The relative role of gonadal sex steroids and gonadotropin-releasing hormone pulse frequency in the regulation of follicle-stimulating hormone secretion in men

OBJECTIVE: Our objective was to determine the importance of testosterone (T), estradiol (E(2)), and GnRH pulse frequency to FSH regulation in men. DESIGN: This was a prospective study with four arms. SETTING: The study was performed at the General Clinical Research Center. PATIENTS OR OTHER PARTICIPANTS: There were 20 normal (NL) men and 15 men with idiopathic hypogonadotropic hypogonadism (IHH) who completed the study. Intervention: Medical castration and inhibition of aromatase were achieved using ketoconazole x 7 d with: 1) no sex steroid addback, 2) T addback starting on d 4, and 3) E(2) addback starting on d 4. IHH men in these arms received GnRH every 120 min. In a further six IHH men receiving ketoconazole with no addback, GnRH frequency was increased to 35 min for d 4-7. Blood was drawn every 10 min x 12 h at baseline, overnight on d 3-4 and 6-7. MAIN OUTCOME MEASURES: Mean FSH was calculated from the pool of each frequent sampling study. RESULTS: In NL men FSH levels increased from 5.1 +/- 0.7 to 8.7 +/- 1.3 and 9.7 +/- 1.5 IU/liter (P < 0.0001). T caused no suppression of FSH. E(2) reduced FSH from 12.4 +/- 1.8 to 9.3 +/- 1.3 IU/liter (P < 0.05). In IHH men on GnRH every 120 min, FSH levels went from 6.0 +/- 1.6 to 9.0 +/- 3.0 and 11.9 +/- 4.3 (P = 0.07). T caused no suppression of FSH. E(2) decreased FSH such that levels on d 6-7 were similar to baseline. Increasing GnRH frequency to 35 min had no impact on FSH. CONCLUSIONS: The sex steroid component of FSH negative feedback in men is mediated by E(2). Increasing GnRH frequency to castrate levels has no impact on FSH in the absence of sex steroids. When inhibin B levels are NL, sex steroids exert a modest effect on FSH.     

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.     

Pulsatile gonadotropin secretion after discontinuation of long term gonadotropin-releasing hormone (GnRH) administration in a subset of GnRH-deficient men

Normal pituitary and gonadal function can be maintained with long term pulsatile GnRH administration in men with idiopathic hypogonadotropic hypogonadism (IHH), and both pituitary and gonadal priming occur during the process of GnRH-induced sexual maturation. Still, the long term effects of discontinuing GnRH therapy in IHH men have not been examined. Therefore, we evaluated the patterns of gonadotropin and alpha-subunit secretion before and after a prolonged period of pulsatile GnRH administration in 10 IHH men. Before exogenous GnRH stimulation, no patient had any detectable LH pulsations. In 6 of these men, who were typical of most of our IHH patients (group I), no LH pulsations were detectable after cessation of GnRH administration. However, in the other 4 men (group II), LH pulsations were easily detectable despite cessation of exogenous GnRH stimulation, and the amplitude (9.3 +/- 3.5 IU/L) and frequency (13.8 +/- 1.7 pulses/day) of these LH pulses were similar to those in 20 normal men (10.6 +/- 0.7 IU/L and 11.0 +/- 0.7 pulses/day). Three of these 4 men in group II maintained normal serum testosterone levels after discontinuation of GnRH delivery. To determine if there were any characteristics that might be useful in predicting which IHH men could maintain normal pituitary-gonadal function after long term GnRH administration, we evaluated various clinical and hormonal parameters at the time of initial presentation. Mean alpha-subunit levels (P less than 0.01) and alpha-subunit pulse amplitude (P less than 0.02) were significantly higher in the group II than the group I men, suggesting that the group II patients had partial, rather than complete, deficiency of endogenous GnRH secretion. None of the other parameters that were assessed distinguished the two groups. We conclude that gonadotropin and sex steroid levels return to their pretreatment state in the majority of IHH men when long term GnRH administration is discontinued. Normal pituitary-gonadal function can be maintained after discontinuation of long term GnRH administration in a rare subset of IHH men who present with higher levels of alpha-subunit. We hypothesize that these latter IHH men have an incomplete GnRH deficiency and that long term exogenous GnRH administration induces pituitary and gonadal priming, which subsequently enables them to sustain normal pituitary and gonadal function in response to their own enfeebled GnRH secretion.     

