The spectrum of abnormal patterns of gonadotropin-releasing hormone secretion in men with idiopathic hypogonadotropic hypogonadism: clinical and laboratory correlations

Several lines of evidence indicate that hypothalamic-pituitary-gonadal activity varies among men with idiopathic hypogonadotropic hypogonadism (IHH). To test the hypothesis that a spectrum of abnormalities of GnRH secretion underlies the syndrome of IHH, we characterized the patterns of GnRH-induced gonadotropin secretion during periods of frequent sampling in 50 consecutive men with IHH and contrasted them with those in 20 normal men. The largest group of IHH patients (n = 42) had no detectable LH or FSH pulsations and could be categorized into 2 subsets according to the presence or absence of evidence of spontaneous puberty. The most severely affected subset (n = 32), who recalled no history of puberty, had testes with a mean volume of 3.3 +/- 0.5 (+/- SEM) ml, with a prepubertal appearance on biopsy, and often were anosmic (n = 17). The second subset of apulsatile IHH men (n = 10) had histories of partial or complete spontaneous sexual development with subsequent isolated loss of sexual function, testes with a mean volume of 13.3 +/- 1.9 ml (P less than 0.01 compared to the first subset), a pubertal or adult appearance of the testes on biopsy, and an intact sense of smell. In a second group of IHH patients (n = 3), LH was secreted predominantly in a nighttime pattern similar to that of normal children during early puberty. These men were aged 18-24 yr, had a mean testicular volume of 10.5 +/- 2.3 ml, pubertal changes on testicular biopsy, and an intact sense of smell. A third group of IHH men (n = 4) had LH pulses of abnormally low amplitude. Only one patient in this group had a history of spontaneous sexual development. The mean testicular volume of these patients was 5.6 +/- 1.9 ml, and the testes appeared prepubertal (n = 3) or pubertal (n = 1) on biopsy. In addition to these groups, another patient had apparent LH pulsations and nearly normal amplitude, but the LH was bioinactive and appeared to consist chiefly of alpha-subunit. Testing of other anterior pituitary hormone functions did not distinguish IHH men from normal men. However, those IHH patients with some evidence of endogenous GnRH secretion had higher basal and stimulated serum PRL levels than IHH men without such evidence (P less than 0.05), suggesting an influence of GnRH on PRL secretion.     

Administration of low dose pulsatile gonadotropin-releasing hormone (GnRH) to GnRH-deficient men regulates free alpha-subunit secretion

Although pharmacological doses of GnRH and TRH stimulate free alpha-subunit (alpha-subunit) secretion from the pituitary, little is known about the pattern and control of alpha-subunit release under physiological circumstances. Euthyroid men with idiopathic hypogonadotropic hypogonadism, a condition of deficient GnRH release, provide a unique opportunity to study alpha-subunit secretion before and during administration of a physiological regimen of GnRH administration. Before GnRH therapy, six euthyroid IHH men with normal endogenous TSH secretion had circulating alpha-subunit levels close to or below assay detection limits, with a mean level less than 0.5 ng/ml. During 12-42 weeks of physiological GnRH replacement, serum alpha-subunit concentrations rose to a mean value of 2.07 +/- 0.3 (+/- SEM) ng/ml (P less than 0.01). After GnRH administration, alpha-subunit was released in a pulsatile pattern following each dose of GnRH and mirrored the secretory pattern of LH. Increases in serum alpha-subunit concentrations during GnRH administration were closely correlated with increases in LH (r = 0.91; P less than 0.01), but not FSH (r = 0.24; P = NS), levels. In addition, a situation in which LH secretion was clearly predominant and FSH levels were barely detectable was created by increasing the frequency of GnRH administration to every 30 min. In this circumstance, free alpha-subunit concentrations increased in conjunction with LH levels in the face of decreased FSH levels. We conclude that replacement of GnRH regulates both the level and pattern of alpha-subunit secretion in GnRH-deficient men, and that there is tight correlation of alpha-subunit with LH, but not with FSH, secretion.     

The nature of the gonadotropin-releasing hormone stimulus-luteinizing hormone secretory response of human gonadotrophs in vivo

To examine the stimulus-secretion response of human pituitary gonadotrophs in vivo, we applied a new multiple parameter deconvolution technique to analyze (1) exogenous GnRH-stimulated LH secretory responses in 10 men with isolated hypogonadotropic hypogonadism (IHH), and (2) endogenous and exogenous GnRH-stimulated LH secretory responses in 8 normal men. The GnRH-deficient men were given 4 bolus doses of synthetic GnRH (7.5, 25, 75, and 250 ng/kg) iv at 2-h intervals in randomized order after long term pulsatile GnRH administration. The normal men were studied by sampling blood at 10-min intervals for 12 h basally and after 2 consecutive 10-micrograms iv GnRH doses. The serum LH peaks in both groups were subjected to quantitative deconvolution to resolve underlying LH secretory and clearance rates simultaneously. Such analyses revealed that exogenous GnRH-induced LH secretory episodes in GnRH-deficient men with IHH could be modeled as algebraically Gaussian distributions of instantaneous LH secretory rates with a mean half-duration of 14 +/- 2 min. The simultaneously resolved half-life of endogenous LH disappearance was 71 +/- 5 min. The log dose-response relationship for GnRH dose vs. maximal LH secretory rate or vs. calculated mass of LH released per secretory burst was linear. In contrast, varying GnRH doses did not alter the duration of LH secretory bursts, the half-time of LH disappearance, or the latency of LH secretory bursts after iv GnRH injections (viz. 7.6 min). Deconvolution analysis of the spontaneous (endogenous GnRH-stimulated) LH peaks in normal men revealed a mean half-duration of secretory bursts of 9.9 +/- 1.5 min, and a mean half-time of endogenous LH disappearance of 76 +/- 5 min. These values were not significantly different from those in the GnRH-treated normal or GnRH-deficient men. In summary, deconvolution analysis of LH release in men with IHH revealed a significant linear relationship between iv doses of pulsed GnRH and computer-resolved LH secretory rate and/or the mass of LH released per secretory event. In contrast, varying doses of GnRH did not alter the lag time between the GnRH stimulus and the LH secretory burst, the duration of LH secretion, or the calculated half-life of the LH released. We conclude that GnRH exerts dose-dependent effects on specific attributes of the secretory response of human gonadotrophs in vivo.     

