1 INSERM U 342, Hôpital Saint-Vincent-de-Paul,82 av Denfert-Rochereau, 75014 Paris and 2 Service d'Endocrinologie, Hôpital Saint-Antoine, Paris, France
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Abstract |
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Key words: FSH/inhibin A/inhibin B/menstrual cycle/oestradiol
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Introduction |
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Inhibins are dimeric proteins secreted by the gonads. Inhibin A and inhibin B share a common subunit while the ß subunit (ß-A or ß-B respectively) is specific for each dimer. It is now well documented that both dimeric inhibins and
subunit precursors account for the so-called immunoreactive inhibin detected in serum samples by means of
-specific immunoassays (Schneyer et al., 1990
; Groome et al., 1995
). Therefore most physiological data established by means of
-specific immunoassays should be reassessed.
During the normal menstrual cycle, inhibin A and inhibin B exhibit strikingly different patterns (Groome et al., 1996). Inhibin A concentrations are very low in the early follicular phase at the time of the FSH follicular rise. They start to rise in the late follicular phase and reach their maximum values in the mid-luteal phase. Conversely inhibin B concentrations begin to rise in the early follicular phase a few days later than FSH concentrations.
We have previously reported (Le Nestour et al., 1993) that the fall in immunoreactive inhibin concentrations as measured by means of an
-specific immunoassay did not seem to be the main factor triggering the intercycle increase in FSH concentrations: transdermal administration of oestradiol mimicking luteal phase concentrations of this hormone delayed FSH rise by 6 days relative to control cycles. On the other hand, the serum pattern of immunoreactive
inhibin was not significantly altered in the treated cycles.
The availability of immunoassays specific for the A or for the B dimeric inhibin has enabled reinvestigation in the same samples of the relationship between gonadotrophins (particularly FSH) and inhibins during the lutealfollicular transition.
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Materials and methods |
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Hormone assays
FSH, LH, oestradiol and progesterone were assessed by radioimmunoassay as previously reported (Le Nestour et al., 1993). In brief FSH and LH were measured by means of immunometric assays (BioMerieux, Marcy-l'Etoile, France). Results were expressed in terms of International Standards 83/575 and 80/552 respectively. Intra-assay coefficients of variation were: FSH: 4.3 and 2.9% at concentrations of 1.5 and 15.3 IU/l respectively, LH: 4.1 and 2.0% at concentrations of 1.5 and 14.9 IU/l respectively. The sensitivity of the assays was 0.2 and 0.1 IU/l for FSH and LH respectively. Oestradiol was measured by radioimmunoassay after ether extraction and chromatography on a Sephadex LH-20 microcolumn (Pharmacia Biotech, St-Quentin en Yvelines, France). The sensitivity of the assay was 28 pmol/l. The intra- and inter-assay coefficients of variation at a concentration of 370 pmol/l were 10 and 12% respectively. Progesterone was measured by radioimmunoassay after ether extraction and chromatography on a Sephadex LH-20 microcolumn. The sensitivity of the assay was 0.16 nmol/l. The intra-assay coefficients of variation at concentrations 0.7 and 35 nmol/l were 8.6 and 5.4% respectively.
Inhibin A and inhibin B were measured by enzyme-linked immunosorbent assays (Serotec, Oxford, UK). Inhibin A assay was a solid phase sandwich assay using a monoclonal antibody raised to the ßA subunit immobilized on microwell plates and a monoclonal antibody specific for the subunit coupled to alkaline phosphatase. This system demonstrates minimal cross-reactivity with the pro-
C subunit and inhibin B. Intra-assay precision in inhibin A assay was 5.4 and 3.2% at concentrations of 14 and 48 pg/ml respectively. The detection limit was 1 pg/ml. Inhibin B assay was a solid phase sandwich assay using a monoclonal antibody raised to ßB subunit immobilized on microwell plates and the same monoclonal anti-
subunit antibody coupled to alkaline phosphatase as in the inhibin A assay. Inhibin A exhibits a 1% cross-reactivity in the inhibin B assay. Intra-assay precision in the inhibin B assay was 7.4 and 4.2% at concentrations of 44 and 225 pg/ml respectively. The detection limit was 6 pg/ml.
Statistics
Comparisons between samples were made by means of analysis of variance for repeated measures. Results of the assays are given as mean ± SEM. Cycle days in luteal and follicular phase were numbered relative to the first day of the succeeding or preceding menses respectively.
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Results |
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Inhibin A
In control cycles, inhibin A concentrations were maximum on day 6 prior to menses: 37.9 ± 7.4 pg/ml, then declined progressively to a minimum on day 2 of the following cycle: 4.9 ± 1.7 pg/ml. Inhibin A concentrations began to rise as early as day 7 of the cycle (Figure 1a).
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On day 10 of the cycle inhibin A concentrations were significantly lower in treated cycles than in controls: 8.5 ± 1.4 versus 23.3 ± 3.4 pg/ml (P < 0.01).
As evidenced by Figure 1a and b, the follicular increase in inhibin A concentrations paralleled that of oestradiol, both in control and in treated cycles, but lagged slightly behind it.