Hypothalamic regulation of cyclic ovulation: evidence that the increase in gonadotropin-releasing hormone pulse frequency during the follicular phase reflects the gradual loss of the restraining effects of progesterone

The luteal-follicular transition is characterized by decreasing plasma levels of E(2), progesterone (P), and inhibin A, with concomitant increases in FSH and LH levels. LH (and by inference GnRH) pulse frequency increases from 1 pulse every 3-4 h during the luteal phase to approximately 1 pulse/h at the midcycle LH surge. To examine the regulation of GnRH pulse frequency, we gave 10 normally cycling women transdermal E(2) and oral P to produce midluteal levels [364 +/- 65.0 pmol/liter (99 +/- 18 pg/ml) and 29.7 +/- 6.8 nmol/liter (9.3 +/- 2.1 ng/ml), respectively] for 10 d after the LH surge (d 0). P was then discontinued, and E(2) was given alone for 3 additional wk. Pulsatile LH secretion and follicular size were assessed on d 10, 17, 24, and 31. Results are presented as the mean +/- SEM. LH pulse frequency was 3.1 +/- 0.5 pulses/12 h after 10 d of E(2) and P, and remained low on d 17 when P had fallen below 1.6 nmol/liter (<0.5 ng/ml). In the continued presence of midluteal levels of E(2) [ approximately 360 pmol/liter (100 pg/ml)], LH pulse frequency increased on d 24 and 31 to 5.5 +/- 0.9 and 5.8 +/- 0.5 pulses/12 h, respectively, whereas pulse amplitude remained unchanged. FSH increased 2-fold, but follicular size did not change. These results are consistent with E(2) potentiating the effects of low concentrations of P on the GnRH pulse generator for at least 7 d, after which pulse frequency increases despite maintenance of E(2) levels. This supports the hypothesis that the increasing GnRH pulse frequency throughout the follicular phase reflects the gradual loss of the inhibitory actions of low concentrations of P.     

Poorly controlled type I diabetes mellitus in young men selectively suppresses luteinizing hormone secretory burst mass

Alterations in the reproductive axis function are present to a variable extent in patients with type 1 diabetes mellitus (IDDM). Results from studies in IDDM men have yielded discrepant findings, which may reflect nonuniform patient selection criteria, age, diabetic status, duration of the disease and differences in sampling protocols. To more clearly define the impact of early diabetic alterations in the male reproductive axis, we applied a combined strategy of patient selection restricted to young men with relatively short duration of IDDM, dual control groups, multiparameter deconvolution analysis to assess LH secretory activity, and assessment of time-dependent changes in human chorionic gonadotropin (hCG)-stimulated serum testosterone concentrations. Three groups of subjects were studied: 11 young men with poorly controlled IDDM, 9 well controlled diabetics, and 9 healthy men. All volunteers underwent blood sampling at 10-min intervals before and after 2 consecutive iv pulses of 10 micro g GnRH. On a separate day, 40 IU/kg hCG were given im, and blood samples were collected before hCG administration, every 60 min thereafter for 6 h, and then 24, 48, and 72 h after the injection. Mean serum LH concentrations across the basal 6-h sampling period were significantly (P < 0.05) decreased in men with poorly controlled IDDM (11 +/- 1.6 IU/liter) compared with those in well controlled diabetics (19 +/- 1.8 IU/liter) and healthy controls (19 +/- 1.5 IU/liter). Multiple parameter deconvolution analysis revealed a 50% reduction in the mass of LH secreted per burst and the pulsatile LH secretion rate in poorly controlled IDDM (mass of LH secreted/burst, 7 +/- 1.1 vs. 12 +/- 2.1 and 13 +/- 1.5 IU/liter; LH secretion rate, 47 +/- 6.3 vs. 78 +/- 10 and 87 +/- 11 IU/liter.6 h; poorly controlled vs. well controlled IDDM and healthy controls, respectively; P < 0.05 for both parameters). Uncontrolled IDDM patients had significantly (P < 0.05) lower integrated serum LH concentrations after the first and second GnRH pulses (first GnRH pulse, 4460 +/- 770 vs. 7250 +/- 1200 and 5120 +/- 910 IU/liter; second pulse, 4700 +/- 615 vs. 7640 +/- 881 and 7100 +/- 1230 IU/liter; poorly controlled vs. well controlled IDDM and healthy men, respectively) and markedly attenuated LH secretory burst mass after the second GnRH stimulus (49 +/- 8.8 vs. 90 +/- 13 and 83 +/- 19 IU/liter; poorly controlled vs. well controlled IDDM and healthy controls, respectively). The biological to immunological ratio of LH released in baseline conditions was higher in uncontrolled IDDM patients (0.81 +/- 0.10) than in controlled IDDM (0.37 +/- 0.08) and healthy controls (0.48 +/- 0.06; P < 0.01), whereas LH released in response to exogenous GnRH exhibited comparable ratios among the three study cohorts. Baseline serum testosterone levels as well as absolute and incremental responses to exogenous hCG did not differ by degree of metabolic control. Collectively, these results indicate that the function of the hypothalamic-gonadotrope axis is compromised in young men with poorly controlled IDDM, such that the amplitude of spontaneous pulsatile and exogenous GnRH-stimulated LH secretion is attenuated. This central hypogonadotropism is paradoxically associated with the presence in the circulation of gonadotropin molecules with enriched biological activity, which is evidently sufficient to temporarily maintain normal total testosterone concentrations in the earlier stages of IDDM.     

