1 Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, 2 Department of Obstetrics and Gynaecology, Haroldwood Hospital, Essex RM3 0BE and 3 School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford, UK
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Abstract |
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Key words: FSH/inhibin/menopause/estradiol/progesterone
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Introduction |
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The objectives of this study were: (i) to confirm the source of inhibin A and B in cycling women and their disappearance after the removal of the source; and (ii) to study the endocrine feedback relationship between FSH, inhibins, E2 and progesterone at surgical menopause.
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Materials and methods |
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The Barking and Havering Health Authority Research Ethics Committee approved the protocol and written consent was obtained from subjects before enrolling for the study. Serum was separated and stored at 20°C for hormone measurements.
Hormone measurements
Inhibin A
Serum concentrations of dimeric inhibin A were measured in duplicate 50 µl aliquots as described elsewhere (Muttukrishna et al., 1994). The mean intra- and inter-assay coefficients of variation (CV) were 4.3 and 5.1% respectively. Minimum detection limit of the assay for human recombinant inhibin A (National Institute for Biological Standards and Control, Potters Bar, Herts, UK) was 2 pg/ml.
Inhibin B
Serum concentrations of dimeric inhibin B were measured in 50 µl duplicates using an enzyme immunoassay as described elsewhere (Muttukrishna et al., 2000). An in-house standard preparation (partially purified human follicular fluid) was standardized against human recombinant inhibin B (Genentech, San Francisco, CA, USA) and was used as the assay standard. Minimum detection limit of the assay for human recombinant inhibin B was 15 pg/ml. The mean intra- and inter-assay CV were 6.2 and 7.2% respectively.
Gonadotrophins and steroids
Serum concentrations of FSH and LH were measured using Immulite chemiluminescent assay kits (DPC, Glyn Rhonwy, Llanberis, Gwynedd, UK). The detection ranges of FSH and LH were 0.1170 and 0.7400 mIU/ml respectively. Progesterone was measured by an in-house radioimmunoassay (following ether extraction and using tritium-labelled tracers) as previously described (Darne et al., 1989). The minimum detection limit of the progesterone assay was 0.1 nmol/l.
Estradiol was measured by a double antibody 125 I sequential radioimmunoassay (DPC). The minimum detection limit of the assay was 4.8 pmol/l (0.0048 nmol/l). The mean intra- and inter- assay CV were <10% for all four assays.
Statistical analysis
Data were log-transformed to obtain a normal distribution. One-way analysis of variance (ANOVA) was carried out to investigate the significance of the time-dependent changes in the parameters studied. Pearsons correlation analysis was carried out to study the relationship between the hormones analysed, using SPSS statistical package (SPSS Inc., USA).
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Results |
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Follicular phase
Mean inhibin A and inhibin B levels were 11 ± 4 and 173.5 ± 46 pg/ml respectively in the early follicular phase. Inhibin A and inhibin B levels decreased rapidly and were below the limit of detection after 12 h [inhibin A <2 pg/ml (P < 0.05) and inhibin B 15 pg/ml; ANOVA: P < 0.01] of bilateral oophorectomy in women who had surgery in their early follicular phase (day 38; Figure 1a). Mean serum E2 level was 0.171 ± 0.06 nmol/l in the patients. Estradiol levels fell rapidly within the first 6 h of surgery and then levels were stable after 6 h (0.0280.042 nmol/l) until the end of the study period (Figure 1b
). Mean progesterone level was 3.76 ± 0.84 nmol/l in the early follicular phase patients. Serum progesterone levels were not significantly altered for the first 6 h and then levels decreased until the end of the study (0.48 ± 0.1 nmol/l) (P < 0.001; Figure 1b
).
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Relationship between hormones
Follicular phase
Correlation analysis shows that serum inhibin A concentrations were positively correlated with inhibin B (r = 0.48, P < 0.001) and progesterone (r = 0.52; P < 0.001), and negatively correlated with FSH (r = 0.319, P < 0.05). Inhibin B was positively correlated with E2 (r = 0.663, P < 0.001) and progesterone (r = 0.66, P < 0.001) and negatively correlated with FSH (r = 0.427, P < 0.01). Estradiol was positively correlated with progesterone (r = 0.41, P = 0.001) and negatively correlated with FSH (r = 0.53, P < 0.01). Serum progesterone was negatively correlated with FSH (r = 0.626, P < 0.001) and FSH was positively correlated with LH (r = 0.64, P < 0.001).
Luteal phase
Correlation analysis shows that serum inhibin A was positively correlated with E2 (r = 0.585; P < 0.001) and progesterone (r = 0.871, P < 0.001), and negatively correlated with FSH (r = 0.8, P < 0.001) and LH (r = 0.5, P < 0.001). Estradiol was positively correlated with progesterone (r = 0.716, P < 0.001) and negatively correlated with FSH (r = 0.593, P < 0.001). Serum progesterone was negatively correlated with FSH (r = 0.791, P < 0.001) and FSH was positively correlated with LH (r = 0.73, P < 0.001).
