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Original Studies |
-Subunit Levels during Pulsatile Gonadotropin-Releasing Hormone Replacement in Women with Idiopathic Hypogonadotropic Hypogonadism1
The National Center for Infertility Research and the Reproductive Endocrine Sciences Center, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Janet E. Hall, The National Center for Infertility Research and the Reproductive Endocrine Sciences Center, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit
(FAS) levels, as reported in GnRH-deficient men, and to determine
whether this pattern is unique to congenital GnRH deficiency or is also
characteristic of patients with hypogonadotropic hypogonadism caused by
other factors. GnRH was administered iv at a physiologic frequency and dose (75–100 ng/kg·bolus) to women with IHH (n = 11; n = 6 with anosmia); acquired GnRH deficiency secondary to treatment for cranial tumors (AHH; n = 7); and secondary hypothalamic amenorrhea (HA; n = 8). Results were compared with 24 normal cycling women. Gonadotropins, sex steroids, and FAS levels were measured in samples drawn daily across induced or normal menstrual cycles in patients or normal women, respectively. Samples were drawn at the same time of day and were collected 45 min after a GnRH bolus in patients.
All women ovulated in response to pulsatile GnRH. There were no differences in the patterns of LH or gonadal steroid secretion between any of the patient groups (IHH, AHH, and HA). The patterns of LH and FSH secretion in the induced patient cycles were not different from normal women, with the exception of lower midcycle FSH levels in IHH women (P < 0.002). However, the daily dynamic secretion of FAS was exaggerated in IHH (compared with AHH, HA, and normal) women (P < 0.002). The increase in FAS levels in IHH was dependent on cycle stage, with the greatest difference observed during the early (P < 0.005) and midfollicular phase (P < 0.05) and the early luteal phase (P < 0.05). There was no difference in FAS between groups during the late follicular phase, at the midcycle, or in the midluteal and late luteal phase. This exaggerated FAS response to GnRH replacement in IHH was demonstrated in repeat cycles in two patients.
Conclusions are: 1) Women with IHH respond to pulsatile GnRH replacement with an exaggerated secretion of FAS, which seems to be modified by gonadal factors; 2) this exaggerated FAS response, which is similar to that seen in GnRH-deficient men, is unique to congenital GnRH deficiency, and it is not observed in patients with acquired or secondary hypogonadotropic hypogonadism, suggesting that IHH patients may be missing a factor, in addition to GnRH, which normally restrains FAS secretion; and 3) the FAS response may prove to be a useful marker to distinguish constitutional delay of puberty from congenital GnRH deficiency.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit, though the ß-subunit is unique and confers specificity
for each hormone (1). Under physiological circumstances, the
-subunit is oversecreted, in relation to the ß-subunit, and a
portion of it remains free in peripheral circulation (2, 3). The
secretion of free -subunit (FAS) is under the dual control of GnRH
and TRH (4, 5, 6, 7, 8, 9, 10, 11, 12, 13). Men with IHH, receiving long-term physiologic replacement with exogenous pulsatile GnRH, present a paradoxical increase in their FAS serum levels despite achievement of normal levels of LH, FSH, testosterone, and normal testicular growth (5, 11). This supraphysiological FAS response to GnRH is seen 3 days after beginning pulsatile GnRH replacement therapy (14). Although increased serum FAS levels have been observed with pituitary desensitization, using a GnRH agonist (15, 16, 17), desensitization seems not to be the mechanism for FAS hypersecretion in GnRH-deficient men, because FAS maintains its pulsatile response to GnRH and the increase is present, even at GnRH doses that result in a subnormal LH response (18). This differential regulation of FAS and LH, in response to GnRH replacement in GnRH-deficient men, suggests that other previously unrecognized factors may participate in the physiologic regulation of FAS. Although a function for FAS has yet to be delineated, if this paradoxical response to pulsatile GnRH replacement is unique to the congenital GnRH deficiency state, it may prove to be useful as a diagnostic tool to distinguish constitutional delay of puberty from congenital GnRH deficiency, a distinction that is critical to the clinical management of these patients. We proposed that examination of the FAS response to pulsatile GnRH replacement in women with GnRH deficiency of varying etiologies would help to address these issues.