Predictors of outcome of long-term GnRH therapy in men with idiopathic hypogonadotropic hypogonadism

GnRH treatment is successful in inducing virilization and spermatogenesis in men with idiopathic hypogonadotropic hypogonadism (IHH). However, a small subset of IHH men, poorly characterized to date, fail to reach a normal testicular volume (TV) and produce sperm on this therapy. To determine predictors of outcome in terms of TV and sperm count, we studied 76 IHH men (38% with anosmia) undergoing GnRH therapy for 12-24 months. The population was stratified according to the baseline degree of prior pubertal development: absent (group 1, n = 52), partial (group 2, n = 18), or complete (adult onset HH; group 3, n = 6). Cryptorchidism was recorded in 40% of group 1, 5% of group 2, and none in group 3. Pulsatile GnRH therapy was initiated at 5-25 ng/kg per pulse sc and titrated to attain normal adult male testosterone (T) levels. LH, FSH, T, and inhibin B (I(B)) levels were measured serially, and maximum sperm count was recorded. A longitudinal mixed effects model was used to determine predictors of final TV. LH (97%) and T (93%) levels were normalized in the majority of IHH men. Groups 2 and 3 achieved a normal adult testicular size (92%), FSH (96%), I(B) levels (93%), and sperm in their ejaculate (100%). However, given their prior complete puberty and thus primed gonadotropes and testes, group 3 responded faster, normalizing androgen production by 2 months and completing spermatogenesis by 6 months. In contrast, group 1 failed to normalize TV (11 +/- 0.4 ml) and I(B) levels (92 +/- 6 pg/ml) by 24 months, despite normalization of their FSH levels (11 +/- 2 IU/liter). Similarly, sperm counts of group 1 plateaued well below the normal range (median of 3 x 10(6)/ml) with 18% remaining azoospermic. The independent predictors of outcome of long-term GnRH therapy were: 1) the presence of some prior pubertal development (positive predictor; group effect (beta) = 4.3; P = 0.003); 2) a baseline I(B) less than 60 pg/ml (negative predictor; beta = -3.7; P = 0.009); and 3) prior cryptorchidism (negative predictor; beta = -1.8; P = 0.05). Notably, anosmia was not an independent predictor of outcome when adjusted for other baseline variables. Our conclusions are: 1) pulsatile GnRH therapy in IHH men is very successful in inducing androgen production and spermatogenesis; 2) normalization of the LH-Leydig cell-T axis is achieved more uniformly than the FSH-Sertoli cell-I(B) axis during GnRH therapy; and 3) favorable predictors for achieving an adult testicular size and consequently optimizing spermatogenesis are prior history of sexual maturation, a baseline I(B) greater than 60 pg/ml, and absence of cryptorchidism.     

Acute effects of testosterone infusion and naloxone on luteinizing hormone secretion in normal men.