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Discussion |
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This is the first study to investigate the dynamic changes in inhibin A and inhibin B after surgical menopause. These data show that inhibin A and B are cleared from circulation within 12 h of bilateral oophorectomy, confirming the ovary as the predominant source for these circulatory proteins in normal cycling women. However, the presence of inhibin/activin subunit proteins in extragonadal tissue has been reported previously (Meunier et al., 1988), suggesting that extragonadal inhibins may act locally without making a significant contribution to the systemic circulation. It is clear that soon after the removal of the ovary, inhibin A and inhibin B rapidly decrease in the circulation. Data show that the time taken to reach half-maximal concentration in the circulation (maximal concentration = the concentration before surgery) after surgical castration for inhibin A, inhibin B and E2 in the follicular phase was 60, 45 and 60 min respectively, whereas in the luteal phase, half-maximal concentrations in the circulation were achieved for inhibin A, E2 and progesterone after 60, 30 and 15 min respectively post castration, suggesting that E2 and progesterone were cleared faster in the luteal phase.
We speculate that after the removal of the ovaries and the fall in inhibins, E2 and progesterone increases the synthesis of FSH followed by increased FSH release. If there was a surge in the release of FSH from a pre-synthesized stored pool, concentrations would have risen with falling concentrations of inhibins and E2 within the first few hours after surgery. However, although the magnitude of rise of FSH in the follicular phase was similar to the rise in the luteal phase, the absolute concentration of FSH was 2-fold higher in women who had surgical castration in their follicular phase, suggesting that the pituitary gland is more sensitive to negative feedback in the follicular phase. Mean LH levels started to rise within the study period but the rise was not significant, confirming a previous study which showed that FSH is more sensitive to negative feedback than LH (Yen and Tsai, 1971).
The acute changes in inhibin concentrations in this study are consistent with our previous observation in first trimester pregnancy termination (Muttukrishna et al., 1997). However, the mechanism(s) by which inhibins are cleared from the circulation is yet to be elucidated. It will be interesting to measure these proteins in urine samples to investigate if they are cleared by the kidney.
We have studied the temporal relationship between FSH rise and fall of inhibins, E2 and progesterone after menopause. The relationship between E2 and FSH/LH (but not inhibins) has been studied by other groups in the past (Kohler et al., 1968; Coble et al., 1969
; Yen and Tsai, 1971
). This is the first study to investigate the relationship between the acute changes in FSH and inhibins in women at surgical menopause.
The correlation analysis shows a significant negative correlation between inhibin A and FSH in both phases of the cycle, whilst inhibin B levels were negatively correlated with FSH in the follicular phase. This indicates that inhibin B is involved in controlling FSH in the follicular phase whereas inhibin A has a negative feedback role on pituitary FSH suppression in both phases of the cycle. Our observation is consistent with previous studies that have suggested a negative feedback role for inhibin B on FSH in the follicular phase (Groome et al., 1996; Klein et al., 1996
). Inhibin B levels were very low in the luteal phase, as expected. Hence, a lack of a demonstrable relationship between FSH and inhibin B in the luteal phase was not surprising. As in the previous studies, falling E2 levels were significantly related to the rising FSH in both phases of the cycle. In the luteal phase, falling progesterone levels were also negatively correlated with the FSH rise, suggesting that FSH is regulated by inhibin A, E2 and progesterone in the luteal phase and by inhibin A and B and E2 in the follicular phase. However, we are aware that correlation analysis should not be used to evaluate a cause-and-effect relationship. A previous study reported that the effect of oral conjugated estrogens (commencing 1 week after surgery) increased the E2 concentrations in circulation but did not suppress FSH to pre-menopausal levels until 2.5 mg (achieving circulating E2 levels above pre-menopausal levels) was administered (Utian et al., 1978
). Our preliminary observations suggest that concentrations of FSH did not fall significantly 1 day after estrogen replacement therapy in our patients. However, these observations are preliminary and future studies have been planned to investigate the changes in FSH with estrogen replacement in the patients who have had surgical menopause and to analyse the relative contribution of inhibins and E2 on controlling pituitary FSH.
In summary, this study confirms the ovary as the predominant source for circulating inhibins in normal cycling women. The rise in FSH after surgical menopause is caused by the fall in inhibins, estradiol and progesterone after ovariectomy. The time lag between the fall of the levels of ovarian inhibitors in the circulation and the rise of serum FSH may be due to the time taken for the up-regulation of FSH synthesis before release.