Thus, the goals of this study were: 1) to determine whether women with idiopathic hypogonadotropic hypogonadism (IHH) demonstrate an increase in serum FAS levels in response to pulsatile GnRH replacement; 2) to determine whether this response is unique to patients with congenital GnRH deficiency or is also seen in patients with secondary hypogonadotropic hypogonadism [GnRH deficiency acquired after treatment for cranial tumor (AHH) or hypothalamic amenorrhea (HA)]; and 3) to determine whether this response is modified by changes in gonadal feedback across GnRH-induced ovulatory cycles.
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Subjects and Methods |
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Congenital GnRH deficiency (IHH). Eleven women with either IHH (n = 5) or Kallmann’s syndrome (n = 6), 17–39 yr old (mean: 26.1 yr), were selected on the basis of the following criteria: primary amenorrhea and absence of spontaneous pubertal development, normal cranial imaging of the hypothalamic-pituitary area, and normal basal and stimulated levels of other anterior pituitary hormones in response to iv insulin and TRH. All patients also had undergone a baseline frequent gonadotropin sampling study before pulsatile GnRH replacement, which confirmed the absence of pulsatile LH secretion. Patients were considered to have Kallmann’s syndrome if the above characteristics were accompanied by anosmia. In two IHH patients, FAS was measured in repeat cycles.
Acquired GnRH deficiency (AHH). Seven women (15–32 yr old, mean: 27.1 yr) with primary or secondary amenorrhea, associated with a history of hypothalamic and/or pituitary surgery and/or irradiation, were studied. Two patients had primary thyroid dysfunction with normal basal and stimulated TSH on replacement, whereas one patient presented, after radiation therapy, with tertiary hypothyroidism diagnosed by low T4 and normal TSH. These patients were included in this study because their FAS levels were not lower than women not receiving thyroid hormone replacement. Two patients had an elevated PRL level at the time of the study. One patient was taking corticosteroid replacement. All had normal basal and stimulated LH and FSH levels, in response to standard GnRH testing.
Hypothalamic amenorrhea (HA). Eight women (25–39 yr old, mean: 32 yr) with HA were selected on the basis of the following criteria: normo- or hypogonadotropic secondary amenorrhea of at least 6 months duration or primary amenorrhea with spontaneous breast development, absence of intensive exercise, no hirsutism or ovarian enlargement, no androgen excess and normal TSH and PRL levels.
All patients ovulated in response to pulsatile GnRH replacement. It was a first exposure to GnRH for 23 of the 26 patients. All had a body mass index (BMI) < 30 kg/m2 and > 18 kg/m2 and had not been exposed to sex steroids for at least 3 months before receiving GnRH. Partial results in 7 IHH or Kallmann’s, 6 AHH, and 8 HA patients have been included in previously published series (19, 20, 21, 22).
Normal controls. Twenty-four women (20–32 yr old, mean: 26.4 yr), in whom gonadotropins, sex steroids, and FAS were measured daily for a complete spontaneous ovulatory cycle, were used to define normal ranges of these parameters. Each subject had a history of regular 25- to 35-day cycles and a proven ovulatory cycle preceding the study. Their BMI and serum TSH and PRL were normal, and the subjects were on no medication. This group was randomly selected from a larger group of normal women described previously (23).
Study protocols
Studies were approved by the Subcommittee on Human Studies of the Massachusetts General Hospital, and informed consent was obtained from each subject before participation.
Before receiving pulsatile GnRH replacement, patients underwent a baseline study to characterize their spontaneous pattern of LH secretion. Blood sampling was performed every 10 min for 8–12 h.
