Main inhibitor of follicle stimulating hormone in the luteal–follicular transition: inhibin A, oestradiol, or inhibin B?

N. Lahlou1,3, N. Chabbert-Buffet2, S. Christin-Maitre2, E. Le Nestour2, M. Roger1 and P. Bouchard2

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The roles of oestradiol, inhibin A and inhibin B in the luteal–follicular transition were assessed by means of specific assays. Six premenopausal women were studied during a control and then a cycle treated with percutaneous oestradiol 0.1 mg/day from day 10 after the luteinizing hormone (LH) surge until day 4 of the following cycle. Inhibin A concentrations decreased similarly in control and treated cycles from day –5 to day 2, then increased in control cycle to 23.3 ± 3.4 pg/ml on day 10 (mean ± SEM). They remained low until day 5 in treated cycles and were lower than controls on day 10 (P < 0.01). Follicle stimulating hormone (FSH) concentrations increased on day 1 in controls and on day 5 in treated cycles when oestradiol concentration fell abruptly. Inhibin B concentrations remained low until day 1 in controls and day 4 in treated cycles. In both, inhibin B concentrations increased 1 day after FSH, peaking at 160 pg/ml. FSH concentrations began to plateau when inhibin B concentrations were >100 pg/ml and oestradiol concentrations below 200 pmol/l. These data suggest that inhibin A is not responsible for FSH suppression in the luteal phase and that the negative control of FSH shifts from oestradiol in the luteal phase to inhibin B in the mid-follicular phase.

Key words: FSH/inhibin A/inhibin B/menstrual cycle/oestradiol


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
At the end of each ovulatory cycle, the corpus luteum undergoes regression through apoptosis (programmed cell death). At this time the endocrine secretion of the corpus luteum drops in a dramatic manner. These hormonal changes are responsible for menstruation and intercycle rise in follicle stimulating hormone (FSH) concentrations. This rise in FSH concentrations allows the terminal growth of follicles. The respective role of the different ovarian signals, progesterone, oestradiol and inhibin A, in the rise in FSH concentrations remains unclear.

Inhibins are dimeric proteins secreted by the gonads. Inhibin A and inhibin B share a common {alpha} 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 {alpha} subunit precursors account for the so-called immunoreactive inhibin detected in serum samples by means of {alpha}-specific immunoassays (Schneyer et al., 1990Go; Groome et al., 1995Go). Therefore most physiological data established by means of {alpha}-specific immunoassays should be reassessed.

During the normal menstrual cycle, inhibin A and inhibin B exhibit strikingly different patterns (Groome et al., 1996Go). 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., 1993Go) that the fall in immunoreactive inhibin concentrations as measured by means of an {alpha}-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 {alpha} 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 luteal–follicular transition.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects and protocol
Six women (age 28–38 years) were enrolled after informed consent. All had ovulatory cycles and all had been pregnant. None was taking medication or had a history of endocrine disease. The cycle length was 25–35 days. Body weight was normal for height. Each volunteer was studied during one untreated (control) cycle and one oestradiol-treated cycle. The day of the luteinizing hormone (LH) surge was monitored by daily blood sampling beginning on the 7th day of the cycle. Daily blood sampling was continued until day 11 of the succeeding cycle. In treated cycles, oestradiol was administered by the percutaneous route (Estraderm TTS®; Ciba-Geigy, Rueil, France) at 0.1 mg/24 h from day 10 after the LH surge until day 4 of the succeeding cycle.

Hormone assays
FSH, LH, oestradiol and progesterone were assessed by radioimmunoassay as previously reported (Le Nestour et al., 1993Go). 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 {alpha} subunit coupled to alkaline phosphatase. This system demonstrates minimal cross-reactivity with the pro-{alpha} 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-{alpha} 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As previously reported (Le Nestour et al., 1993Go), oestradiol concentrations fell in the control group to a nadir of 100–130 pmol/l in the early days of the following follicular phase, whilst concentrations similar to those of a normal luteal phase were maintained until day 4 in treated cycles (mean concentrations 338 ± 38 to 382 ± 65 pmol/l during treatment). In treated cycles, mean oestradiol concentration fell abruptly from 338 ± 38 to 103 ± 29 pmol/l after completion of oestrogen administration. By contrast similar progesterone patterns were seen in control and in treated cycles (not shown).