Aromatase inhibition in the human male reveals a hypothalamic site of estrogen feedback

The preponderance of evidence states that, in adult men, estradiol (E2) inhibits LH secretion by decreasing pulse amplitude and responsiveness to GnRH consistent with a pituitary site of action. However, this conclusion is based on studies that employed pharmacologic doses of sex steroids, used nonselective aromatase inhibitors, and/or were performed in normal (NL) men, a model in which endogenous counterregulatory adaptations to physiologic perturbations confound interpretation of the results. In addition, studies in which estrogen antagonists were administered to NL men demonstrated an increase in LH pulse frequency, suggesting a potential additional hypothalamic site of E2 feedback. To reconcile these conflicting data, we used a selective aromatase inhibitor, anastrozole, to examine the impact of E2 suppression on the hypothalamic-pituitary axis in the male. Parallel studies of NL men and men with idiopathic hypogonadotropic hypogonadism (IHH), whose pituitary-gonadal axis had been normalized with long-term GnRH therapy, were performed to permit precise localization of the site of E2 feedback. In this so-called tandem model, a hypothalamic site of action of sex steroids can thus be inferred whenever there is a difference in the gonadotropin responses of NL and IHH men to alterations in their sex steroid milieu. A selective GnRH antagonist was also used to provide a semiquantitative estimate of endogenous GnRH secretion before and after E2 suppression. Fourteen NL men and seven IHH men were studied. In Exp 1, nine NL and seven IHH men received anastrozole (10 mg/day po x 7 days). Blood samples were drawn daily between 0800 and 1000 h in the NL men and immediately before a GnRH bolus dose in the IHH men. In Exp 2, blood was drawn (every 10 min x 12 h) from nine NL men at baseline and on day 7 of anastrozole. In a subset of five NL men, 5 microg/kg of the Nal-Glu GnRH antagonist was administered on completion of frequent blood sampling, then sampling continued every 20 min for a further 8 h. Anastrozole suppressed E2 equivalently in the NL (136 +/- 10 to 52 +/-2 pmol/L, P < 0.005) and IHH men (118 +/- 23 to 60 +/- 5 pmol/L, P < 0.005). Testosterone levels rose significantly (P < 0.005), with a mean increase of 53 +/- 6% in NL vs. 56 +/- 7% in IHH men. Despite these similar changes in sex steroids, the increase in gonadotropins was greater in NL than in IHH men (100 +/- 9 vs. 58 +/- 6% for LH, P = 0.07; and 85 +/- 6 vs. 41 +/- 4% for FSH, P < 0.002). Frequent sampling studies in the NL men demonstrated that this rise in mean LH levels, after aromatase blockade, reflected an increase in both LH pulse frequency (10.2 +/- 0.9 to 14.0 +/- 1.0 pulses/24 h, P < 0.05) and pulse amplitude (5.7 +/- 0.7 to 8.4 +/- 0.7 IU/L, P < 0.001). Percent LH inhibition after acute GnRH receptor blockade was similar at baseline and after E2 suppression (69.2 +/- 2.4 vs. 70 +/- 1.9%), suggesting that there was no change in the quantity of endogenous GnRH secreted. From these data, we conclude that in the human male, estrogen has dual sites of negative feedback, acting at the hypothalamus to decrease GnRH pulse frequency and at the pituitary to decrease responsiveness to GnRH.     

Stimulation of serum inhibin concentrations by gonadotropin-releasing hormone in men with idiopathic hypogonadotropic hypogonadism

Inhibin is a gonadal hormone thought to be important in FSH regulation. We investigated the effects of the hypogonadotropic state and subsequent GnRH-induced increases in gonadotropin levels on inhibin secretion. Serum levels of inhibin, LH, FSH, and testosterone (T) as well as sperm concentrations were measured in 5 men with idiopathic hypogonadotropic hypogonadism (IHH) before (baseline) and during 8 weeks of GnRH therapy (5 micrograms, sc, every 2 h). Baseline and peak inhibin levels were compared to those in a group of 19 normal men. Before GnRH administration, the mean serum inhibin level was significantly lower in the IHH men than in the normal men [166 +/- 56 (+/- SE) vs. 588 +/- 30 U/L; P less than 0.001]. Serum inhibin levels rose after 1 week of GnRH therapy (P less than 0.05) and remained higher than the baseline level thereafter. The mean peak inhibin level during GnRH administration was lower than the mean value in normal men (485 +/- 166 vs. 588 +/- 30 U/L; P less than 0.05). Serum LH and FSH levels rose promptly to the midnormal range or slightly above it. Serum T levels did not significantly increase until 4-5 weeks of GnRH administration and remained in the low normal range. All IHH men were azoospermic throughout the study. These data are consistent with the hypothesis that inhibin is produced by the testis under gonadotropin control. They also suggest the possibility of defective Sertoli and Leydig cell function in men with IHH, since the men's serum inhibin and T levels did not rise to the same extent as did their normalized serum gonadotropin levels during GnRH administration.     

