Research Institute for Endocrinology, Reproduction and Metabolism, Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynaecology,Vrije Universiteit Medical Centre, PO Box 7057, 1007 MB Amsterdam, The Netherlands
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: gonadotrophin pulsatility/imminent ovarian failure/inhibin/reproductive ageing
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is generally assumed that early follicular phase FSH, at the time of recruitment of follicles, is elevated due to diminished ovarian feedback of steroids and inhibins (Sherman and Korenman 1975; Lee et al., 1988
; MacNaughton et al., 1992
). Inhibins are dimeric proteins that selectively inhibit FSH secretion. Inhibin A is composed of a common
subunit and a ßA subunit, and inhibin B consists of an
subunit combined with a ßB subunit. In recent years, studies on the differential secretion of inhibin A and inhibin B in the menstrual cycle (Groome et al., 1996
) and in-situ hybridization studies (Roberts et al., 1993
) have shown that inhibin A appears to be primarily secreted by the mature follicle and corpus luteum. Inhibin B appears to be secreted by smaller pre-ovulatory follicles. Decreased concentrations of both inhibin A and inhibin B can theoretically contribute to high FSH concentrations in the early follicular phase.
It is not fully clear, however, what compounds, attributing to the rate of pituitary FSH secretion, are altered in patients with IOF. Hypothalamic causes for elevated FSH concentrations could induce an altered release mode (more pulses) of pulsatile gonadotrophin-releasing hormone (GnRH) as in mothers of dizygotic twins (Lambalk et al., 1998). At the level of the pituitary a higher sensitivity to the GnRH pulses might explain the elevation of FSH. FSH and LH are released in a pulsatile manner. Studying episodic gonadotrophin release enables observation of dynamic changes in the FSH release of patients with IOF. Since pulsatile LH is considered to be a good representation of the episodic activity of the hypothalamic GnRH pulse generator, a detailed analysis of its episodic gonadotrophin secretion in combination with information on the LH and FSH response to a GnRH challenge, will potentially reveal responsible mechanisms.
The aim of this study was to evaluate the hypothalamic and pituitary contribution to the elevated FSH concentrations in women with IOF. Therefore, the pulsatile release of FSH and LH on day 3 of the menstrual cycle was examined and the subsequent pituitary response to GnRH in patients with IOF and controls was evaluated. The role of the ovary as a potential cause of the differences in gonadotrophin secretion was also evaluated by concomitant measurement of the concentrations of oestradiol, inhibin A and inhibin B.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Study design
Serial blood samples were collected on day 3 of a later menstrual cycle (study cycle). An indwelling catheter was placed in a forearm vein for 6 h. Blood was drawn into heparinized tubes every 10 min. Sampling started between 08:00 and 09:00. Immediately after the last sample a GnRH challenge with an i.v. injection of 100 µg GnRH (HRF; Wyeth, Hoofddorp, The Netherlands) was given and three additional blood samples were taken after 30, 60 and 90 min. The basal body temperature (BBT) of all subjects was measured during the study cycle.
Hormone measurements
LH and FSH were measured in duplicate by commercially available immunometric assays (Amerlite; Amersham, Bucks, UK). The lower limit of detection was 0.3 IU/l for LH and 0.5 IU/l for FSH. The assays were calibrated against the 1st International Reference Preparation (IRP) 68/40 and 2nd IRP 78/549 for LH and FSH respectively. Of each individual, all samples were analysed in the same run for each hormone. The inter- and intra-assay coefficients of variation (CV) were <9 and 5% for LH and FSH.
Inhibin A and inhibin B were measured in duplicate by ultra-sensitive two-site enzyme immunoassays (Serotec, Oxford, UK). The development of these commercially available assays was based on the work of Groome et al. (Groome et al., 1994, 1996
). The lower limit of detection was 3 pg/ml for inhibin A and 15 pg/ml for inhibin B. The inter-assay CV was <9% for both inhibin A and inhibin B. Oestradiol was measured by radioimmunoassay (Sorin Biomedical, Sallugia, Italy) with a lower limit of detection of 18 pmol/l and an inter-assay CV of <11%. For data analysis, values below the lower limits of detection were assigned the value of assay sensitivity.