Inhibin B
Inhibin B concentrations were very low (<20 pg/ml) from mid-luteal phase to day 1 of menses both in control and treated cycles. In control cycles, inhibin B concentrations started to rise on day 2 and high values were attained between day 5 and day 11 (Figure 1a). In treated cycles the first significant rise was seen on day 5 and values similar to that of control were reached from day 6 onward (Figure 1b
).
Relationship between gonadotrophins and inhibins
A close relationship was seen between the rise in inhibin B concentrations and that of FSH.
In control cycles FSH concentrations increased significantly as early as day 1 of the cycle, LH concentrations as early as day 3 (data not shown). Inhibin B concentrations started to rise on day 2, i.e. only one day later than FSH concentrations.
In treated cycles, the increase in FSH concentrations was significant on day 5, the increase in inhibin B concentrations was significant on day 6 (P < 0.02 and P < 0.01 respectively). It is interesting to note that the more acute rise in FSH seen in treated cycles was followed by a more dramatic rise in inhibin B.
In both control and treated cycles, FSH concentrations plateaued as soon as inhibin B concentrations reached 100 pg/ml, while oestradiol concentrations were still below 200 pmol/l. However the decrease in FSH concentrations did not seem to occur before oestradiol rose.
The increase in inhibin A concentrations occurred both in control and in treated cycles 67 days later than that of FSH or LH. This increase was very progressive and delayed relative to that of oestradiol as shown in Figure 1.
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Discussion |
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The decrease in inhibin A concentrations (like the decrease in progesterone concentrations) at the time of luteolysis has no effect on FSH secretion in the treated cycle. This confirms our previous assumption (Le Nestour et al., 1993) that the fall in oestradiol concentrations is the main trigger of FSH resumption in the lutealfollicular transition. On the other hand, administration of recombinant human inhibin A in female rhesus monkeys for the first 5 days of the cycle showed that supraphysiological concentrations of inhibin A suppressed in part FSH concentrations (but not LH) and delayed ovulation (Molskness et al., 1996
). That is consistent with a putative role of inhibin A in the luteal phase as a modulator of gonadotrophin pulse frequency as shown for progesterone (Soules et al., 1984
; Hall et al., 1992
).
The maintenance for the first days of the cycle of high oestradiol concentrations identical to those observed in luteal phase also altered the kinetics of inhibin A secretion in the mid-follicular phase. In control cycles, inhibin A concentrations began to rise on day 7 of the cycle and values comparable to luteal phase concentrations were reached on day 11. In contrast inhibin A concentrations in treated cycles increased progressively from day 7 onward, but the slope of the increase was very low and the mean concentration on day 11 was far lower than that in the luteal phase. Therefore, the dependency of inhibin A on gonadotrophins is not as obvious as that of inhibin B. It is clear from the figures that the increase in inhibin A concentrations paralleled (although with a 2 day delay) that in oestradiol concentrations. Therefore the increase in inhibin A concentrations seems to give evidence of follicular growth. This assumption is consistent with the data reported by Muttukrishna et al. (1994). Daily administration of human menopausal gonadotrophins (HMG) in women undergoing in-vitro fertilization for tubal infertility increased inhibin A concentrations by 10 to 20-fold in 6 days. Although FSH concentrations increased within one day of commencing treatment with HMG, inhibin A concentrations increased progressively according to kinetics very similar to that of oestradiol during follicular development. It should be noted that the urinary gonadotrophin preparation used in this experiment has both intrinsic FSH and LH bioactivity. On the other hand, administration of human chorionic gonadotrophin (HCG) in the late luteal phase increased significantly inhibin A concentrations but not inhibin B concentrations (Illingworth et al., 1996). However such an experiment does not demonstrate a direct stimulatory effect of HCG (or LH) on inhibin A secretion because it only induced a rescue of the corpus luteum.
In this study, the increase in inhibin B concentrations occurred only 1 day later than that in FSH concentrations both in control and treated cycles. A similar pattern was shown by Schipper et al. (1998) in spontaneous cycles. This is a closer relationship than that found by Groome et al. (1996) who reported that the rise in inhibin B concentrations occurred not earlier than the FSH follicular peak, i.e. approximately on day 6 of the cycle.
The positive effect of FSH on inhibin B secretion as evidenced by the pharmacological manipulation of the early follicular phase is in keeping with previous observations in women and in children. In hypogonadal women treated with pulsatile gonadotrophin-releasing hormone (GnRH), the increase in inhibin B is clearly dependent on the GnRH pulse frequency: slow pulse frequency is associated with slower rise in FSH secretion and lower inhibin B concentrations (Welt et al., 1997). Similarly on day 5 of normal cycles, a close temporal relationship has been reported between changes in concentrations of inhibin B and FSH, inhibin B variations occurring 50 min after FSH variations (Lockwood et al., 1996
). In girls undergoing pubertal development, the increases in inhibin B and FSH concentrations are strongly correlated (Crofton et al., 1997
).