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Acknowledgements |
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Notes |
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5 Present address: Section of Reproductive and Developmental Medicine, Jessop Wing, Tree root walk, Sheffield
6 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Royal FreeUniversity College London Medical School, 8696 Chenies Mews, London WC1E 6HX, UK. E-mail: s.muttukrishna{at}ucl.ac.uk
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References |
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Burger, H.G., Cahir, N., Robertson, D.M., Groome, N.P., Dudley, E., Green, A. and Dennerstein, L. (1998) Serum inhibin A and B fall differentially as FSH rises in perimenopausal women. Clin. Endocrinol., 48, 808813.
Cobellis, L., Luisi, S., Pezzani, I., Reis, F.M., De Leo, V. and Petraglia, F. (2002) Serum inhibin A, inhibin B and pro alpha C levels are altered after surgically or pharmacologically induced menopause. Fertil. Steril., 77, 745749.[ISI][Medline]
Coble, Y. D. Jr, Kohler, P.O. Cargille, C.M. and Ross, G.T. (1969) Production rate and metabolic clearance rates of human follicle stimulating hormones in pre-menopausal and post menopausal women. J. Clin. Invest., 48, 359363.[ISI][Medline]
Danforth, D.R., Arbogast, L.K., Mroueh, J., Kim, M.H., Kennard, E.A., Seifer, D.B. and Friedman, C.I. (1998) Dimeric inhibin: a direct marker of ovarian aging. Fertil. Steril., 70, 119123.[ISI][Medline]
Darne, F.J., McGarrigle, H.H. and Lachelin, G.C.L. (1989) Diurnal variation of plasma and saliva oestrogen, progesterone, cortisol and plasma dehydroepiandrosterone sulphate in late pregnancy. Eur. J. Obstet. Gynecol. Reprod. Biol., 32, 5766.[ISI][Medline]
Groome, N.P., Illingworth, P.J., OBrien, M., Pai, R., Rodger, F.E., Mather, J. and McNeilly, A.S. (1996) Measurement of dimeric inhibin-B throughout the human menstrual cycle. J. Clin. Endocrinol. Metab., 81, 14011405.[Abstract]
Klein, N.A., Illingworth, P.J., Groome, N.P., McNeilly, A.S., Battaglia, D.E. and Soules, M.R. (1996) Decreased inhibin B secretion is associated with the monotropic FSH rise in older, ovulatory women: a study of serum and follicular fluid levels of dimeric inhibin A and B in spontaneous menstrual cycles. J. Clin. Endocrinol. Metab., 81, 27422745.[Abstract]
Kohler, P.O., Ross, G.T. and Odell, W.D. (1968) Metabolic clearance and production rates of human luteinising hormone in pre and post menopausal women. J. Clin. Invest., 47, 3843.[ISI]
Meunier, H., Rivier, C., Evans, R.M. and Vale, W. (1988) Gonadal and extra gonadal expression of inhibin , ßA, ßB subunits in various tissues predicts diverse functions. Proc. Natl Acad. Sci. USA, 85, 247251.[Abstract]
Muttukrishna, S., Fowler, P.A, Groome, N.P., Mitchell, G.G., Robertson, W.R. and Knight, P.G. (1994) Serum concentrations of dimeric inhibin during the spontaneous human menstrual cycle and after treatment with exogenous gonadotrophin. Hum. Reprod., 9, 16341642.[Abstract]
Muttukrishna S., Child, T.J., Groome, N.P. and Ledger, W.L. (1997) Source of circulating levels of inhibin A, pro alpha C-containing inhibins and activin A in early pregnancy. Hum. Reprod., 12, 10891093.[ISI][Medline]
Muttukrishna, S., Child, T., Lockwood, G.M., Groome, N.P., Barlow, D.H. and Ledger, W.L. (2000) Serum concentrations of dimeric inhibins, activin A, gonadotrophins and ovarian steroids during the menstrual cycle in older women. Hum. Reprod., 15, 549556.
Santoro, N., Tovaghgol, A. and Shunrick, J.H. (1999) Decreased inhibin tone and increased activin A secretion characterize reproductive aging in women. Fertil. Steril., 71, 658661.[ISI][Medline]
Seifer, D.B., Scott, R.T., Bergh, P.A., Abrogast, L.K., Friedman, C.I., Mack, C.K. and Danforth, D.R. (1999) Women with declining ovarian reserve may demonstrate a decrease in day 3 serum inhibin B before a rise in day 3 FSH. Fertil. Steril., 72, 6365.[ISI][Medline]
Utian, W.H., Katz, M., Davey, D.A. and Carr, P.J. (1978) Effect of premenopausal castration and incremental dosages of conjugated equine estrogens on plasma follicle-stimulating hormone, luteinizing hormone and estradiol. Am. J. Obstet. Gynecol., 132, 297302.[ISI][Medline]
Yen, S.S.C. and Tsai, C.C. (1971) The effect of ovariectomy on gonadotrophin release. J. Clin. Invest., 50, 11491153.[ISI][Medline]
Submitted on October 16, 2001; resubmitted on May 28, 2002; accepted on July 12, 2002.