Pulse analysis
Pulse analysis was carried out with a computerized version of a previously developed and validated method (Lambalk et al., 1985; Scheele et al., 1987
). The algorithm is valid for replicate repeated measurements of LH and FSH with a chance of <5% to indicate non-existing pulses as a pulse in series of 100 samples taken from pooled serum. This method is particularly of value in detecting episodic secretion of hormones with relatively long half-lives because pulses are indicated when a significant rise occurs without the requirement of a subsequent decline. Nadirs preceding the pulses are indicated as marker points in the hormone patterns rather than the pulses themselves.
Statistical analysis
The patients had to have elevated day 3 FSH in the screening as well as in the study cycle. The controls had normal FSH concentrations in both cycles. For each subject, the mean concentrations of LH and FSH, the mean pulse amplitude over the 6 h period and the frequency of LH and FSH pulses per 6 h were calculated. The maximal gonadotrophin increment was taken as a parameter for the response to the GnRH challenge. The non-parametric MannWhitney U-test was used for differences between groups. P < 0.05 was considered to be statistically significant. Spearman rank correlations were calculated for overall relation between mean FSH concentrations and inhibin A and inhibin B.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
These findings contrast with a previous study of the episodic FSH secretion in mothers of hereditary dizygotic twins. In those we found elevated FSH concentrations in association with an increased number of FSH pulses, without changes in the response to GnRH and without alterations in LH and feedback (Lambalk et al., 1998). This indicates that in twin mothers the origin of elevated early follicular phase FSH is pituitary or supra-pituitary, whereas elevated FSH in imminent ovarian failure is of ovarian origin. These contrasting findings, in seemingly identical conditions, i.e. elevated early follicular phase FSH, underscore the importance of investigating the dynamics of the episodically secreted gonadotrophins.
Only a few others have studied the dynamics of gonadotrophin secretion in relation to the luteal follicular transition (Hall et al., 1992) and in reproductive ageing (Wilshire et al., 1995
; Klein et al., 1996b
; Reame et al., 1996
). The increase in LH pulse frequency in this stage of the menstrual cycle, due to increased GnRH pulsatility, shows the importance of GnRH action in the increase of FSH in the early follicular phase (Hall et al., 1992
).
The age-related increase in FSH concentration is associated with enhancement of pulsatile LH secretion, particularly in the LH pulse amplitudes as previously reported (Reame et al., 1996). This is in full agreement with the current observations and greater pituitary responsiveness to GnRH is probably responsible for this. In a smaller pulsatility study (Klein et al., 1996b
), no differences were detected in the endocrinology of the early follicular phase in older and younger cycling women. Moreover, other authors (Wilshire et al., 1995
) reported no differences in LH pulse amplitudes and number of pulses during the early follicular phase in younger versus older women. In a frequent sampling study of LH in the late follicular phase (Matt et al., 1998
), a lower number of LH pulses was found in older women, which may indicate a slowing of the GnRH pulse generator in that phase. This is in line with our earlier observations in younger versus older post-menopausal women (Lambalk et al., 1997
). Whether a slowing of the GnRH pulse generator demonstrated in the late follicular phase is responsible for a paradoxical increased concentration of FSH in the early follicular phase is highly questionable (Lambalk et al., 1989
).
The GnRH challenge tests have been used by a few other authors to study gonadotrophin secretion dynamics in reproductive ageing. In contrast to our findings, one study (Fujimoto et al., 1996) described a lower gonadotrophin responses to GnRH in older women. This apparent discrepancy may be explained by the fact that Fujimoto used age as a primary variable, while we compared the results of IOF patients with a control group. Muasher et al. found that the FSH/LH response to GnRH was correlated with results in IVF (Muasher et al., 1988
). In this study, identical to our results, higher gonadotrophin responses in patients with elevated FSH were observed. Finally, an increased response of both LH and FSH to GnRH was described in a study on perimenopausal women but with irregular cycles of 1090 days (Schmidt et al., 1996
), while no differences in response to GnRH were observed in older subjects with a regular cycle.