Sex steroid control of gonadotropin secretion in the human male. I. Effects of testosterone administration in normal and gonadotropin-releasing hormone-deficient men

The precise sites of action of the negative feed-back effects of gonadal steroids in men remain unclear. To determine whether testosterone (T) administration can suppress gonadotropin secretion directly at the level of the pituitary, the pituitary responses to physiological doses of GnRH were assessed in six men with complete GnRH deficiency, whose pituitary-gonadal function had been normalized with long term pulsatile GnRH delivery, before and during a 4-day continuous T infusion (15 mg/day). Their responses were compared with the effects of identical T infusions on spontaneous gonadotropin secretion and the response to a 100-micrograms GnRH bolus in six normal men. Both groups were monitored with 15 h of frequent blood sampling before and during the last day of the T infusion. In the GnRH-deficient men, the first three GnRH doses were identical and were chosen to produce LH pulses with amplitudes in the midphysiological range of our normal men (i.e. a physiological dose), while the last four doses spanned 1.5 log orders (7.5, 25, 75, and 250 ng/kg). The 250 ng/kg dose was always administered last because it is known to be pharmacological. In the GnRH-deficient men, mean LH (P less than 0.02) and FSH (P less than 0.01) levels as well as LH pulse amplitude (P less than 0.05) decreased significantly during T infusion, demonstrating a direct pituitary-suppressive effect of T and/or its metabolites. Mean LH levels were suppressed to a greater extent in the normal than in the GnRH-deficient men (58 +/- 15% vs. 28 +/- 7%; P less than 0.05). In addition, LH frequency decreased significantly (P less than 0.01) during T administration in the normal men. These latter two findings suggest that T administration also suppresses hypothalamic GnRH release. T was unable to suppress gonadotropin secretion in one GnRH-deficient and one normal man. In both groups, the suppressive effect of T administration was present only in response to physiological doses of GnRH. Because the pituitary- and hypothalamus-suppressive effects of T could be mediated by its aromatization to estrogens, five GnRH-deficient and five normal men underwent identical T infusions with concomitant administration of the aromatase inhibitor testolactone (TL; 500 mg, orally, every 6 h). As an additional control, four GnRH-deficient and four normal men received TL alone. TL administration completely prevented the effect of T administration to suppress gonadotropin secretion in both the normal and GnRH-deficient men, and mean LH levels increased significantly in both the GnRH-deficient (P less than 0.01) and the normal (P less than 0.001) men who received TL alone. The increase in mean LH levels was greater (P less than 0.01) in the normal men who received TL alone than in the normal men who received T plus TL, thus revealing a direct effect of androgens in normal men. Measurements of T and estradiol production rates in three men demonstrated that TL effectively blocked aromatization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sex steroid control of gonadotropin secretion in the human male. II. Effects of estradiol administration in normal and gonadotropin-releasing hormone-deficient men

Although prior studies have suggested that estrogens exert their negative feedback effect at the pituitary level in men, these conclusions have been based on models that evaluate changes in LH pulse amplitude and frequency and, therefore, only provide indirect information concerning the site of action of estrogens. To assess whether estradiol (E2) inhibits gonadotropin secretion directly and solely at the pituitary level in men, we determined the pituitary responses to physiological doses of GnRH in six men with complete GnRH deficiency, whose pituitary-gonadal function had been normalized with long term pulsatile GnRH delivery, before and during a 4-day continuous E2 infusion (90 micrograms/day). To deduce whether E2 has an additional inhibitory effect on hypothalamic GnRH secretion, their responses were compared with the effects of identical E2 infusions on spontaneous gonadotropin secretion and the responses to a 100-micrograms GnRH bolus in six normal men. Both groups were monitored with 15 h of frequent blood sampling before and during the last day of the E2 infusion. In the GnRH-deficient men, the first three GnRH doses were identical and chosen to produce LH pulses with amplitudes in the midphysiological range of values in our normal men (i.e. a physiological dose), while the last four doses spanned 1.5 log orders (7.5, 25, 75, and 250 ng/kg). The 250-ng/kg dose was always administered last because it is known to be pharmacological. In the GnRH-deficient men, mean LH and FSH levels as well as LH pulse amplitude all decreased significantly (P less than 0.02) during E2 infusion, demonstrating a direct pituitary-suppressive effect of E2. Mean LH (P less than 0.01) and FSH (P less than 0.05) levels and LH pulse amplitude (P less than 0.01) also decreased significantly in the normal men. The degree of suppression of mean LH (52 +/- 3% vs. 42 +/- 12%) and FSH (49 +/- 10% vs. 37 +/- 10%) levels was similar in the two groups. These results provide direct evidence that E2 inhibits gonadotropin secretion at the pituitary level in men and suggest that the pituitary is the most important, and possibly the sole, site of negative feedback of estrogens in men.     