To evaluate the role of endogenous opioid pathways in the acute suppression of LH secretion by testosterone (T) infusion in men, we studied eight normal healthy volunteers who received a saline infusion, followed 1 week later by a T infusion (960 nmol/h) starting at 1000 h and lasting for 33 h. After 2 h of infusion (both saline and T), four iv boluses of saline were given hourly, and after 26 h of infusion, four hourly iv boluses of naloxone were given. Blood was obtained every 15 min for LH and every 30 min for T. T infusion increased the mean plasma T concentration 2.1-fold (18.7 +/- 2.1 to 39.5 +/- 3.5 nmol/L, saline vs. T infusion, P < 0.01). The mean plasma LH concentration was 7.9 +/- 0.5 IU/L during the saline control study and was decreased to 6.9 +/- 0.6 IU/L by the infusion of T (P < 0.05). LH pulse frequency was similar during both saline and T infusions (0.48 +/- 0.02 vs. 0.43 +/- 0.04 pulses/man.h, saline vs. T infusion). The mean LH pulse amplitude decreased from 4.3 +/- 0.4 IU/L during saline infusion to 3.3 +/- 0.2 IU/L during T infusion (P < 0.05). The administration of naloxone increased the mean plasma LH concentration significantly during saline infusion (7.6 +/- 0.4 to 10.0 +/- 0.9 IU/L, saline vs. naloxone boluses, P < 0.01), but not during T infusion (6.9 +/- 0.6 vs. 7.3 +/- 0.6 IU/L). LH pulse frequency increased significantly after the administration of naloxone during both saline and T infusions (0.54 +/- 0.04 to 0.71 +/- 0.08 pulses/man.h, saline vs. naloxone boluses during saline infusion, and 0.46 +/- 0.08 to 0.60 +/- 0.07 pulses/man.h during T infusion; P < 0.05). LH pulse amplitude was suppressed by T infusion, but administration of naloxone did not reverse this suppression. The mean amplitude of the LH response to exogenous GnRH (250 ng/kg) was decreased by T infusion from 48 +/- 13.5 to 31.2 +/- 8.5 IU/L (P < 0.01). Therefore, in men, the administration of naloxone increases LH pulse frequency during both saline and T infusions, but the acute suppression of LH pulse amplitude seen with T infusion was not reversed by naloxone. This pattern contrasts sharply with the effects of T infusion in pubertal boys, as elucidated by our earlier studies. The negative feedback effects of T on LH secretion are primarily hypothalamic in early pubertal boys and change to pituitary suppression in men.     

Importance of inhibin B in the regulation of FSH secretion in the human male.

Regulation of FSH secretion in the male involves a complex balance between stimulation by GnRH from the hypothalamus, inhibitory feedback by sex steroids (T and E2) and inhibin B (Inh B) from the gonads, and autocrine/paracrine modulation by activin and follistatin within the pituitary. The aim of the present study was to delineate the feedback control of FSH in the human male with specific reference to the relative roles of sex steroids vs. Inh B. Two experimental human models were used: 1) normal (NL) men subjected to acute sex steroid withdrawal (-T, -E2, + Inh B), and 2) functional castrate males (-T, -E2, -Inh B). Nine NL men (age range, 25-45 yr) and three castrate males (age range, 23-47 yr) were studied. The NL men underwent acute sex steroid suppression using high dose ketoconazole (1-g loading dose, followed by 400 mg, orally, four times daily for 150 h). Gonadotropin secretion was characterized by frequent blood sampling every 10 min for 12 h at baseline and on d 3 and 6 of sex steroid ablation. In the three castrate subjects, blood sampling was performed every 5 min for 24 h 8 wk after discontinuing androgen replacement therapy. In the NL men, treatment with ketoconazole resulted in a decline to castrate levels in T (451 +/- 20 to 38 +/- 7 ng/dl; P < 0.0005) and E2 (39 +/- 4 to 15 +/- 2 pg/ml; P < 0.005) and a modest, but significant, decline in Inh B levels, which remained within the normal range (183 +/- 19 to 136 +/- 13 pg/ml; P < 0.005). This suppression of sex steroids was associated with a more marked increase in mean LH (9.5 +/- 0.9 to 24.9 +/- 2.0 IU/liter; P < 0.0001) than FSH levels (5.1 +/- 0.7 to 10.0 +/- 1.5 IU/liter; P < 0.005), with the latter not exceeding the normal adult male range. The castrate subjects had a mean T level of 66 +/- 8 ng/dl, an E2 level of 20 +/- 1 pg/ml, and undetectable Inh B levels. Despite a similar sex steroid milieu, the mean FSH levels observed in NL men after acute sex steroid ablation were approximately 6-fold lower than those seen in the castrate subjects (10.0 +/- 1.5 vs. 59.5 +/- 17.7 IU/liter; P < 0.0005). In contrast, mean LH levels in the NL men were less than 3-fold lower than those in castrate subjects (24.9 +/- 2.0 vs. 66.8 +/- 20.1 IU/liter; P < 0.005). From this human model of acute sex steroid withdrawal, we conclude that Inh B is likely to be the major feedback regulator of FSH secretion in the human male.     

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.     

