 0.05) and 455% at 200 IU FSH (P < 0.01).

View larger version (17K): [in this window] [in a new window]   Figure 1. Levels of INH-A (upper panel) and INH-B (lower panel) in normal subjects given saline (control), 100 IU FSH (solid triangle), or 200 IU FSH (solid circle).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The present data show that exogenously induced increases in circulating FSH levels in the follicular phase of the human menstrual cycle resulted in dose-dependent increases in the circulating concentrations of each of the inhibin dimers, A and B. The previously described increase (2) in INH concentrations in response to FSH is thus contributed to by both INH-A and INH-B.

Recent observations (4) have shown that there is a marked contrast in the patterns of circulating concentrations of INH-A and INH-B in the follicular phase of the normal menstrual cycle. After a decline in INH-A during the luteal-follicular transition, levels of that dimer remain relatively constant until a few days before the midcycle gonadotropin peak, when its levels rise in parallel with increasing concentrations of E₂. The readily demonstrable messenger ribonucleic acid for inhibin - and ß_A-subunits in the granulosa cells of the dominant follicle are consistent with that structure being a major source of INH-A in the circulation at that time (5). In contrast, INH-B peaks during the midfollicular phase of the cycle, having increased progressively from the beginning of the cycle and subsequently falling before a midcycle peak (4). Falling levels late in the follicular phase contrast with the rising levels of INH-A and E₂. The demonstration that small antral follicles are rich in inhibin - and ß_B-subunit messenger ribonucleic acid (5) is consistent with the recruited cohort of antral follicles being the source of circulating INH-B concentrations in the early and midfollicular phases of the cycle. Rigorous proof of these postulates for the sources of INH-A and INH-B during the follicular phase of the cycle is not yet available.

The present study supports the hypothesis that FSH is a physiological regulator of both INH-A and INH-B during the follicular phase of the cycle, even though the origins of those inhibins in the ovary may be different. The data are consistent with the recent observations of Welt et al. (6) that FSH stimulates INH-B in the luteal-follicular transition and of Anderson et al. (7) that serum INH-B levels are elevated after FSH treatment. The data suggest that INH-B may be more sensitive to the stimulus of FSH than is INH-A in this early phase of the menstrual cycle and are also consistent with previous publications that quantitatively INH-B concentrations are substantially higher than those of INH-A during the follicular phase of the cycle (4). The close correlation among INH-A, INH-B, and E₂ confirm earlier data observed with INH and E₂ (2).

In conclusion, the present data indicate that variations in circulating FSH within the physiological range during the follicular phase of the menstrual cycle give rise to dose-dependent increases in the levels of INH-A and INH-B as measured by specific assays. FSH can thus be considered to be an important physiological regulator of each of these inhibin species.

	Acknowledgments

 
The authors thank Nick Cahir and Enid Pruysers for excellent technical assistance.

	Footnotes

 
¹ This work was supported by Program Grant 943208/983212 from the National Health and Medical Research Council of Australia.

Received February 20, 1998.

Revised July 9, 1998.

Accepted July 29, 1998.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

1. Burger HG. 1993 Clinical review–clinical utility of inhibin measurements. J Clin Endocrinol Metab. 76:1391–1396.[Abstract]
2. Hee JP, MacNaughton J, Bangah M, et al. 1993 Follicle-stimulating hormone induces dose-dependent stimulation of immunoreactive inhibin secretion during the follicular phase of the human menstrual cycle. J Clin Endocrinol Metab. 76:1340–1343.[Abstract]
3. Groome NP, Illingworth PJ, O’Brien M, et al. 1994 Detection of dimeric inhibin throughout the human menstrual cycle by two-site enzyme immunoassay. Clin Endocrinol (Oxf). 40:717–723.[Medline]
4. Groome NP, Illingworth PJ, O’Brien M, et al. 1996 Measurement of dimeric inhibin B throughout the human menstrual cycle. J Clin Endocrinol Metab. 81:1401–1405.[Abstract]
5. Roberts VJ, Barth S, Elroeiy A, Yen SSC. 1993 Expression of inhibin/activin subunits and follistatin messenger ribonucleic-acids and proteins in ovarian follicles and the corpus-luteum during the human menstrual cycle. J Clin Endocrinol Metab. 77:1402–1410.[Abstract]
6. Welt CK, Martin KA, Taylor AE, et al. 1997 Frequency modulation of follicle-stimulating hormone (FSH) during the luteal-follicular transition: evidence for FSH control of inhibin B in normal women. J Clin Endocrinol Metab. 82:2645–2652.[Abstract/Free Full Text]
7. Anderson RA, Groome NP, Baird DT. 1997 Basal concentrations of inhibin B are higher in women with PCOS and show an abnormal pattern during monovulation in response to low-dose FSH. J Endocrinol. 153:P234.

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