Pituitary glycoprotein hormone alpha-subunit secretion after thyrotropin-releasing hormone stimulation in normal men and men with idiopathic hypogonadotropic hypogonadism.

Although TRH stimulates the release of uncombined alpha-subunit into the circulation in patients with primary hypothyroidism, it is not clear whether alpha-subunit is released from the thyrotrophs in euthyroid subjects. We hypothesized that spontaneous fluctuations in circulating alpha-subunit released from gonadotrophs by GnRH in normal adults could obscure the detection of small changes in alpha-subunit after TRH administration. We, therefore, examined alpha-subunit responses to TRH in five euthyroid men with idiopathic hypogonadotropic hypogonadism (IHH), who produce little or no GnRH, five normal men, and four postmenopausal women. Mean (+/- SEM) basal serum alpha-subunit levels were significantly (P less than 0.05) less in men with IHH (0.26 +/- 0.07 microgram/L) than in the normal men (0.80 +/- 0.20 microgram/L) or postmenopausal women (3.54 +/- 0.60 microgram/L). alpha-Subunit levels rose after TRH administration in all men with IHH to a peak level of 0.86 +/- 0.25 ng/ml; TSH levels also increased from 1.9 +/- 0.4 to 13.0 +/- 5.6 mU/L. The increment in TSH and alpha-subunit levels was highly positively correlated (r = 0.96). alpha-Subunit levels also increased 2-fold in normal men given TRH, whereas alpha-subunit levels in postmenopausal women were unchanged. We conclude that thyrotrophs release alpha-subunit into the circulation in normal men and euthyroid men with IHH. Thus, both thyrotrophs and gonadotrophs appear to contribute to circulating alpha-subunit in men with IHH; however, most of the uncombined alpha-subunit in normal men appears to be from gonadotrophs.     

Effects of increasing the frequency of low doses of gonadotropin-releasing hormone (GnRH) on gonadotropin secretion in GnRH-deficient men

The effects of increasing the frequency of pulsatile GnRH administration on LH and FSH responsiveness were studied in five GnRH-deficient men who had achieved normal sex steroid levels during prior long term GnRH replacement. Intravenous doses of GnRH were employed that had previously been demonstrated to produce LH and FSH levels in each subject similar to those in normal men. Both acute and chronic changes in pituitary responses were studied after progressive increases in GnRH frequency (from every 120 to 60 min, from 60 to 30 min, and from 30 to 15 min) during three 12-h admissions, each separated by 7 days. During the two intervals between the studies GnRH frequency was 60 and 30 min, respectively. Pituitary responses were characterized by determining the mean serum LH and FSH levels, LH pulse amplitudes, and mean LH and FSH levels which were normalized for the frequency of GnRH administration (nLH and nFSH). As the frequency of GnRH stimulation was increased acutely, mean serum LH levels rose progressively, in contrast to both LH pulse amplitude and nLH levels which decreased, while serum testosterone (T) concentrations remained constant. No further evidence of gonadotroph desensitization occurred after chronic GnRH administration at either 60- or 30-min intervals. At higher frequencies of GnRH stimulation, discrete pulses of LH were not always apparent after injections of GnRH, and in two men, marked destabilization of the gonadotroph responses occurred. Even without detectable LH pulses, serum T levels did not decline during administration of GnRH at intervals as rapid as 15 min. In contrast, there was no change in mean FSH concentrations, although nFSH values decreased progressively as the GnRH frequency was increased. nFSH levels fell to a greater degree than nLH after each increase in GnRH frequency. Thus, pituitary gonadotroph responsiveness to a fixed dose of GnRH decreased as the frequency of GnRH stimulation increased. FSH responsiveness decreased to a greater degree than did LH. Gonadotropin secretory responses are destabilized at higher frequencies of GnRH administration. Pulsatile LH stimulation of the testes does not appear necessary to maintain T secretion.     

Utility of free alpha-subunit as an alternative neuroendocrine marker of gonadotropin-releasing hormone (GnRH) stimulation of the gonadotroph in the human: evidence from normal and GnRH-deficient men.

To examine the hypothesis that the secretion of free alpha-subunit (FAS) can serve as an alternative to LH as a neuroendocrine marker of gonadotroph stimulation by GnRH in euthyroid humans, we have investigated the relationship of pulsatile FAS secretion in euthyroid GnRH-deficient men (n = 10) before and after exogenous GnRH stimulation and in normal men under the influence of endogenous GnRH secretion (n = 18). Before GnRH exposure, the GnRH-deficient men showed a complete absence of both LH and FAS pulses. During the initial 7 days of GnRH exposure, all GnRH-deficient men exhibited pulsatile release of FAS by the third day, whereas the appearance of pulsatile release of LH and FSH was more variable. Long term administration of GnRH led to pulses of LH and FAS that were 100% concordant with a demonstrable dose-response relationship between GnRH and FAS, which was quantitatively similar to but more exuberant than that for LH. All doses of GnRH that produced LH pulses within the normal adult range yielded supraphysiological FAS pulses. Analysis of distribution histograms of interpulse intervals and pulse amplitudes of LH and FAS in both normal and GnRH-deficient subjects demonstrated no significant difference between these glycoproteins in interpulse intervals in either the normal or GnRH-deficient groups or in the pulse amplitudes in the GnRH-deficient subjects. There was, however, a significant difference (P less than 0.01) between the distribution histogram of LH and FAS pulse amplitudes in normal men. We conclude that the pulsatile secretion of FAS in euthyroid men 1) is determined by GnRH secretion, 2) is the initial glycoprotein to be secreted in a pulsatile fashion from the gonadotroph during early GnRH exposure in GnRH-deficient men, 3) demonstrates a dose-response relationship to exogenous GnRH which is more robust than that of LH in GnRH-deficient men receiving GnRH, and 4) can, therefore, serve as a complementary and powerful tool with LH for the study of GnRH neurosecretory dynamics.     