Muting of androgen negative feedback unveils impoverished gonadotropin-releasing hormone/luteinizing hormone secretory reactivity in healthy older men

Plasma bioavailable testosterone concentrations decline in healthy older men without a uniformly commensurate rise in serum LH concentrations, which disparity is consistent with a hypothesis of relative hypogonadotropism. Likewise, preserved gonadotrope responsiveness to exogenous GnRH stimulation, despite an attenuated amplitude of endogenous LH pulses, points to reduced hypothalamic GnRH feedforward signaling in aging males. To appraise GnRH/LH secretory reserve more directly in older men, we have compared daily LH secretion, driven by profound short-term blockade of androgen biosynthesis by oral ketoconazole administration, in nine young (ages, 18-32 yr) and seven older (ages, 60-73 yr) volunteers. The ability to unleash endogenous GnRH-driven LH secretion in response to acute testosterone withdrawal was quantitated by sampling blood every 10 min, for 24 h, followed by high-precision immunoradiometric assay. The resultant serum LH concentration profiles were analyzed by: 1) model-free discrete peak detection (Cluster) analysis; 2) the approximate entropy statistic to quantitate pattern regularity; and 3) 24-h rhythmic (cosinor) analysis. At baseline, mean and integrated (24-h) serum LH concentrations were similar in both age cohorts. However, Cluster analysis established an elevated LH peak frequency [18 +/- 0.86 (older) vs. 13 +/- 1.3 pulses/24 h (young), P = 0.0028] and a reduced incremental LH pulse area [37 +/- 6.9 (older) vs. 106 +/- 20 (young) IU/L x min, P = 0.016] in older men. Approximate entropy calculations also revealed more irregular LH release patterns in older men before intervention (P = 0.00089). Feedback stress, achieved by ketoconazole-induced androgen deprivation, unmasked further neuroregulatory defects in older volunteers, who failed to equivalently increase the: 1) mean (24-h) serum LH concentration [i.e. to 5.0 +/- 0.99 (older men) vs. 9.0 +/- 1.1 (young) IU/L, P = 0.000071]; 2) maximal LH peak height (to 6.1 +/- 1.1 vs. 10.4 +/- 1.2 IU/L, P = 0.00043); 3) incremental LH pulse area (to 41 +/- 8.8 vs. 87 +/- 20 IU/L x min, P = 0.016); 4) interpeak nadir serum LH concentration (to 4.0 +/- 0.77 vs. 7.9 +/- 1.0 IU/L, P < 10(-6)); 5) the quantitable irregularity of LH release (P = 0.00089); and 6) the mesor of 24-h rhythmic LH secretion (P = 0.000062). In summary, experimental imposition of a novel hypoandrogenemic open-loop feedback stressor, for 48 h, to heighten hypothalamic GnRH feedforward drive, unveils impoverished augmentation of LH pulse mass, impaired orderliness of LH release, and diminished 24-h rhythmic LH secretion in older men. The foregoing trilogy of neuroregulatory defects identifies unequivocally attenuated hypothalamo-pituitary reactivity to muting of androgen negative feedback in the aging male.     

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.     

Middle-aged men secrete less testosterone at night than young healthy men

Aging men largely maintain their testicular androgen production. Cross-sectional studies have demonstrated that after the age of 40 yr a 0.2-2% annual decline is observed in morning total testosterone. In elderly males, the coordinate release of LH and testosterone became asynchronous despite normal serum levels of these hormones. The aim of this study was to test the reproductive hormone rhythm at night in middle-aged men. We studied seven healthy middle-aged (46.6 +/- 6.7 yr) and six healthy young (23.9 +/- 2.4 yr) men by determining their serum levels of LH and testosterone levels every 15 min from 1900-0700 h with simultaneous sleep recordings. The nocturnal rise in testosterone occurred earlier in young men (2235 +/- 0022 h) and at 2331 +/- 0057 h in middle-aged men (P < 0.04). In young men, the mean testosterone level at night (5.0 +/- 1.3 ng/ml; 17.4 +/- 4.4 nmol/liter) and the integrated nocturnal secretion [area under the curve (AUC); 60.6 +/- 8.9 ng/ml.h; 210 +/- 31 nmol/liter.h] were significantly higher compared with the values (3.6 +/- 1.1 and 31.1 +/- 7.2 ng/ml.h; 12.6 +/- 3.8 and 108 +/- 24.8 nmol/liter.h, respectively) observed in middle-aged men (P < 0.04 and P < 0.01, respectively). The mean (3.5 +/- 0.3 mIU/ml; 3.5 +/- 0.3 IU/liter) and AUC (43.4 +/- 8.3 mIU/ml.h; 43.4 +/- 8.3 IU/liter.h) LH values in middle-aged men were significantly higher than the values observed in young men (2.0 +/- 0.7 and 30.8 +/- 6.1 mIU/ml.h; 2.0 +/- 0.7 and 30.8 +/- 6.1 IU/liter.h; P < 0.05 and P < 0.01, respectively). Young men had significantly more testosterone pulses at night (6.7 +/- 1.6/12 vs. 3.8 +/- 1.1/12 h in middle-aged men; P < 0.005) of shorter interpulse interval (88.5 +/- 23.6 vs. 137.4 +/- 46.4 min; P < 0.02). LH pulse characteristics and sleep quality were similar in both groups. However, the first rapid eye movement (REM) sleep episode occurred earlier in middle-aged men (2303 +/- 0034 h) vs. young men (0010 +/- 0054 h; P < 0.04). As a consequence, the testosterone rise antedated the first REM episode by 90 min in young men. The link between testosterone rise and REM sleep episode was not observed in middle-aged men. Linear regression analysis revealed that the LH AUC was significantly related to age (P < 0.02). Analysis of covariance revealed that the two groups differed significantly in testosterone AUC (P < 0.04). Comparison of LH and testosterone concentrations showed significant and positive cross-correlations between LH and testosterone only in young men, with the testosterone rise lagging 60 min after the rise in LH. Our findings suggest that in middle-aged men, less pulsatile testosterone and more LH are secreted at night than in young men, with disruption of the association between testosterone rhythm and REM sleep. The decline in nocturnal testosterone secretion appears to involve a combination of testicular and pituitary hypogonadism.     

