1 Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU and 2 School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford, UK
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Abstract |
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Key words: activin A/inhibin A/inhibin B/ovary/perimenopause
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Introduction |
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Inhibins are glycoprotein hormones consisting of two dissimilar subunits ( and ß) linked by disulphide bridges and are secreted by the ovarian granulosa and luteal cells during the menstrual cycle. Inhibin A is an
-ßA dimer and inhibin B is an
-ßB dimer. Inhibins are a part of the pituitaryovarian endocrine loop, specifically having an inhibitory effect on pituitary FSH synthesis and secretion (Muttukrishna and Knight, 1990
). Pro alpha C consists of higher molecular weight inhibins containing the pro region of the alpha subunit and the biologically inert monomeric pro alpha subunits. Activins are dimers of ß subunits, linked by disulphide bridges. Activin A is a ßA-ßA dimer, activin AB is a ßA-ßB dimer and activin B is a ßB-ßB dimer. In the pituitaryovarian axis, activin is a physiological antagonist to inhibin and specifically stimulates pituitary FSH synthesis and secretion (Muttukrishna and Knight, 1991
).
A study measuring FSH, LH, oestradiol and immunoreactive (ir) inhibin in women of reproductive age has shown a significant fall in concentrations of ir-inhibin with increasing age in the follicular phase (day 47) and luteal phase (7 days prior to the next anticipated menses) (MacNaughton et al., 1992). In that study, ir-inhibin was measured using the Monash radioimmunoassay (Robertson et al., 1985
; Burger et al., 1995
) which cannot distinguish between biologically active dimeric inhibin A, inhibin B and the inert monomeric alpha subunit that is in abundance in circulation.
The recent development of highly specific and sensitive enzyme immunoassays (EIA) for dimeric inhibin A (Groome et al., 1994; Muttukrishna et al., 1994
), inhibin B (Groome et al., 1996
), pro alpha C-containing inhibins (Groome et al., 1995
) and activin A (Knight et al., 1995) has enabled us to measure the individual dimeric proteins specifically. Using these two-site EIA, cyclical patterns of inhibin A, inhibin B, pro alpha C and activin A have been reported in young women. In young women (age 2532), the cyclical patterns of inhibin A and pro alpha C are similar to that of oestradiol with a pre-ovulatory and a mid-luteal peak (Muttukrishna et al., 1994
; Groome et al., 1995
). Inhibin B concentrations rise in early follicular phase and peak around day 5 of the cycle (Groome et al., 1996
). Activin A concentrations fall during the early follicular phase and rise during the mid-follicular phase and late luteal phase (Muttukrishna et al., 1996
).
A previous study using these new assays for inhibin A and inhibin B to assess the follicular phase (FP) in older women with raised FSH concentrations (Klein et al., 1996) has been reported. In that study, serum inhibin A and inhibin B during the follicular phase in a control group of young women (2025 years) and a group of older women (4045 years) were measured. Serum measurements in the older raised FSH group of women showed a significant fall in inhibin B concentrations and a significant rise in oestradiol concentrations in early follicular phase with no difference in concentrations of inhibin A. The authors concluded that the rise in FSH gonadotrophin alone (monotropic) was due to declining inhibin B. Recent cross-sectional studies have reported concentrations of dimeric inhibins throughout the cycle in reproductive ageing (Santoro et al., 1999
; Welt et al., 1999
). The authors have concluded that inhibin A and inhibin B concentrations are lower in older women with raised FSH. However, there is a group of older women (>40 years) who have normal day 3 FSH concentrations. None of the published studies has analysed the pattern of dimeric inhibins in older women with normal and raised day 3 FSH concentrations.
This longitudinal study was designed to compare the pattern of secretion of dimeric inhibin A, inhibin B, pro alpha C-containing inhibins and `total' activin A throughout the menstrual cycle in regularly cycling older women (4050 years) with normal and raised day 3 serum FSH and normally cycling younger women (2532 years). The relationship between inhibins, activin A, ovarian steroids and gonadotrophins was also investigated during the menstrual cycle.
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Materials and methods |
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Group 1: ON-FSH (older normal FSH group); day 3 FSH <8 mIU/ml (n = 10) (mean age = 43.4 years).
Group 2: R-FSH (raised FSH group); day 3 FSH >8 mIU/ml (n = 6) (mean age = 46 years).
Group 3: YN-FSH (young normal FSH group); day 3 FSH <8 mIU/ml (n = 6) (mean age = 29.6 years).
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 (NIBSC), Potters Bar, 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 in detail elsewhere (Groome et al., 1996). An in-house standard preparation (partially purified human follicular fluid) was standardized against human recombinant inhibin B (Genentech Inc., 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.
Pro alpha C
Serum concentrations of pro alpha C-containing inhibins and free pro alpha subunits were measured in duplicate 50 µl aliquots as previously described (Groome et al., 1995). The minimum detection limit of the assay for pro alpha C standard was 5 pg/ml. The mean intra- and inter-assay CV were 6.8 and 8.6% respectively.