Pituitary luteinizing hormone responses to intravenous and subcutaneous administration of gonadotropin-releasing hormone in men

Although differences in plasma GnRH concentrations have been identified after iv and sc injection of this peptide, differences in pituitary LH responses to iv and sc GnRH have not been evaluated in detail. We studied the magnitude and contour of plasma GnRH and LH responses after low doses of iv and sc GnRH administered to men with idiopathic hypogonadotropic hypogonadism and compared them to LH pulses in normal men after endogenous GnRH secretion. Mean areas under the LH response curves differed significantly (P less than 0.01) after 25 ng/kg, but not 250 ng/kg, iv and sc GnRH doses. The mean time from basal to peak plasma LH concentrations was significantly longer with sc than iv GnRH (P less than 0.02). In addition, individual LH responses were more variable with sc GnRH. Intravenous administration produced greater GnRH amplitude (P less than 0.001) and area under the curve (P less than 0.005) and shorter time to peak (P less than 0.01) GnRH concentrations. When plasma LH responses of similar area and amplitude were compared, the contour of LH responses after iv GnRH more closely simulated the LH pulses in normal men. These data demonstrate that 1) significant differences exist in the amplitude, contour, and variability of plasma LH and GnRH pulses after iv and sc GnRH; and 2) iv GnRH elicits LH secretory episodes which closely resemble endogenous pulsations of normal men. These results suggest that iv GnRH administration may be preferred in physiological studies and, if the data can be extrapolated to women, may account for the greater success of ovulation induction reported with iv GnRH.     

Circadian rhythm of plasma testosterone in men with idiopathic hypogonadotrophic hypogonadism before and during pulsatile administration of gonadotrophin-releasing hormone

OBJECTIVE: The aim was to investigate whether a pulsatile discharge of LH from the pituitary is necessary to achieve the circadian secretion of testosterone. DESIGN: The daily rhythm of the androgen has been studied in patients with idiopathic hypogonadotrophic hypogonadism (IHH) both in the absence of therapy and during pulsatile administration of gonadotrophin releasing hormone (GnRH). PATIENTS: Six patients with IHH and ten normal subjects were analysed. Blood sampling was performed at 2-hourly intervals, for 24 hours. The IHH patients then received synthetic GnRH i.v. at the rate of one pulse every 2 hours (10 micrograms/pulse). On day 11 of treatment, blood samples were taken for the rhythm analysis every 2 hours, for 24 hours. MEASUREMENTS: Plasma testosterone and LH were measured in the individual samples by radioimmunoassay. Evaluation of the rhythm was performed by cosinor analysis. RESULTS: A significant circadian rhythm of plasma testosterone was statistically validated in the normal subjects, whereas no rhythm was detected in the IHH patients in the absence of therapy. On day 11 of GnRH pulsatile administration the IHH patients showed normal testosterone levels and a statistically significant circadian rhythm of the androgen was evident, with acrophase between 0700 and 0800 h. Moreover, the amplitude, acrophase and mesor of testosterone rhythm in IHH patients in the course of treatment were statistically indistinguishable from the corresponding values in the normal subjects. Plasma LH did not show statistically significant circadian variations, either in the control group or in the IHH patients before or during therapy. CONCLUSIONS: We conclude that a physiological circadian rhythm of plasma testosterone can be obtained, in IHH men, by treatment with GnRH. Since the pulsatile administration of exogenous GnRH at constant doses induced a circadian rhythm in testosterone and no daily variations in LH were evident, we suggest that, although a pulsatile secretion of LH is probably necessary for the synchronization of the circadian rhythm with acrophase in the morning, the testosterone variations might be the results of a local testicular modulation of LH action.     

Bio- and immunoactive luteinizing hormone responses to low doses of gonadotropin-releasing hormone (GnRH): dose-response curves in GnRH-deficient men

