1 Department of Pediatrics/Endocrinology and Reproductive Sciences Program, University of Michigan, Ann Arbor, MI 48109-0718, USA
2 To whom correspondence should be addressed. Email: cmfoster{at}umich.edu
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Abstract |
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Key words: activin/follistatin/FSH/inhibin/puberty
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Introduction |
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FSH concentrations are greater than LH concentrations in prepubertal and early pubertal girls and increase at night coincident with the onset of sleep (Apter et al., 1993). The nocturnal increase in FSH concentrations can be reduced with GnRH antagonist treatment, but FSH concentrations are not reduced to the same extent as are LH concentrations (Apter et al., 1993
), suggesting that FSH secretion has GnRH-independent control. We speculated that the difference in regulation of FSH, compared with LH, secretion might be due to the fact that FSH is also regulated by the FSH-regulatory peptides. If circulating changes in activins and inhibin-A or inhibin-B affect FSH secretion, one might expect relatively high activin or low inhibin concentrations several hours before the nocturnal increase in FSH secretion. The purpose of this research was to test the hypothesis that diurnal changes in FSH can be accounted for by changes in circulating concentrations of activin-A, inhibin-A, inhibin-B and follistatin during puberty in girls. To test this hypothesis, we studied day/night changes in FSH and FSH-regulatory peptide concentrations in pubertal girls, girls with gonadal dysgenesis or ovarian failure (GD/OF) with little ovarian contribution of FSH-regulatory peptides, and girls with idiopathic hypogonadotrophic hypogonadism (IHH) with little GnRH drive.
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Subject and methods |
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Girls with IHH. Subjects 8 and 9, who were originally studied because of delayed adolescence, have had failure of progression of pubertal development and persistently low gonadotrophin concentrations consistent with IHH. Both girls had normal GH secretory profiles and the absence of other hormone abnormalities. Both girls were otherwise healthy, and neither had eating disorders.
Girls with GD/OF. Eight girls had elevated FSH concentrations indicating gonadal failure. Seven girls, subjects 2228, had GD confirmed by karyotype analysis. None had a Y chromosome. Subject 29 had OF associated with radiation and chemotherapy for treatment of acute myelocytic lymphoma which has remained in remission for >7 years. All eight girls had marked elevation of FSH by the time of study. GH concentrations were normal in all girls with the exception of subjects 26 and 27, both with Turner syndrome, who had mean 12 h GH concentrations of 0.8 and 0.5 pg/ml.
Protocol
The University of Michigan Institutional Review Board approved all research protocols. A parent provided written informed consent, and the subject provided written assent prior to study. The girls who had 24 h blood sampling were admitted to the General Clinical Research Center of the University of Michigan at 17:00 h the day prior to study. An i.v. access was established in a forearm vein at 06:00 h the following day. Blood was obtained every 15 min beginning at 08:00 h for 24 h. For the purposes of this study, an equal aliquot of each sample for a 3 h time block beginning at 08:00 h was pooled to make eight samples covering the 24 h period of sampling. The time blocks were 08:0010:45, 11:0013:45, 14:0016:45, 17:0019:45, 20:0022:45, 23:0001:45, 02:0004:45 and 05:0008:00 h. Each of the eight 3 h pools was assayed for LH, FSH, E2, activin-A, inhibin-A, inhibin-B and follistatin 288.
The girls who had 12 h blood sampling were admitted at 17:00 h and had an i.v. access established at 18:00 h. Blood was drawn every 15 min from 20:00 to 08:00 h. An equal aliquot of each sample over a 3 h period beginning at 20:00 h was pooled to make four samples in a manner similar to that for the 24 h study. Each of the four pools was analysed for LH, FSH, E2, inhibin-A, inhibin-B and activin-A. Follistatin 288 concentrations could not be assessed in these girls because of insufficient sample volume.
Hormone determinations
LH and FSH were determined by immunofluorometric assay using kits purchased from Wallac-PerkinElmer (Gaithersburg, MD). The assay sensitivities were 0.05 IU/l for both LH and FSH, and the intra- and inter-assay coefficients of variation (CVs) were 3.1 and 6.1%, respectively, for LH, and 3.9 and 4.8%, respectively, for FSH. E2 was determined by radioimmunoassay using kits purchased from Diagnostic Products Corporation (Los Angeles, CA). The assay sensitivity was 18 pmol/l and the intra- and inter-assay Cvs were 5 and 9%, respectively. Activin-A, inhibin-A and inhibin-B were determined by two-site enzyme-linked immunosorbent assays (ELISAs) using kits purchased from Serotec (Raleigh, NC). The assay sensitivity for activin-A was 39 pg/ml and the intra- and inter-assay Cvs were 6 and 16%, respectively. The assay sensitivity for inhibin-A was 7.8 pg/ml and the intra- and inter-assay Cvs were 6 and 13%, respectively. The assay sensitivity for inhibin-B was 8 pg/ml for the 24 h studies and 15 pg/ml otherwise. The intra- and interassay Cvs for inhibin-B were 10 and 16%, respectively. Follistatin 288 ELISA reagents were the gift of Nigel Groome, PhD, Oxford Brookes University, Oxford, UK. The assay was performed as described by Evans et al. (1998). The assay sensitivity was 37 pg/ml, and the intra- and interassay Cvs were 8 and 16%, respectively.