Kallmann's syndrome: a comparison of the reproductive phenotypes in men carrying KAL1 and FGFR1/KAL2 mutations

CONTEXT: Kallmann's syndrome (KS) is a genetically heterogeneous disorder consisting of congenital hypogonadotropic hypogonadism (CHH) with anosmia or hyposmia. OBJECTIVE: Our objective was to compare the reproductive phenotypes of men harboring KAL1 and FGFR1/KAL2 mutations. DESIGN AND PATIENTS: We studied the endocrine features reflecting gonadotropic-testicular axis function in 39 men; 21 had mutations in KAL1 and 18 in FGFR1/KAL2, but none had additional mutations in PROK-2 or PROKR-2 genes. RESULTS: Puberty failed to occur in the patients with KAL1 mutations, all of whom had complete CHH. Three patients with FGFR1/KAL2 mutations had normal puberty, were eugonadal, and had normal testosterone and gonadotropin levels. Cryptorchidism was more frequent (14 of 21 vs. 3 of 15; P<00.1) and testicular volume (2.4+/-1.1 vs. 5.4+/-2.4 ml; P<0.001) was smaller in CHH subjects with KAL1 mutations than in subjects with FGFR1/KAL2 mutations. The mean basal plasma FSH level (0.72+/-0.47 vs. 1.48+/-0.62 IU/liter; P<0.05), serum inhibin B level (19.3+/-10.6 vs. 39.5+/-19.3 pg/ml; P<0.005), basal LH plasma level (0.57+/-0.54 vs. 1.0+/-0.6 IU/liter; P<0.01), and GnRH-stimulated LH plasma level (1.2+/-1.0 vs. 4.1+/-3.5 IU/liter; P<0.01) were significantly lower in the subjects with KAL1 mutations. LH pulsatility was studied in 13 CHH subjects with KAL1 mutations and seven subjects with FGFR1/KAL2 mutations; LH secretion was nonpulsatile in all the subjects, but mean LH levels were lower in those with KAL1 mutations. CONCLUSION: KAL1 mutations result in a more severe reproductive phenotype than FGFR1/KAL2 mutations. The latter are associated with a broader spectrum of pubertal development and with less severe impairment of gonadotropin secretion.     

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.     

Effect of improved assay sensitivity on luteinizing hormone pulse detection after gonadotropin-releasing hormone antagonist treatment in man.

When serum levels of a hormone are at or below the detection limit of the measurement system, accurate characterization and quantitation of pulsatile hormone secretion may be difficult. This point is well illustrated in this study, which used two immunoassays with markedly different assay sensitivities to quantitate pulsatile LH secretion in men in whom serum LH levels had been suppressed by the administration of a GnRH antagonist. Five normal men received 5 mg Nal-Glu, sc, daily for 21 days. Blood was drawn at 10-min intervals over 8 h on days 0 and 21. Samples were assayed in triplicate in both a conventional LH RIA with a sensitivity of 0.6-1.0 IU/L and a two-site-directed ultrasensitive immunofluorometric assay (IFMA) with assay sensitivity ranging from 0.05-0.125 IU/L. LH pulses were analyzed by Cluster analysis (C) and were corroborated by the Detect algorithm (D). Nal-Glu suppressed LH (C) pulse amplitude from 4.0 +/- 0.7 to 0.40 +/- 0.03 IU/L by LH RIA and from 7.8 +/- 2.1 to 0.21 +/- 0.04 IU/L by LH IFMA. An apparent reduction in LH pulse number was observed in the RIA data on day 21 by both pulse detection methods [4.2 +/- 0.4 vs 2.4 +/- 0.2/8 h on days 0 and 21 by C (P < 0.005); 5.8 +/- 0.8 vs. 2.8 +/- 0.7 by D (P < 0.05)]. However, the IFMA measurements in the same samples using the more sensitive LH IFMA showed no difference in pulse number between days 0 and 21. The RIA data correlated well with the IFMA data, with a concordance coefficient ranging from 0.66-0.9. In summary, the Nal-Glu GnRH antagonist markedly decreases LH pulse amplitude, but not pulse frequency. These observations are consistent with competitive inhibition of GnRH action at the pituitary site by the Nal-Glu antagonist; they indicate that assay characteristics can significantly affect quantitation of hormone pulse pattern and underscore the need for ultrasensitive LH assays for accurate assessment of LH pulse characteristics when LH levels are low or suppressed.     