Ovarian feedback seems to play a role in alterations of early follicular phase FSH secretion. There were no differences in oestradiol concentration between the groups in our study. This is in agreement with previous observations (Sherman and Korenman, 1975; Lee et al., 1988
; Buckler et al., 1991
). In the current study, the elevated cycle day 3 FSH concentrations were found to be associated with lower concentrations of inhibin A and inhibin B. A number of studies have indicated an inverse relationship between inhibin B and calender age (Klein et al., 1996a
) and a relationship between low inhibin B and poor outcome in assisted reproduction (Seifer et al., 1997
). One study (Reame et al., 1998
), also showed older cycling women to have lower follicular phase inhibin B. Recently, inhibin B was reported to be lower in the early follicular phase of older women (Welt et al., 1999
), together with lower inhibin A on the day after the LH peak. In that study FSH concentrations were slightly higher in the early follicular phase of older (>35 years) women. It is believed that lower inhibin B concentrations signify a decline in size of the available cohort of follicles, and increased early follicular phase FSH concentrations probably represent the same. By focusing on differentiation between high and normal FSH concentrations the results presented here clearly indicate a role of deficient inhibin A. Inhibin A is predominantly secreted in the luteal phase (Groome et al., 1996
). Therefore, the early follicular phase rise of FSH may, at least in part, result from some luteal phase deficiency of the previous cycle (Danforth et al., 1998
). These authors observed a good inverse relationship between luteal inhibin A concentrations and day 3 FSH values. In addition, it has been shown (Seifer et al., 1996
) that cultured luteinized granulosa cells of women with high FSH concentrations produce less inhibin A.
Activins were not measured in this study. Inconsistent data are available on the role of activins in pituitary stimulation. Some authors (Ying et al., 1988) report that activins are capable of direct pituitary FSH stimulation, whereas others (Katayama and Conn, 1994
) question this. Nevertheless, slightly higher activin A concentrations are found in older pre-menopausal women (Reame et al., 1998
). These data were confirmed by another study (Santoro et al., 1999
). So far, the role of activin A as a classical hormone involved in gonadal function has remained unclear (Harada et al., 1996
).
Based on the current understanding of inhibin physiology (Hayes et al., 1998), it is not possible to explain fully the exaggerated GnRH-induced gonadotrophin response in patients with IOF. Inhibins selectively inhibit FSH secretion, so the decreased inhibin A and inhibin B may account for the increased FSH response, but not for the higher LH response. The concentrations of oestradiol and progesterone (data not shown) were not lower in IOF patients. Therefore, a decline in the activity of other ovarian regulators might be involved in the loss of negative feedback across the cycle. We speculate that a possible mediator of reproductive ageing is loss of gonadotrophin surge inhibiting factor (GnSIF) activity. This ovarian peptide keeps the pituitary in a low state of responsiveness to GnRH (de Koning, 1995
; Fowler et al., 1995
; Balen, 1996
). Changes in GnSIF activity are thought to play a role in the generation of the midcycle LH surge. So far, there is no reliable assay available to measure GnSIF serum concentrations. GnSIF bioactivity is present in human follicles (Fowler et al., 1995
) and concentrations vary across the follicular phase (Fowler and Templeton, 1996
).