Statistical analysis
The hormone values were non-Gaussian in distribution and were therefore transformed logarithmically prior to performance of repeated measures analysis of variance. Post hoc testing was performed using Fisher's PLSD at a 5% alpha value to ascertain where differences between time blocks occurred within each analysis.
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Results |
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IHH (n=1). The girl with IHH who had 24 h blood sampling had no nocturnal increase in LH or FSH concentrations. Her inhibin-A, inhibin-B and follistatin 288 concentrations were less than the mean concentrations for the pubertal girls.
12 h overnight LH, FSH, inhibin-A, inhibin-B, activin-A and E2 concentrations in pubertal girls, girls with IHH and girls with GD/OF
Mean LH, FSH and activin-A concentrations between 20:00 and 08:00 h are shown in Figure 2 for 19 pubertal girls, including the 20:0008:00 h data from the seven pubertal girls who underwent the 24 h blood sampling protocol, two girls with IHH including the girl who underwent 24 h blood sampling, and eight girls with GD/OF.
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IHH girls (n=2). LH and FSH concentrations exhibited no nocturnal increase. Inhibin-A, inhibin-B and E2 concentrations had no night-time variability and were lower than in the pubertal girls. Activin-A concentrations tended to be greater in the girls with IHH than in the pubertal girls, and activin-A appeared to decrease during the night, but the small sample precluded analysis.
Girls with GD/OF (n=8). LH concentrations increased significantly after 23:00 h compared with the 20:0022:45 h time period (P=0.002). Although FSH concentrations demonstrated a trend toward a late night increase, the change was not significant (P=0.12). E2 concentrations were near assay sensitivity. Inhibin-A concentrations were less than the assay sensitivity in all of the girls. Seven of the eight girls had inhibin-B concentrations that were less than the assay sensitivity. Subject 27, who had GD, had a mean overnight inhibin-B concentration of 75 pg/ml and a mean overnight FSH of 131 IU/l. Her E2 concentrations were at assay sensitivity. When her results were removed from the analysis, the mean FSH concentrations were 54.5±10.7, 59.2±11.4, 64.8±10.8 and 66.6±10.8 IU/l and the mean activin-A concentrations were 295±61, 273±53, 249±33 and 273±51 pg/ml in the time blocks beginning at 20:00, 23:00, 02:00 and 05:00 h, respectively. There were no differences between time blocks for either FSH or activin-A concentrations.
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Discussion |
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Although the increase in nocturnal FSH may occur as a consequence of GnRH-induced secretion, it is of interest that inhibin-B concentrations achieve a nadir just prior to the nocturnal increase in FSH secretion in girls with spontaneous onset of puberty. This decline in inhibin-B, in concert with an increase in GnRH pulse generator activity, may contribute towards the night-time increase in FSH concentrations. The girls with GD/OF demonstrated a strong night-time GnRH drive, as indicated by their brisk nocturnal increase in LH concentrations. They did not exhibit a significant increase in FSH secretion, however. Thus, removal from a daytime inhibin-B threshold may play a role in the amplification of a nocturnal increase of FSH in girls.
Activin-A concentrations are greater in the daytime than at night in girls with spontaneous puberty. The decline in activin-A concentration occurs at the same time as the increase in FSH concentrations. The higher daytime activin-A concentrations could contribute towards the increase in FSH secretion during the night, since there appears to be a considerable time lag between a change in activin environment and a change in pituitary FSH secretion (Padmanabhan et al., 2002). Studies in sheep support an endocrine role for activin in control of FSH secretion (Padmanabhan et al., 2002
). A similar decline in activin-A concentration could not be discerned in the girls with IHH, but the small sample number precludes analysis. There was no nocturnal decline of activin-A in the girls with GD/OF. Thus, the decline in activin-A concentrations in the girls with spontaneous puberty probably represents a decrease in ovarian contribution of activin-A to the circulation. Our findings are also consistent with the notion that an increase in pituitary FSH secretion may account for the decline in ovarian activin-A secretion. This interpretation fits well with our previous observation that, when FSH secretion is blunted by administration of E2, activin-A concentrations increase in girls who have ovaries but not in girls with GD/OF (Foster et al., 2004
). These data together support a role for FSH in governing ovarian activin-A secretion in pubertal girls.
In summary, pubertal girls exhibit a night-time increase in FSH concentrations that is probably a consequence of the nocturnal increase in GnRH secretion, a decline in inhibin-B concentrations during the afternoon and evening, and a daytime increase in activin-A concentrations. The increase in FSH secretion is accompanied by a decline in activin-A secretion in pubertal girls but not in girls with GD/OF, suggesting that this decrease stems from a decline in ovarian activin-A release into the circulation. Our data support the possibility that a closed feedback loop exists between FSH-regulatory proteins and circulating FSH in pubertal girls.
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Acknowledgements |
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References |
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Submitted on April 22, 2004; resubmitted on August 16, 2004; accepted on October 15, 2004.
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