Previous investigations of the effects of GnRH on pituitary LH responses in normal men required pharmacological doses of GnRH to avoid the confounding effects of endogenous GnRH secretion and employed nonphysiological dose intervals. To examine the role of GnRH in determining both the qualitative and quantitative nature of physiological LH responses, we studied five GnRH-deficient men in whom pituitary and gonadal function had been normalized with GnRH replacement. Both bio- and immunoactive LH responses were evaluated in these men after a wide range of GnRH doses (7.5-250 ng/kg) administered at a physiological frequency (every 2 h), while gonadal steroid levels were within the normal adult male range. In addition, the amplitude and contour of the immunoactive LH pulses were compared to those of 15 normal men to assure that these experiments achieved physiological pituitary responses. The relationship between bio- and immunoactive LH was compared between patients, between doses as the amount of GnRH was increased, and within pulses of LH. As the dose of GnRH was increased, both bio- and immunoactive LH responses increased in a log-linear fashion when assessed by both amplitude (r = 0.96 for bioactive LH and r = 0.98 for immunoactive LH) and area under the curve (r = 0.99 for bioactive LH and r = 0.97 for immunoactive LH). GnRH doses of 7.5 and 25 ng/kg produced LH responses with amplitudes similar to those in normal men. The relationship between bio- and immunoactive LH between patients and between differing doses of GnRH was analyzed by comparing the slopes of lines fit to individual bioactive vs. immunoactive LH plots after each dose of GnRH in each patient. There was a marked variation in the relationship of bio- to immunoactive LH between patients (P less than 0.005). No change was found in the biopotency of LH as the dose of GnRH was increased (P less than 0.10). Finally, no variation of the bioactivity of LH was evident within individual pulses. We conclude that a log-linear relationship exists between doses of GnRH that produce physiological LH pulses and both bio- and immunoactive LH responses; the bioactivity of secreted LH varies markedly between patients; the relative bioactivity of LH in an individual does not change as the dose of GnRH is increased; and no change in bioactivity of LH responses was demonstrated within pulses of LH.     

Inhibition of luteinizing hormone secretion by testosterone in men requires aromatization for its pituitary but not its hypothalamic effects: evidence from the tandem study of normal and gonadotropin-releasing hormone-deficient men

CONTEXT: Studies on the regulation of LH secretion by sex steroids in men are conflicting. OBJECTIVE: Our aims were to determine the relative contributions of testosterone (T) and estradiol (E2) to LH regulation and localize their sites of negative feedback. DESIGN: This was a prospective study with three arms. SETTING: The study was conducted at a General Clinical Research Center. PATIENTS OR OTHER PARTICIPANTS: Twenty-two normal (NL) men and 11 men with GnRH deficiency due to idiopathic hypogonadotropic hypogonadism (IHH) participated. INTERVENTION: Medical castration and inhibition of aromatase were achieved using high-dose ketoconazole (KC) for 7 d with 1) no sex steroid add-back; 2) T enanthate 125 mg im starting on d 4; or 3) E2 patch 37.5 microg/d starting on d 4. Blood sampling was performed every 10 min for 12 h at baseline, overnight on d 3-4 and d 6-7. MAIN OUTCOME MEASURES: Mean LH levels, LH pulse amplitude, and GnRH pulse frequency were assessed at baseline, d 3-4, and d 6-7. RESULTS: In NL men, KC caused a 3-fold increase in mean LH on d 3-4, which was stable on d 6-7 with no add-back. Addition of T reduced LH levels (34.6+/-3.9 to 17.4+/-3.6 IU/liter, P<0.05) by slowing GnRH pulse frequency (13.3+/-0.4 to 6.7+/-1.0 pulses/12 h, P<0.005). LH amplitude increased (6.9+/-1.0 to 12.1+/-1.4 IU/liter, P<0.005). E2 add-back suppressed LH levels (36.4+/-5.6 to 19.0+/-2.4 IU/liter, P<0.005), by slowing GnRH pulse frequency (11.4+/-0.2 to 8.6+/-0.4 pulses/12 h, P<0.05) and had no impact on LH pulse amplitude. In IHH men, restoring normal T levels caused no suppression of mean LH levels or LH amplitude. E2 add-back normalized mean LH levels and decreased LH amplitude from 14.7+/-1.7 to 12+/-1.5 IU/liter (P<0.05). CONCLUSIONS: 1) T and E2 have independent effects on LH. 2) Inhibition of LH by T requires aromatization for its pituitary, but not hypothalamic effects. 3) E2 negative feedback on LH occurs at the hypothalamus.     

Gonadotropin and alpha-subunit responses to chronic gonadotropin-releasing hormone analog administration in patients with glycoprotein hormone-secreting pituitary tumors

Pituitary tumors secreting intact glycoprotein hormones (LH, FSH, and TSH) and/or alpha-subunit are being increasingly recognized. Because chronic administration of GnRH analogs decreases gonadotropin secretion in normal subjects, we investigated gonadotropin and alpha-subunit responses to chronic GnRH analog administration in five men with glycoprotein hormone-secreting pituitary tumors. Two patients (patients A and B) received the GnRH agonist analog (D-Trp6-Pro9-NEt-LHRH) for 4 weeks as a daily sc dose (8 micrograms/kg.day). In both, secretion of LH and/or alpha-subunit increased markedly. Subsequently, three patients received a higher analog dose (32 micrograms/kg.day) for a longer duration (8 weeks). One patient with a LH- and FSH-secreting tumor (patient C) had a highly significant (P less than 0.001) fall in serum LH and FSH concentrations; however, alpha-subunit secretion increased. During a subsequent study, when this patient received a lower dose (8 micrograms/kg.day) for 8 weeks, gonadotropin suppression also occurred. In two additional patients who received this dose (32 micrograms/kg.day), it had a persistent agonist effect on FSH beta (patient D) and alpha-subunit secretion (patient E). A marked increase in alpha-subunit secretion occurred in all five patients, regardless of whether basal serum alpha-subunit concentrations were elevated. These patients received the GnRH analog at doses 2-8 times greater than those that suppress gonadotropin secretion in normal men. Serum LH and FSH concentrations decreased in only one patient with a gonadotropin-secreting adenoma. The serum LH and FSH responses to acute GnRH stimulation did not predict the gonadotropin responses to chronic GnRH analog administration. Thus, gonadotropin and alpha-subunit production by most pituitary adenomas is augmented during chronic GnRH analog administration, consistent with defective GnRH desensitization in the adenomatous tissue. Despite the heterogeneous gonadotropin responses to the GnRH analog in these patients, serum alpha-subunit levels increased in all patients, indicating dissociation in the secretion of intact gonadotropins and alpha-subunit.     