In conclusion, elevated day 3 FSH concentrations in women with IOF result from a pituitary more sensitive to GnRH, leading to higher FSH and LH pulse amplitudes and an increased response to GnRH. Decreased feedback by ovarian inhibin may play a central role.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Buckler, H.M., Evans, C.A., Mamtora, H. et al. (1991) Gonadotropin, steroid, and inhibin levels in women with incipient ovarian failure during anovulatory and ovulatory rebound cycles. J. Clin. Endocrinol. Metab., 72, 116124.[Abstract]
Cameron, I.T., O'Shea, F.C., Rolland, J.M. et al. (1988) Occult ovarian failure: a syndrome of infertility, regular menses, and elevated follicle-stimulating hormone concentrations. J. Clin. Endocrinol. Metab., 67, 11901194.[Abstract]
Danforth, D.R., Arbogast, L.K., Mroueh, J. et al. (1998) Dimeric inhibin: a direct marker of ovarian aging. Fertil. Steril., 70, 119123.[ISI][Medline]
de Koning, J. (1995) Gonadotrophin surge-inhibiting/attenuating factor governs luteinizing hormone secretion during the ovarian cycle: physiology and pathology. Hum. Reprod., 10, 28542861.[Abstract]
Fowler, P.A., Fahy, U., Culler, M.D. et al. (1995) Gonadotrophin surge-attenuating factor bioactivity is present in follicular fluid from naturally cycling women. Hum. Reprod., 10, 6874.[Abstract]
Fowler, P.A. and Templeton, A. (1996) The nature and function of putative gonadotropin surge-attenuating/inhibiting factor (GnSAF/IF). Endocr. Rev., 17, 103120.[ISI][Medline]
Fujimoto, V.Y., Klein, N.A., Battaglia, D.E. et al. (1996) The anterior pituitary response to a gonadotropin-releasing hormone challenge test in normal older reproductive-age women. Fertil. Steril., 65, 539544.[ISI][Medline]
Groome, N.P., Illingworth, P.J., O'Brien, M. et al. (1994) Detection of dimeric inhibin throughout the human menstrual cycle by two-site enzyme immunoassay. Clin. Endocrinol., 40, 717723.[ISI][Medline]
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., Schoenfeld, D.A., Martin, K.A. and Crowley, W.F. (1992) Hypothalamic gonadotropin-releasing hormone secretion and follicle-stimulating hormone dynamics during the luteal-follicular transition. J. Clin. Endocrinol. Metab., 74, 600607.[Abstract]
Hayes, F.J., Hall, J.E., Boepple, P.A. et al. (1998) Clinical review 96: Differential control of gonadotropin secretion in the human: endocrine role of inhibin. J. Clin. Endocrinol. Metab., 83, 18351841.
Harada, K., Shintani, Y., Sakamoto, Y. et al. (1996) Serum immunoreactive activin A levels in normal subjects and patients with various diseases. J. Clin. Endocrinol. Metab., 81, 21252130.[Abstract]
Jones, G.S., Muasher, S.J., Rosenwaks, Z. et al. (1986) The perimenopausal patient in in vitro fertilization: the use of gonadotropin-releasing hormone. Fertil. Steril., 46, 885891.[ISI][Medline]
Katayama, T. and Conn, P.M. (1994) Activin modulates the intracellular signaling system activated by gonadotropin-releasing hormone: dual effect on calcium messenger system and protein kinase-C pathway. Endocrinology, 134, 119125.[Abstract]
Klein, N.A., Illingworth, P.J., Groome, N.P. et al. (1996a) 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]
Klein, N.A., Battaglia, D.E., Clifton, D.K. et al. (1996b) The gonadotropin secretion pattern in normal women of advanced reproductive age in relation to the monotropic FSH rise. J. Soc. Gynecol. Invest., 3, 2732.[ISI][Medline]
Lambalk, C.B., de Koning, J., Van Kessel, H. et al. (1985) Calculation of the intra-assay variation per assay, and its relevance to LH pulse detection. IRCS Med. Sci., 13, 11831184.[ISI]
Lambalk, C.B., Schoemaker, J., van Rees, G.P. et al. (1989) The frequency of pulsatile LHRH treatment and LH and FSH secretion in women with amenorrhea of suprapituitary origin. Fertil. Steril., 51, 416422.[ISI][Medline]
Lambalk, C.B., de Boer, L., Schoute, E. et al. (1997) Post-menopausal and chronological age have divergent effects on pituitary and hypothalamic function in episodic gonadotrophin secretion. Clin. Endocrinol., 46, 439443.[ISI][Medline]
Lambalk, C.B., Boomsma, D.I., De Boer, L. et al. (1998) Increased levels and pulsatility of Follicle-Stimulating Hormone in mothers of hereditary dizygotic twins. J. Clin. Endocrinol. Metab., 83, 481486.