Pulsatile GnRH was administered using a closed iv system, as previously described (19). A dose of 75 (n = 23) or 100 ng/kg·bolus (n = 3, 2 IHH and 1 AHH) was employed, and the frequency of GnRH administration was adjusted during the cycle to mimic the frequency changes that occur during the normal menstrual cycle. Briefly, a 90-min frequency interval was employed for the first 5–7 days, followed by an increase to 60 min with the appearance of a dominant follicle on ultrasound (>11 mm) and maintained until clinical and/or ultrasonographic evidence of ovulation (disappearance of a dominant follicle by ultrasound, a LH surge by urinary monitoring, and/or a basal body temperature shift). The frequency was then slowed to 90 min for 1 week after ovulation and then to 4 h for the remainder of the luteal phase until menses or a positive pregnancy test intervened.
Blood samples for LH, FSH, FAS, estradiol (E2), and progesterone (P) were drawn daily in patients and normal women for the duration of each cycle. Samples were drawn at the same time of day and were collected 45 min after a GnRH bolus in the patients.
To determine both the time course of appearance of this exaggerated FAS response and its pattern in response to a single GnRH bolus, blood samples for LH and FAS were drawn 10 min before and every 10 min after a single GnRH bolus, for up to 80 min during each of the first 7 days of pulsatile GnRH replacement in two IHH and five AHH patients. Again, samples were drawn at the same time of the day.
FAS also was measured during the initial GnRH test (100 µg iv) in five IHH and two AHH patients, to determine whether the exaggerated FAS response was present at baseline.
Assays
All blood samples for each cycle were measured in duplicate in a
single assay. Serum LH and FSH were assayed in specific ß- and
dimer-directed RIAs, respectively, as previously described (23). Both
hormones are expressed in international units per liter, as equivalents
of the Second International Reference Preparation of human menopausal
gonadotropin (World Health Organization, 71/223). The sensitivities of
the assays were 0.8 IU/L for LH and FSH, and 30 ng/L for FAS. The
intraassay coefficients of variation (CV) for LH and FSH were 6.1% and
7.0%, respectively, and the interassay CV were 8.5% and 11.6%. FAS
concentrations were determined by a previously described monoclonal
antibody RIA using highly purified -subunit of hCG as the assay
calibrator (24, 25). Assay performance was monitored on approximately
150 assays during the time this study was conducted. Intra- and
interassay variance was determined using aliquots of 3 pools of human
serum containing 234 (
80% B/B0), 444 (50%
B/B0), and 851 (20% B/B0) ng/L. The intraassay
CV was 7–8%, and the interassay CV was <15% for all 3 levels of
quality control sera. Cross-reactivity of FAS in the LH assay was
4.9%, caused entirely by the known FAS contamination of the LH
standard and label, as previously described (24). The FAS assay had
cross-reactivities of 0.67% for human LH (hLH), 2.32% for hFSH, and
0.36% for hTSH. Serum E2 and P were measured using RIA
methods previously described (26, 27).
Data analysis
Daily serum levels of LH, FSH, E2, P, and FAS during each cycle were analyzed, in relation to the day of ovulation, using hormonal criteria as previously described (21). Multiple groups were compared using ANOVA with post hoc Newman-Keuls testing. Mean follicular- and luteal-phase FAS levels were significantly elevated in IHH, compared with AHH, HA, and normal women. Cycles were then standardized to a 28-day length for each subject and divided into seven phases, in relation to the day of ovulation; the early follicular phase (EFP), day -13 to -9; the midfollicular phase, day -8 to -5; the late follicular phase, day -4 to -1; the midcycle surge (MCS), day 0; the early luteal phase, days +1 to +4; the MLP, days +5 to +9; and the late luteal phase, days +10 to +14, as previously described (23). The data from five patients who conceived in response to GnRH were not included after day +7 of the luteal phase.
To further define the timing of the supraphysiological increase in serum FAS level in IHH women, serum levels of LH, FSH, and FAS also were analyzed in relation to the first 7 days of GnRH administration. LH, FSH, FAS, E2, and P mean values for each of the 7 cycle phases and for the first 7 days of GnRH exposure were compared between IHH women and AHH, HA, and normal women, using ANOVA. Values are expressed as the mean ± SEM unless otherwise specified, and a P < 0.05 was accepted as significant.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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There was no difference in age and BMI between the three patient groups and the normal women. LH pulses were absent in all IHH, and in four AHH patients during the baseline studies, although all HA and three AHH patients demonstrated various patterns of diminished pulsatile secretion of LH at baseline in comparison with normal EFP women, as previously described (21, 28).