EFFECTS OF CHANGING GONADOTROPHIN-RELEASING HORMONE PULSE FREQUENCY ON GONADOTROPHIN SECRETION IN MEN

To investigate the effects of alterations in GnRH pulse frequency on gonadotro-phin secretion, we administered low dose GnRH pulses (25 ng/kg) at hourly or 2-hourly frequencies to eight normal men. All subjects received GnRH pulses i.v. every 2 h for 88 h. Following this, exogenous GnRH was discontinued in four normal men (Group A, GnRH withdrawal), and the frequency of GnRH injections was increased to one pulse every hour for 24 h in the other four normal men (Group B, hourly GnRH). Blood samples were obtained every 20 min for LH and FSH and every 12 h for testosterone (T) and oestradiol (E2). Plasma LH increased in all subjects during injection of GnRH pulses every 2h. Withdrawal of GnRH pulses in Group A men was accompanied by a fall in mean LH, reductions in LH pulse amplitude (± SEM: control 6.5±1.0; GnRH withdrawal 4.0 ± 0.5 mlU/ml) and pulse frequency (control 5.5 ± 0.2; GnRH withdrawal 3.5 ± 0.7 pulses/12 h), and an increase in plasma E₂ (control 122 ± 15; GnRH withdrawal 340 ± 37 pmol/l). Gonadotrophin responses to GnRH (25 ng/kg) were normal when tested 32 h after GnRH withdrawal. Injection of hourly GnRH pulses in Group B men was accompanied by a time-dependent change in mean LH, which transiently rose, then fell, and subsequently rose to a plateau during the second 12 h period of hourly GnRH. The final rise in LH was accompanied by an increase in LH frequency to 11.8 ± 0.3 pulses/12 h. These data suggest that: (1) increases in gonadal steroids decrease LH secretion by reducing the amplitude and frequency of endogenous GnRH pulses; and (2) the normal adult male pituitary requires approximately 12 h to initiate a sustained increase in LH secretion in response to a doubling in GnRH pulse frequency.     

Does the type II glucocorticoid receptor mediate cortisol-induced suppression in pituitary responsiveness to gonadotropin-releasing hormone?

Stress-like elevations in plasma cortisol suppress LH pulse amplitude in ovariectomized ewes by inhibiting pituitary responsiveness to GnRH. Here we sought to identify the receptor mediating this effect. In a preliminary experiment GnRH and LH pulses were monitored in ovariectomized ewes treated with cortisol plus spironolactone, which antagonizes the type I mineralocorticoid receptor (MR), or with cortisol plus RU486, which antagonizes both the type II glucocorticoid receptor (GR) and the progesterone receptor (PR). Cortisol alone reduced LH pulse amplitude, but not pulsatile GnRH secretion, indicating that it reduced pituitary responsiveness to endogenous GnRH. RU486, but not spironolactone, reversed this suppression. We next tested whether RU486 reverses the inhibitory effect of cortisol on pituitary responsiveness to exogenous GnRH pulses of fixed amplitude, frequency, and duration. Hourly GnRH pulses were delivered to ovariectomized ewes in which endogenous GnRH pulses were blocked by estradiol during seasonal anestrus. Cortisol alone reduced the amplitude of LH pulses driven by the exogenous GnRH pulses. RU486, but not an antagonist of PR (Organon 31710), prevented this suppression. Thus, the efficacy of RU486 in blocking the suppressive effect of cortisol is attributed to antagonism of GR, not PR. Together, these observations imply that the type II GR mediates cortisol-induced suppression of pituitary responsiveness to GnRH.     

Influence of sodium valproate on late follicular phase pulsatile LH secretion in normal women

OBJECTIVE: The present study was designed to further assess the mechanism of action of GnRH and GnRH analogues. DESIGN AND PATIENTS: Both the Nal-Glu GnRH antagonist and the D-Trp6 GnRH agonist were administered sequentially to nine normal, post-menopausal women. MEASUREMENTS: A baseline study of pulsatile LH, FSH and free alpha-subunit secretion was performed, with sampling every 10 min for 8 h, and then repeated 8 h after a single subcutaneous injection of Nal-Glu GnRH antagonist (5 mg). Sampling was repeated 21 days after the intramuscular injection of a depot preparation of D-Trp6 GnRH (3.75 mg) in the same women. RESULTS: The baseline sampling period showed synchronous pulses of LH and free alpha-subunit. The antagonist Nal-Glu decreased plasma LH (71%) and free alpha-subunit (43%). However, with the single dose of 5 mg, pulsatile LH and free alpha-subunit release were not completely suppressed and remained temporally correlated. The GnRH agonist had a potent inhibitory action on plasma immunoreactive LH (IRMA) (93%). In contrast, it increased the mean plasma levels of free alpha-subunit from 1.66 +/- 0.01 to 5.06 +/- 0.02 micrograms/l (205%). The pulsatile secretory patterns of both LH and free alpha-subunit were abolished by the agonist. Immunoreactive FSH levels were decreased by the antagonist (24%) and suppressed by the agonist (93%). CONCLUSIONS: The pulsatile study confirms the different mechanism of action of GnRH analogues. Following antagonist administration, low amplitude free alpha-subunit pulses persist and are synchronous with residual LH pulses. In contrast, LH and free alpha-subunit are not maintained under agonist treatment. These data provide evidence for the differential regulation of LH and free alpha-subunit by GnRH.     