Activin A
Serum concentrations of `total' activin A were measured using an EIA specific for `total' activin A as described in detail elsewhere (Knight et al., 1996). The mean intra- and inter-assay CV were 6.5 and 7.7% respectively. The minimum detection limit of the assay for human recombinant activin A (Genentech) was 50 pg/ml.
Gonadotrophins and steroids
Serum concentrations of FSH, LH, oestradiol and progesterone were measured using Immulite-chemiluminescent assay kits (Diagnostic Products Ltd, Llanberis, Gwynedd, UK). The mean intra- and inter-assay CV were <10% for all four assays. The detection range of FSH, LH, oestradiol and progesterone were 0.1170 mIU/ml, 0.7400 mIU/ml, 0.0737.32 nmol/l and 0.73127 nmol/l respectively.
Statistical analysis
General linear model (GLM) multivariate analysis was carried out to investigate the difference in the pattern of individual hormones in the menstrual cycle between the groups. Unpaired Student's t-tests were carried out to compare the serum concentrations of normal and raised FSH groups of women at a particular time point in the cycle. Correlation analysis was carried out to investigate the relationship between different hormones. SPSS statistical package for social sciences (SPSS Inc., Chicago, IL, USA) was used for all statistical analysis.
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Results |
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Circulatory pattern of inhibins and activin A in the menstrual cycle
Serum inhibin A concentrations in the YN-FSH and ON-FSH group increased steadily during the follicular phase and peaked (42.96 ± 5.8 pg/ml; 39.1 ± 4.6 pg/ml respectively; Table I) prior to the pre-ovulatory LH surge (Figure 1a
). Concentrations fell immediately after ovulation and then rose steadily during the luteal phase forming a peak (54.66 ± 19 pg/ml; 82.63 ± 18.8 pg/ml respectively; Table I
) 46 days after the LH surge. Inhibin A concentrations fell in the late luteal phase before luteolysis along with oestradiol and progesterone. In the R-FSH group, concentrations of inhibin A were significantly lower (P = 0.001) than in the ON-FSH group. Whilst there were no distinct pre-ovulatory (30 ± 6.9 pg/ml) and mid-luteal peaks (27 ± 10 pg/ml) of inhibin A in the R-FSH group the concentrations were higher during this period of the cycle (Figure 1a
).
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Pro alpha C exhibited a similar cyclical pattern to inhibin A. Concentrations rose steadily in the follicular phase forming a peak prior to ovulation and in the mid-luteal phase of the cycle. The cyclical pattern of pro alpha C was similar in all three groups. However, the pre-ovulatory and mid-luteal peaks in the ON-FSH group (343 ± 41.4 and 525 ± 19.7 pg/ml respectively) were observed to be higher than the R-FSH (205 ± 39.5 and 383 ± 79.5 pg/ml respectively) (Figure 2a). Compared to the pro alpha C in the YN-FSH group the ON-FSH group tended to have higher follicular (343 ± 41.4 and 143.15 ± 16.3 pg/ml respectively) and luteal peak concentrations (525 ± 19.7 and 294.4 ± 10.3 pg/ml respectively) (Table I
). However, the apparent difference did not reach statistical significance.
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Concentrations of LH and FSH in the serum from women in the ON-FSH group shared the typical pattern seen in YN-FSH women with an early follicular rise in FSH followed by a pronounced LH surge in mid-cycle. Whilst women in the R-FSH group had a similar LH surge, the pattern of FSH secretion was disordered, with fluctuations in the follicular and early luteal phases of the cycle in the R-FSH women with high early follicular phase concentrations (Figure 3).
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Discussion |
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A reduction in the circulating concentrations of dimeric inhibins is not unexpected in R-FSH women, as it is well established that dimeric inhibins are undetectable in postmenopausal women. Serum dimeric inhibin B is regarded as a measure of ovarian reserve and follicle number as it is mainly secreted by pre-antral follicles (Klein et al., 1996), whereas inhibin A is mainly produced by the dominant follicle. In a previously reported study, the authors had investigated the secretion of inhibin A by granulosa-luteal cells obtained at oocyte retrieval from IVF patients with raised day 3 serum FSH (>8 mIU/ml) and normal day 3 serum FSH (<8 mIU/ml) (Seifer et al., 1996
). It was reported that luteinized granulosa cells cultured in vitro from patients with raised FSH secreted lower concentrations of inhibin A compared to cells obtained from patients with normal FSH. The finding in the current study that the follicular phase peak of inhibin A was significantly lower in the older women with raised FSH is in agreement with that in-vitro study.
Activin A concentrations were similar in all three groups of women during the follicular phase. In the luteal phase, concentrations of activin A in the R-FSH group were elevated compared to the ON-FSH group. As activin A has a stimulatory effect on pituitary FSH production (Muttukrishna and Knight, 1991), activin A could have an endocrine effect on the pituitary that contributes to the early follicular phase increase in FSH secretion of the following cycle.