Increasing insulin resistance is associated with a decrease in Leydig cell testosterone secretion in men

Insulin resistance is associated with low testosterone (T) levels in men, the mechanism of which is unclear. Thus, the aim of this study was to evaluate the hypothalamic-pituitary-gonadal axis in men with a spectrum of insulin sensitivity. Twenty-one men (aged 25-65 yr) had a glucose tolerance test and assessment of insulin sensitivity using a hyperinsulinemic-euglycemic clamp. Insulin sensitivity, expressed as the M value (milligrams per kilograms(-1) per minute(-1)), was calculated from the glucose disposal rate during the final 30 min of the clamp. Eighteen subjects had blood sampling every 10 min for 12 h to assess LH pulsatility. Hypogonadism was then induced with a GnRH antagonist, followed by sequential stimulation testing with GnRH (750 ng/kg, iv) and human chorionic gonadotropin (hCG; 1000 IU, im) to assess pituitary and testicular responsiveness, respectively. Nine subjects had normal glucose tolerance, nine had impaired glucose tolerance, and three had diabetes mellitus. There was a positive relationship between M and T levels (r = 0.46; P < 0.05). No relationship was seen between M and parameters of LH secretion, including mean LH levels, LH pulse amplitude, LH pulse frequency, and LH response to exogenous GnRH administration. In contrast, a strong correlation was observed between M and the T response to hCG (r = 0.73; P < 0.005). Baseline T levels correlated with the increase in T after hCG administration (r = 0.47; P < 0.05). During the clamp, T levels increased from a baseline level of 367 +/- 30 to 419 +/- 38 ng/dl during the last 30 min (P < 0.05). From these data we conclude that insulin resistance is associated with a decrease in Leydig cell T secretion in men. Additional studies are required to determine the mechanism of this effect.     

Cosecretion of estrogen and inhibin B by a feminizing adrenocortical adenoma: impact on gonadotropin secretion

We describe the first reported case of a feminizing adrenocortical adenoma cosecreting estrogens and inhibin B. A 39-yr-old man, with no previous history of disease and free of treatment, complained of gynecomastia without any clinical abnormality. Plasma E2 and T were 496 pmol/liter and 8.7 nmol/liter, respectively. Testicular echography was normal, and abdominal computed tomography scan showed a 28-mm right adrenal tumor. hCG (5000 IU, im) induced a rise in plasma T levels (20.7 nmol/liter) without any change in plasma E2 levels. Basal plasma LH and FSH levels were undetectable. GnRH (100 microg, i.v.) induced an increase in plasma LH levels without a change in plasma FSH levels. The mean plasma inhibin B level was 330 +/- 45 pg/ml (normal range, 94-327). Pulsatile GnRH administration (20 microg/pulse every 90 min for 3 d) stimulated LH secretion, whereas FSH secretion remained blunted. The patient underwent surgery to remove a 12-g adrenal adenoma. Six months later, plasma E2 and T levels were normalized. LH showed a spontaneous pulsatile pattern, and the mean plasma FSH level was 4.8 U/liter. The secretion of both gonadotropins was stimulated during a pulsatile GnRH administration performed in the same manner as before surgery. The mean plasma inhibin B level was 210 +/- 25 pg/ml. Immunohistochemical studies revealed the presence of aromatase in clusters of tumor cells. Incubation of tumor sections with anti-beta(B)-inhibin antibody revealed intense staining in groups of cells that were also labeled with anti-alpha-inhibin antibody. These data show that the tumor cosecreted E2 and inhibin B, which were both responsible for inhibition of gonadotropin secretion. Tumor secretions appeared to be much more potent in suppressing FSH than LH levels.     