A preponderance of circulating basic isoforms is associated with decreased plasma half-life and biological to immunological ratio of gonadotropin-releasing hormone-releasable luteinizing hormone in obese men

Hormonal abnormalities of the reproductive axis have been described in obesity. In men, extreme obesity is associated with low serum testosterone (T) and high estrogen [estrone and estradiol (E(2))] levels. As changes in the sex steroid milieu may profoundly affect the carbohydrate heterogeneity and thus some of the biological and physicochemical properties of the LH molecule, we analyzed the relative distribution of LH isoforms circulating under baseline conditions (endogenous GnRH drive) as well as the forms discharged by exogenous GnRH stimulation from putative acutely releasable and reserve pituitary pools in overweight men. Secondarily, we determined the impact of the changes in LH terminal glycosylation on the in vitro bioactivity and endogenous half-life of the gonadotropin. Seven obese subjects with body mass indexes ranging from 35.7-45.5 kg/m(2) and seven normal men with body mass indexes from 22.5-24.2 kg/m(2) underwent blood sampling at 10-min intervals for a total of 10 h before and after the iv administration of 10 and 90 microg GnRH. Basally released and exogenous GnRH-stimulated serum LH isoforms were separated by preparative chromatofocusing and identified by RIA of eluent fractions. Serum pools of successive samples collected across 2-h intervals (five serum pools per subject) containing LH released under baseline and exogenous GnRH-stimulated conditions were tested for bioactivity employing a homologous in vitro bioassay. Mean serum T and E(2) levels were significantly lower and higher, respectively, in the obese men than in the control group [serum T, 13.5 +/- 2.4 vs. 19.4 +/- 1.4 nmol/L (mean +/- SEM; P: = 0.01); serum E(2), 0.184 +/- 0.01 vs. 0.153 +/- 0.01 nmol/L (P: < 0.05)]. Mean baseline serum LH levels were similar in obese subjects and normal controls (13.3 +/- 1.3 and 12.2 +/- 1.2 IU/L). Although multiple parameter deconvolution of the exogenous GnRH-induced LH pulses revealed that the magnitude of the pituitary response in terms of secretory burst mass, secretory amplitude, and half-duration of the LH pulses was similar in obese and control subjects, the apparent endogenous half-life of LH was significantly (P: < 0.05) shorter in the obese group (98 +/- 11 min) than in the normal controls (132 +/- 10 min). Under all conditions studied, the relative abundance of basic isoforms (those with pH >/=7.0) was significantly (P: < 0.05) increased in the obese subjects compared with the controls (percentages of LH immunoactivity recovered at pH >/=7.0: obese subjects, 34-57%; normal controls, 22-46%). The biological to immunological ratio of LH released in baseline and low dose (10 microg) GnRH-stimulated conditions were similar in obese subjects and normal controls, whereas LH released by obese subjects in response to the high (90 microg) GnRH dose exhibited significantly lower ratios than those detected in normal individuals (0.62 +/- 0.07 and 0.45 +/- 0.09 vs. 1.01 +/- 0.10 and 0.81 +/- 0.09 for LH released within 10-120 min and 130-240 min after GnRH administration in obese and controls, respectively; P: < 0.05). Collectively, these results indicate that the altered sex steroid hormone milieu characteristic of extreme obesity provokes a selective increase in the release of less acidic LH isoforms, which may potentially modify the intensity and duration of the blood LH signal delivered to the gonad. Altered glycosylation of LH may therefore represent an additional mechanism modulating the hypogonadal state prevailing in morbid obesity.     

Gonadotropin-releasing hormone pulsatile administration restores luteinizing hormone pulsatility and normal testosterone levels in males with hyperprolactinemia

Hyperprolactinemia in men is frequently associated with hypogonadism. Normalization of serum PRL levels is generally associated with an increase in serum testosterone (T) to normal. To determine the mechanism of the inhibitory effect of hyperprolactinemia on the hypothalamic-pituitary-gonadal axis, we studied the effect of intermittent pulsatile GnRH administration on LH pulsatility and T levels in four men with prolactinomas. All patients had high PRL values (100-3000 ng/ml), low LH (mean +/- SEM, 2.2 +/- 0.1 mIU/ml), and low T values (2.3 +/- 0.3 ng/ml), with no other apparent abnormality of pituitary function. GnRH was administered iv using a pump delivering a bolus dose of 10 micrograms every 90 min for 12 days. No LH pulses were detected before treatment. Pulsatile GnRH administration resulted in a significant increase in basal LH levels (6.7 +/- 0.6 mIU/ml; P less than 0.001) and restored LH pulsatility. In addition, T levels increased significantly to normal values in all patients (7.8 +/- 0.4 ng/ml; P less than 0.001) and were normal or supranormal as long as the pump was in use, although PRL levels remained elevated. These data, therefore, suggest that hyperprolactinemia produces hypogonadism primarily by interfering with pulsatile GnRH release.