Responses to pulsatile GnRH
All cycles were ovulatory, and gonadotropin levels, as well as sex
steroids, were equivalent to normal cycling women (Fig. 1
). The only differences observed between
groups was a lower FSH at the MCS in IHH women, compared with AHH, HA,
and normal women (10.8 ± 1.3, 19.6 ± 5.1, 20.2 ± 3.2,
and 25.3 ± 4.4 IU/L, respectively; P < 0.002);
and higher luteal-phase estrogen levels in IHH, AHH, and HA, compared
with normal women (258.7 ± 47.7, 263.6 ± 73.2, 281.7
± 67 vs. 129.9 ± 10.8 pg/ml, respectively;
P < 0.05).
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and Table 1). This exaggerated FAS pattern was seen
in the absence of differences in the LH, E2, and P response
to pulsatile GnRH among the three patient groups.
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Examination of the data, in relation to the first day of pulsatile GnRH
in patients or the onset of menses in normal women, indicated that FAS
was increased above the normal range by day 3 of GnRH replacement in
IHH patients and was higher than in AHH and HA patients, whereas LH and
FSH remained within the normal range (Fig. 3). E2 and P also were within
the normal range in all patient groups at that time (data not
shown).
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).
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The increase of FAS levels, in response to GnRH, was rapid (peaking at
10 min), with a fast decrease to baseline (Fig. 4B
). These data suggest
that the FAS degree of hypersecretion in IHH patients is likely
underestimated in the daily samples drawn 45 min after the GnRH
dose.
An exaggerated FAS response was not consistently seen during
standardized GnRH tests in IHH women during their baseline evaluation
(Fig. 5), even with the use of the
100-µg pharmacological dose of GnRH.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Previous studies in IHH men undergoing long-term physiologic GnRH replacement have shown that, in the face of normal gonadotropin levels, FAS levels are elevated (5, 18). In this study, we have now shown that IHH women, with or without anosmia, also respond to exogenous GnRH with an exaggerated serum FAS level. In addition, we have shown that this exaggerated FAS response is unique to congenital GnRH deficiency, because it is not a feature of the response to pulsatile GnRH replacement in women with other causes of GnRH deficiency (AHH and HA).
The importance of the timing of blood sampling was highlighted in the studies in which blood was sampled more frequently after administration of a bolus of GnRH during the first 7 days of GnRH replacement. These studies demonstrated the rapid clearance of FAS and suggest that the results obtained from daily sampling 45 min after a GnRH bolus, if anything, underestimate the magnitude of the FAS overresponsiveness in IHH women.
We have shown that IHH women respond to pulsatile GnRH replacement with an exaggerated FAS response that is evident within 3 days of the beginning of pulsatile GnRH replacement, as in IHH men (14). In contrast, developmental studies in GnRH-deficient men have shown that the FAS hyperresponsiveness became apparent only after 4 weeks of therapy (18). However, direct comparisons cannot be made between those results in IHH men and IHH women, because of protocol differences. In IHH women, the physiologic replacement dose is relatively constant between subjects, and thus, GnRH replacement was instituted with this physiologic dose; whereas in IHH men, the physiologic replacement dose is less constant between individuals, and the above study was designed to gradually increase this dose over a period of several months. Importantly, in these developmental studies in IHH men, FAS levels in response to GnRH replacement were increased not only in relation to those in adult men, but also in relation to the somewhat higher levels that are seen in normal midpubertal boys.