It has been reported that (ir) inhibin concentrations decrease with increasing age and an inverse relationship was found between FSH and ir-inhibin (MacNaughton et al., 1992). However, the reported studies used the Monash radioimmunoassay (Robertson et al., 1985
; Burger et al., 1995
) that measured both dimeric inhibins and inert monomeric alpha subunit, rendering it non-specific for biologically active dimers. It has recently been reported (Burger et al., 1998
) that inhibin B concentrations fall substantially with no significant change in inhibin A and oestradiol in the early follicular phase in the early peri-menopausal phase of the menopausal transition. These observations were made on a single sample taken from women between days 38. The inhibin A and inhibin B data of the current study are consistent with the above report at that given time point.
The present study showed that the absolute concentrations of inhibin A and inhibin B are similar in YN-FSH (2532 years) (Table I) and in ON-FSH (4050 years) women. This is the first study to compare the concentrations of inhibins in young controls with older women with normal FSH, and suggests that inhibin secretion is maintained at a relatively consistent amount throughout the cycle despite the steady decline in ovarian follicle number that occurs throughout reproductive life (Faddy and Gosden, 1996
).
It has been reported (Klein et al., 1996) that older women with elevated early follicular phase concentrations of FSH (day 3 FSH >8 mIU/ml) have significantly lower concentrations of inhibin B, with no difference in the concentration of inhibin A and oestradiol compared to younger women (2025 years, day 3 FSH <8 mIU/ml). This study was restricted to women with raised FSH concentrations during the early follicular phase. Consistent with the reported study, the present study found that circulating concentrations of dimeric inhibin B were significantly lower in women of 4050 years with raised FSH. However, it has been shown by Welt et al. (Welt et al., 1999
) and in this study that inhibin A concentrations during the FP peak and luteal phase peak were also significantly lower in the R-FSH group of women. The data presented here suggest that a reduction in the number and quality of the follicles in perimenopause results in a decrease in circulating concentrations of both inhibins A and B resulting in the rise in FSH seen at all stages of the cycle in R-FSH women.
The secretory pattern of oestradiol during the cycle remains similar in both the N-FSH and R-FSH groups, indicating that the elevations in FSH seen throughout the cycle in the R-FSH group were not the result of alterations in the secretion of oestradiol by the dominant follicle and the corpus luteum. The aim of this work was to study concentrations of inhibins in ovular women entering menopause, hence ovulatory patterns of oestradiol and progesterone were documented in all cases. However, in the R-FSH group, this ovular pattern was maintained against a background of disordered and generally elevated concentrations of FSH.
Correlation analysis suggested that during the follicular phase, FSH had a negative relationship to inhibin A (r = 0.49, not significant) and inhibin B (r = 0.67, not significant) in the R-FSH group. This confirms previous observations (Santoro et al., 1999) and suggests that both dimeric inhibins (A and B) may influence the rise in early follicular phase FSH. However, consistent with a previous study (Schipper et al., 1998
) there was no relationship between inhibin A and/or inhibin B and FSH in the follicular phase of the YN-FSH and ON-FSH group of women, suggesting that in women with normal FSH, other factor(s) may be mainly responsible for the FSH regulation.
A recent study has investigated the predictive value of measurement of inhibin A and B in IVF cycles (Hall et al., 1999). The data showed that concentrations of day 14 inhibin A and or inhibin B were not better than age and number of oocytes in predicting the IVF outcome, although lower FSH and higher inhibin B were associated with greater chance of pregnancy. However, during ovarian stimulation, higher concentrations of inhibin A and B in serum were associated with successful IVF. The lack of relationship between day 14 concentrations of inhibins and IVF outcome (Hall et al., 1999
) may be because inhibin A concentrations are very low and inhibin B concentrations steadily rise at this time of the cycle. Therefore, measurement of inhibin B around day 57 (the time of early follicular phase peak) would be more precise and useful in predicting IVF outcome.
Activin A concentrations in the late luteal phase of the raised FSH group are marginally higher until luteolysis. This suggests that higher concentrations of activin A in the late luteal phase may have an endocrine effect in increasing pituitary FSH production. However, further detailed studies with more subjects should be carried out during luteo-follicular transition to confirm this endocrine effect of activin A on pituitary FSH production.
Pro alpha C is mainly a luteal product and it appeared to show a positive relationship to progesterone in the ON-FSH (r = 0.5, not significant) and R-FSH (r = 0.65, not significant) groups of women. However, it is interesting to note that pro alpha C concentrations in YN-FSH group are considerably lower (Table I) than in the ON-FSH group, suggesting that pro alpha C concentrations increase with age. The similar pro alpha C-containing inhibin concentrations throughout the cycle in the ON-FSH and R-FSH groups of women suggest that the higher molecular weight inhibin dimers and the monomeric pro alpha C subunit do not have any significant endocrine effect during reproductive ageing. Slightly altered concentrations of oestradiol, progesterone and inhibin A in the luteal phase of the R-FSH group suggests that the corpus luteum function may also be marginally altered in women with raised FSH.
In summary, this study has shown that inhibin B concentrations are significantly altered in the follicular phase and inhibin A concentrations are significantly altered in the follicular and luteal phase of older women during the early stage of perimenopause with no significant alterations in oestradiol. The rise in FSH in the older R-FSH group of women may be explained by the decrease in the negative feedback on the pituitary due to declining inhibin A and B production by the ovaries.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 1, 1999; accepted on November 3, 1999.