A single luteinizing hormone determination 2 hours after depot leuprolide is useful for therapy monitoring of gonadotropin-dependent precocious puberty in girls

Long-acting GnRH analogs represent the standard treatment for gonadotropin-dependent precocious puberty. The aim of this study was to determine the hormonal parameters for monitoring the adequacy of depot leuprolide acetate treatment in girls with clinical and hormonal diagnosis of gonadotropin-dependent precocious puberty. Eighteen girls were treated monthly with 3.75 mg depot leuprolide acetate. Adequate hypothalamic-pituitary-gonadal axis suppression during treatment was achieved in 16 of the 18 girls according to the clinical parameters and prepubertal LH levels. In these 16 well-controlled girls, the LH peak after a classical GnRH test was compared with a single LH measurement obtained 2 h after depot leuprolide acetate administration before and during GnRH analog treatment. Before therapy, the mean +/- sd LH peak after a classical GnRH test was 18.4 +/- 11.2 IU/liter (ranging from 7-41.5 IU/liter), and it was 22.6 +/- 8.3 IU/liter 2 h after the first depot leuprolide dose (ranging from 10-35.3 IU/liter). During therapy, the mean +/- sd of LH peak after classical GnRH test was 1.4 +/- 0.6 IU/liter (ranging from <0.6 to 2.3 IU/liter), and it was 2.7 +/- 1.9 IU/liter (ranging from 0.7-6.6 IU/liter) 2 h after depot leuprolide. The LH peak after a classical GnRH test and that 2 h after depot leuprolide administration correlate significantly before and during treatment. In conclusion, we established the LH cut-off values for an adequate depot leuprolide therapy as an LH peak below 2.3 IU/liter after a classical GnRH test or below 6.6 IU/liter 2 h after depot leuprolide. The latter measurement may replace the classical GnRH test as a reliable and convenient tool for monitoring therapy in female gonadotropin-dependent precocious puberty.     

Testosterone differentially modulates gonadotropin subunit messenger ribonucleic acid responses to gonadotropin-releasing hormone pulse amplitude.

Testosterone (T) inhibits GnRH secretion and can also modulate the effects of GnRH on gonadotropin synthesis and secretion. To assess the effect of T on GnRH stimulation of alpha, LH beta, and FSH beta mRNA expression, we replaced T at three levels to reproduce low (1.5 +/- 0.5 ng/ml), medium (3.5 +/- 0.3 ng/ml), and high (6.2 +/- 0.6 ng/ml) physiological plasma concentrations. Additionally, as peripheral conversion to dihydrotestosterone (DHT) or estradiol (E2) may mediate T action, the effects of GnRH pulses in the presence of DHT and E2 were also studied. Male rats were castrated, and steroids were replaced via implants containing either T (three doses) or DHT or E2 (two doses each). GnRH pulses (10-250 ng/pulse) were administered iv at 30-min intervals for 48 h. Pituitary subunit mRNA concentrations, gonadotropin content, and LH and FSH secretion were determined. The patterns of alpha, LH beta, and FSH beta mRNA responses to increasing GnRH pulse amplitude were similar at all concentrations of plasma T. Alpha mRNA concentrations were increased 2- to 4-fold by GnRH pulses. At the same plasma T concentration, all doses of GnRH produced similar increases in alpha mRNA, but the response tended to be lower at the higher (6.2 ng/ml) levels of T. LH beta mRNA showed a clear dependence on GnRH pulse amplitude, with the maximum responses (2- to 3-fold) occurring after 10- to 25-ng GnRH pulses. At the higher (3.5 and 6.2 ng/ml) T concentrations, the dose-response curve was shifted to the left. The lowest GnRH pulse dose (10 ng) produced maximum responses, and LH beta mRNA increments in response to the higher GnRH doses were suppressed. FSH beta mRNA concentrations were increased by T in saline-pulsed controls. FSH beta mRNA responses were similar (2- to 3-fold) after all GnRH doses and at all concentrations of T. Increasing GnRH pulse doses reduced the pituitary content of both LH and FSH at all levels of T. Acute LH secretion was maximal after 10- and 25-ng pulses of GnRH when plasma T was low, but increased progressively with GnRH dose at the highest plasma T concentrations. Plasma FSH did not show any differential responsiveness to GnRH pulse dose or to increasing plasma T. Thus, LH synthesis and secretion are affected more than those of FSH by changing plasma concentrations of T. T may modulate posttranslational events in LH secretion. The higher GnRH doses effected LH release without increasing LH beta mRNA in the presence of higher physiological concentrations of T.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.