Lee, S.J., Lenton, E.A., Sexton, L. et al. (1988) The effect of age on the cyclical patterns of plasma LH, FSH, oestradiol and progesterone in women with regular menstrual cycles. Hum. Reprod., 3, 851855.[Abstract]
Licciardi, F.L., Liu, H.C. and Rosenwaks, Z. (1995) Day 3 estradiol serum concentrations as prognosticators of ovarian stimulation response and pregnancy outcome in patients undergoing in vitro fertilization. Fertil. Steril., 64, 991994.[ISI][Medline]
MacNaughton, J., Banah, M., McCloud, P. et al. (1992) Age related changes in follicle stimulating hormone, luteinizing hormone, oestradiol and immunoreactive inhibin in women of reproductive age. Clin. Endocrinol., 36, 339345.[ISI][Medline]
Matt, D.W., Kauma, S.W., Pincus, S.M. et al. (1998) Characteristics of luteinizing hormone secretion in younger versus older premenopausal women. Am. J. Obstet. Gynecol., 178, 504510.[ISI][Medline]
Muasher, S.J., Oehninger, S., Simonetti, S. et al. (1988) The value of basal and/or stimulated serum gonadotropin levels in prediction of stimulation response and in vitro fertilization outcome. Fertil. Steril., 50, 298307.[ISI][Medline]
Reame, N.E., Wyman, T.L., Phillips, D.J. et al. (1998) Net increase in stimulatory input resulting from a decrease in inhibin B and an increase in activin A may contribute in part to the rise in follicular phase follicle-stimulating hormone of aging cycling women. J. Clin. Endocrinol. Metab., 83, 33023307.
Reame, N.E., Kelche, R.P., Beitins, I.Z. et al. (1996) Age effects of follicle-stimulating hormone and pulsatile luteinizing hormone secretion across the menstrual cycle of premenopausal women. J. Clin. Endocrinol. Metab., 81, 15121518.[Abstract]
Roberts, V.J., Barth, S., El-Roiey A. et al. (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, 14021410.[Abstract]
Santoro, N., Adel, T., Skurnick, J.H. (1999) Decreased inhibin tone and increased activin A secretion characterize reproductive aging in women. Fertil. Steril., 71, 658662.[ISI][Medline]
Scheele, F., Lambalk, C.B., Schoemaker, J. et al. (1987) Patterns of LH and FSH in men during high-frequency blood sampling. J. Endocrinol., 114, 153160.[Abstract]
Schmidt, P.J., Gindoff, P.R., Baron, D.A. et al. (1996) Basal and stimulated gonadotropin levels in the perimenopause. Am. J. Obstet. Gynecol., 175, 643650.[ISI][Medline]
Scott, R.T. and Hofmann, G.E. (1995) Prognostic assessment of ovarian reserve. Fertil. Steril., 63, 111.[ISI][Medline]
Scott, R.T., Toner, J.P., Muasher, S.J. et al. (1989) Follicle-stimulating hormone levels on cycle day 3 are predictive of in vitro fertilization outcome. Fertil. Steril., 51, 651654.[ISI][Medline]
Seifer, D.B., Gardiner, A.C., Lambert-Messerlian, G. et al. (1996) Differential secretion of dimeric inhibin in cultured luteinized granulosa cells as a function of ovarian reserve. J. Clin. Endocrinol. Metab., 81, 736739.[Abstract]
Seifer, D.B., Lambert-Messerlian, G., Hogan, J.W. et al. (1997) Day 3 serum inhibin-B is predictive of assisted reproductive technologies outcome. Fertil. Steril., 67, 110114.[ISI][Medline]
Sherman, B.M. and Korenman, S.G. (1975) Hormonal characteristics of the human menstrual cycle throughout reproductive life. J. Clin. Invest., 55, 699706.[ISI][Medline]
Welt, C.K., McNicholl, D.J., Taylor, A.E. et al. (1999) Female reproductive aging is marked by decreased secretion of dimeric inhibin. J. Clin. Endocrinol. Metab., 84, 105111.
Wilshire, G.B., Loughlin, J.S., Brown, J.R. et al. (1995) Diminished function of the somatotropic axis in older reproductive-aged women. J. Clin. Endocrinol. Metab., 80, 608613.[Abstract]
Ying, S.Y. (1988) Inhibins, activins and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocrin. Rev., 9, 267293.[Abstract]
Submitted on January 5, 2000; accepted on April 25, 2000.