This exaggerated and early increase in FAS levels was not limited to the first cycle of GnRH replacement in IHH women, because it was shown to be similar in repeat cycles. Importantly, this response is limited to congenital GnRH deficiency, because it is not seen in patients in whom GnRH deficiency is caused by other factors, even when the baseline pattern of LH secretion is apulsatile, implying a relatively complete degree of GnRH deficiency. The specificity of the FAS response to GnRH in congenital GnRH deficiency suggests that this may be a useful marker to distinguish this disorder from other causes of delayed puberty. This distinction is important in determining whether a patient will need long-term treatment for hypogonadism and fertility or whether maturation of the reproductive axis will occur spontaneously. In addition, this unique FAS response may prove to be useful in phenotyping patients in the genetic search for the genes responsible for Kallmann’s syndrome. However, an exaggerated FAS response was not consistently seen during standardized 100-µg iv GnRH tests in IHH women, even when a pharmacological dose of GnRH is used. Thus, a short duration of pituitary priming of at least 3 days with physiological pulsatile GnRH seems to be required to reveal this abnormality.
Our observation that the increase in FAS is not uniform across the menstrual cycle suggests that it is modulated by gonadal factors. However, it is unclear how this modulation is mediated, because the pattern of changes in FAS and the gonadal secretory products is not consistent with a role for estradiol, progesterone, inhibin A, or inhibin B. The increase in E2 levels during the luteal phase occurred after the increase in FAS in IHH women. Only one follicle developed in all patients. Other, as yet unidentified, factors may be involved.
An increase in serum levels of FAS may be caused by alterations in gonadotropin subunit synthesis, dimerization, or secretion, and may also result from a decrease in the metabolic or renal clearance. Although LH and FAS are secreted in parallel in normal men and women, the potential for differential regulation of LH and FAS has been observed in two other settings. Glycoprotein-secreting pituitary tumors are characterized by unbalanced glycoprotein subunit synthesis and secretion (30, 31, 32). In contrast, the paradoxical increase in FAS levels observed in children with precocious puberty treated with a GnRH agonist is associated with a decrease in its renal clearance (33). In studies in IHH men and women, it is unlikely that desensitization plays a role in the increased levels of FAS, because the dose and frequencies used resulted in physiologic LH and FSH levels.
The association of the abnormal FAS response with a congenital
deficiency in GnRH raises the possibility that these patients may lack
an additional hypothalamic factor involved in gonadotropin synthesis
and secretion that would normally inhibit FAS secretion. GAP, the GnRH
associated peptide, which is a PRL-inhibiting factor produced from the
proteolytic cleavage of the GnRH prohormone, may be a candidate (34),
because its addition to pulsatile GnRH replacement in the mutant hpg
mouse, at the time of LH surge, decreases to a greater degree than
LHß and FSHß expression (35, 36). Several other possible
intrapituitary modulators of GnRH action (which include galanin,
neuropeptide Y, activin, inhibin, and follistatin) have not been
evaluated, in this regard (37, 38, 39, 40, 41, 42).
The potential impact of this exaggerated FAS response to physiologic GnRH replacement in IHH patients is unclear, because little is known about the physiologic role of FAS in the circulation. FAS is not active at the hCG/LH receptor (43), and no specific FAS receptor has been isolated to date. However, there is evidence that FAS plays a role in lactotroph differentiation in the rat (44). In addition, FAS stimulates PRL secretion in cultured human decidua, myometrium, and leiomyoma cells, and acts synergistically with progesterone to induce more rapid decidualization of human stromal cells (45, 46, 47, 48). Taken together, these studies suggest that FAS may act as a growth factor in the pituitary and the endometrium and may thus play an important physiologic role in human reproduction.
In conclusion, IHH women, with or without anosmia, respond to exogenous pulsatile GnRH replacement with an exaggerated FAS response, despite otherwise normal levels of gonadotropins and sex steroids. This excessive response is unique to the congenital GnRH deficiency state in men and women, because it is not observed in women with other causes of GnRH deficiency and thus may be used to design specific tests to aid in the diagnosis and management of patients with pubertal delay.
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Acknowledgments |
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Footnotes |
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2 Recipient of fellowship support from the Samuel R. McLaughlin
Foundation, the Royal College of Physicians of Canada (Detweiler
Award), and Ferring Pharmaceuticals Canada, Germany.
Received July 11, 1997.
Revised September 2, 1997.
Accepted September 23, 1997.
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; n = 7), and
HA (
; n = 8). The shaded areas represent the
mean ± SD of 24 normal women.