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 1aGo).



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Figure 1. (a). Mean changes (±SEM) in inhibin A and oestradiol concentrations (top) and in inhibin B and follicle stimulating hormone (FSH) concentrations (bottom) in control cycles. (b) Mean changes (±SEM) in inhibin A and oestradiol concentrations (top) and in inhibin B and FSH concentrations (bottom) in treated cycles. Oestradiol treatment = 0.1 mg of percutaneous oestradiol per day.

 
In treated cycles, inhibin A concentrations did not differ significantly from control cycles from mid-luteal phase to day 2 of cycle. By contrast the follicular phase pattern differed markedly from that of controls: concentrations continued to decrease and remained very low until day 5 (2.1 ± 0.6 pg/ml) and the late follicular phase rise was delayed (Figure 1bGo).

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 bGo, 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 1aGo). 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 1bGo).

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 6–7 days later than that of FSH or LH. This increase was very progressive and delayed relative to that of oestradiol as shown in Figure 1Go.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study demonstrates that inhibin A concentration in luteal phase does not exert the main negative control of FSH secretion in normal women. It also shows that the rise in inhibin B concentrations in the early follicular phase is closely dependent on FSH secretion. It clearly dissociates the role of inhibin B and that of oestradiol as physiological regulators of FSH secretion.

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., 1993Go) that the fall in oestradiol concentrations is the main trigger of FSH resumption in the luteal–follicular 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., 1996Go). 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., 1984Go; Hall et al., 1992Go).

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., 1996Go). 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., 1997Go). 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., 1996Go). In girls undergoing pubertal development, the increases in inhibin B and FSH concentrations are strongly correlated (Crofton et al., 1997Go).

Although the experiment was initially designed with the aim to study the interrelationship of FSH and inhibins in the luteal–follicular transition, it is obvious from Figure 1Go 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 7–11 (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., 1996Go). 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., 1996Go). 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.


    Acknowledgments
 
This study was supported in part by a grant from Assistance Publique-Hôpitaux de Paris (Contrat de Recherche Clinique number 94274) and by Institut de Recherche Endocrinienne et Metabolique (IREM), Paris. The authors are indebted to Dr A.F.Parlow and The National Hormone and Pituitary Program (Torrance, CA, USA) and to The National Institute for Biological Standards and Control (Potters Bar, UK) for the generous gift of reference preparations for gonadotrophin immunoassays. They also wish to thank Dr N.Groome for stimulating discussions. The skilful technical assistance of Mrs Josiane Le Fourn and Mrs Nathalie Robert is gratefully acknowledged.


    Notes
 
3 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Crofton, P.M., Illingworth, P.J., Groome, N.P. et al. (1997) Changes in dimeric inhibin A and B during normal early puberty in boys and girls. Clin. Endocrinol., 46, 109–114.[ISI][Medline]

Groome, N.P., Illingworth, P.J., O'Brien, M. et al. (1995) Quantification of inhibin pro-{alpha}C-containing forms in human serum by a new ultrasensitive two-site enzyme-linked immunosorbent assay. J. Clin. Endocrinol. Metab., 80, 2926–2932.[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, 1401–1405.[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 luteal–follicular transition. J. Clin. Endocrinol. Metab., 74, 600–607.[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, 1471–1475.[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, 2742–2745.[Abstract]

Le Nestour, E., Marraoui, J., Lahlou, N. et al. (1993) Role of oestradiol in the rise of follicle-stimulating hormone concentrations during the luteal–follicular transition. J. Clin. Endocrinol. Metab., 77, 439–442.[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, 4002–4006.[Abstract]

Muttukrishna, S., Fowler, P.A., Groome, N.P. et al. (1994) Serum concentrations of dimeric inhibin during spontaneous human menstrual cycle and after treatment with exogenous gonadotrophin. Hum. Reprod., 9, 1634–1642.[Abstract]

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, 1442–1448.[Abstract]

Schneyer, A.L., Mason, A.J., Burton, L.E. et al. (1990) Immunoreactive inhibin {alpha}-subunit in human serum: implications for radioimmunoassay. J. Clin. Endocrinol. Metab., 70, 1208–1212.[Abstract]

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, 378–383.[Abstract]

Welt, C.K., Martin, K.A., Taylor, A.E. 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]

Submitted on October 13, 1998; accepted on February 4, 1999.