Although the experiment was initially designed with the aim to study the interrelationship of FSH and inhibins in the lutealfollicular transition, it is obvious from Figure 1 that mean FSH concentrations plateaued when inhibin B concentrations were at maximum and before a significant rise in oestradiol concentrations occurred. This phenomenon was particularly evident in treated cycles, where mean FSH concentrations began to decrease on day 8 of the cycle whilst inhibin B concentrations remained high and not significantly changed from days 711 (end of the study). This gives further evidence of the inhibitory role of inhibin B on FSH secretion in the follicular phase, as previously suggested (Groome et al., 1996
). The data presented here are consistent with those of Schipper et al. (1988) who showed that the cycle day of oestradiol rise was not correlated with the maximum FSH concentration, while the day of maximum inhibin B was correlated significantly with the day of maximum FSH concentrations. However the fall in FSH concentrations was concomitant with the exponential increase in oestradiol concentrations.
The inhibitory role of inhibin B upon FSH secretion is also consistent with the opposite observation made in older, ovulatory women (Klein et al., 1996). In these women, FSH concentrations increased significantly despite higher oestradiol concentrations than in younger women. Since inhibin B concentrations were significantly lower in older women, it seems that the decrease in inhibin B is a feedback signal able to overcome the effect of increased oestradiol concentrations.
In conclusion, these findings show that inhibin A is not responsible for the inhibition of FSH secretion during the luteal phase. They do not support a tight dependency of inhibin A on FSH secretion, but confirm that the follicular increase in inhibin A concentrations could be an index of follicular growth. On the other hand, inhibin B is strongly dependent on FSH secretion and in turn it seems to be a regulator of FSH secretion. That suggests that inhibin B is the secretory product of granulosa cells under the control of FSH and that it may also play a role as a negative control of FSH secretion in the mid-follicular phase, while oestradiol seems to exert the main negative control in the late follicular phase and in the luteal phase.
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Acknowledgments |
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Notes |
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References |
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Groome, N.P., Illingworth, P.J., O'Brien, M. et al. (1995) Quantification of inhibin pro-C-containing forms in human serum by a new ultrasensitive two-site enzyme-linked immunosorbent assay. J. Clin. Endocrinol. Metab., 80, 29262932.[Abstract]
Groome, N.P., Illingworth, P.J., O'Brien, M. et al. (1996) Measurement of dimeric inhibin B throughout the human menstrual cycle. J. Clin. Endocrinol. Metab., 81, 14011405.[Abstract]
Hall, J.E., Shoenfeld, D.A., Martin, K.A. and Crowley, W.F. Jr (1992) Hypothalamic gonadotrophin-releasing hormone secretion and follicle-stimulating hormone dynamics during the lutealfollicular transition. J. Clin. Endocrinol. Metab., 74, 600607.[Abstract]
Illingworth, P.J., Groome, N.P., Duncan, W.C. et al. (1996) Measurement of circulating inhibin forms during the establishment of pregnancy. J. Clin. Endocrinol. Metab., 81, 14711475.[Abstract]
Klein, N.A., Illingworth, P.J., Groome, N.P. et al. (1996) Decreased inhibin B secretion is associated with the monotropic FSH rise in older, ovulatory women: a study of serum and follicular fluid concentrations of dimeric inhibin A and B in spontaneous menstrual cycle. J. Clin. Endocrinol. Metab., 81, 27422745.[Abstract]
Le Nestour, E., Marraoui, J., Lahlou, N. et al. (1993) Role of oestradiol in the rise of follicle-stimulating hormone concentrations during the lutealfollicular transition. J. Clin. Endocrinol. Metab., 77, 439442.[Abstract]
Lockwood, G.M., Muttukrishna, S., Groome, N.P. et al. (1996) Mid-follicular phase pulses of inhibin B may provide a mechanism regulating emergence of the dominant follicle. Simpson's Symposium, Edinburgh, August 1996 (abstract).
Molskness, T.A., Woodruff, T.K., Hess, D.L. et al. (1996) Recombinant human inhibin-A administered early in the menstrual cycle alters concurrent pituitary and follicular, plus subsequent luteal, function in rhesus monkeys. J. Clin. Endocrinol. Metab., 81, 40024006.[Abstract]
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Schipper, I., de Jong, F.H. and Fauser, B.C.J.M. (1998) Lack of correlation between early follicular phase serum follicle stimulating hormone concentrations and menstrual cycle characteristics in women under the age of 35 years. Hum. Reprod., 13, 14421448.[Abstract]
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Soules, M.R., Steiner, R.A., Clifton, D.K. et al. (1984) Progesterone modulation of pulsatile luteinizing hormone in women. J. Clin. Endocrinol. Metab., 58, 378383.[Abstract]
Welt, C.K., Martin, K.A., Taylor, A.E. et al. (1997) Frequency modulation of follicle-stimulating hormone (FSH) during the lutealfollicular transition: evidence for FSH control of inhibin B in normal women. J. Clin. Endocrinol. Metab., 82, 26452652.
Submitted on October 13, 1998; accepted on February 4, 1999.