Decrease in hypothalamic Kiss1 and Kiss1r expression: a potential mechanism for fasting-induced suppression of the HPG axis in the adult male rhesus monkey (Macaca mulatta).

Fasting suppresses functioning of the hypothalamic-pituitary-gonadal (HPG) axis by mechanisms that are incompletely understood. In 2003, hypothalamic kisspeptin-Kiss1r signaling was discovered to play a significant role in regulating the HPG axis. We have recently shown that in adult male macaques, short-term fasting attenuates the response of the HPG axis to an exogenous kisspeptin challenge. In the present study, we explored the mechanism underlying this attenuated response by examining the modulation of the hypothalamic expression of KISS1 and KISS1R under short-term fasting and normal feeding conditions in the adult male macaques. Hypothalamic mRNA was extracted from normal fed (n=3) and 48-h fasted (n=3) monkeys. KISS1, KISS1R, and GNRH1 mRNA were quantified by reverse transcription followed by real-time polymerase chain reaction. In addition, blood samples were collected for measurement of plasma concentrations of glucose, cortisol, leptin, and testosterone. In contrast to fed animals, plasma glucose, leptin, and testosterone levels decreased and cortisol levels increased in fasted animals. The hypothalamic expression of KISS1 and KISS1R mRNA was significantly lower (p<0.05) in fasted monkeys compared to fed monkeys while hypothalamic GNRH1 mRNA expression was comparable between the 2 groups. Thus, our results demonstrate that expression of hypothalamic KISS1 and KISS1R decrease after a short-term fasting in monkeys. This decrease may contribute to the suppression of the HPG axis during fasting conditions in primates. In addition, our finding of lower expression of KISS1R in fasted monkeys provides an explanation for the attenuation in the HPG axis response to peripheral kisspeptin challenge during short-term fasting.

Effect of treatment with cyproterone acetate on uterine bleeding at the beginning of GnRH analogue therapy in girls with idiopathic central precocious puberty.

BACKGROUND: The flare-up effect of GnRH analogues may cause transient uterine bleeding in girls affected with idiopathic central precocious puberty (ICPP). AIMS: To assess the incidence of endometrial bleeding and verify whether pretreatment with cyproterone acetate could counteract it. METHODS: Fifty-four girls affected by ICPP were divided into 2 groups. The first group (30 girls) was treated with triptorelin (3.75 mg, i.m. injection) every 28 days. The second group (24 girls) was treated with cyproterone acetate and triptorelin: cyproterone acetate (50 mg/m(2)) was administered every day for 8 weeks, and triptorelin (3.75 mg) was commenced 4 weeks after starting the cyproterone, then the intramuscular injection of triptorelin was repeated every 28 days. RESULTS: Eight of 54 girls (15%) had mild withdrawal bleeding. There were no differences in incidence between groups 1 and 2. Girls with pubertal uterus at pelvic ultrasound had a higher incidence of uterine bleeding than girls with infantile uterus (25 vs. 7%), but this difference was not significant. CONCLUSION: Co-administration of cyproterone acetate and GnRH analogues does not significantly decrease the incidence of uterine bleeding.

Kisspeptin/Gpr54-independent gonadotrophin-releasing hormone activity in Kiss1 and Gpr54 mutant mice.

The kisspeptin/Gpr54 signalling pathway plays a critical role in reproduction by stimulating the secretion of gonadotrophin-releasing hormone (GnRH), yet mice carrying mutations in Kiss1 (which encodes kisspeptin) or Gpr54 exhibit partial sexual maturation. For example, a proportion of female Kiss1(-/-) and Gpr54(-/-) mice exhibit vaginal oestrus, and some male Kiss1(-/-) and Gpr54(-/-) mice exhibit spermatogenesis. To characterise this partial sexual maturation, we examined the vaginal cytology of female Kiss1(-/-) and Gpr54(-/-) mice over time. Almost all mutant mice eventually enter oestrus, and then spontaneously transition from oestrus to dioestrus and back to oestrus again. These transitions are not associated with ovulation, and the frequency of these transitions increases with age. The oestrus exhibited by female Kiss1(-/-) and Gpr54(-/-) mice was disrupted by the administration of the competitive GnRH antagonist acyline, which also resulted in lower uterine weights and, in Kiss1(-/-) mice, lower serum follicle-stimulating hormone (FSH) and luteinising hormone (LH) concentrations. Similarly, male Kiss1(-/-) and Gpr54(-/-) mice treated with acyline had smaller testicular sizes and an absence of mature sperm. In addition to examining intact Kiss1(-/-) and Gpr54(-/-) mice, we also assessed the effects of acyline on gonadotrophin concentrations in gonadectomised mice. Gonadectomy resulted in a significant increase in serum FSH concentrations in male Gpr54(-/-) and Kiss1(-/-) mice. Acyline administration to gonadectomised Kiss1(-/-) and Gpr54(-/-) male mice lowered serum FSH and LH concentrations significantly. By contrast to males, gonadectomy did not result in significant gonadotrophin changes in female Kiss1(-/-) and Gpr54(-/-) mice, but acyline administration was followed by a decrease in LH concentrations. These results demonstrate that, although kisspeptin signalling is critical for the high levels of GnRH activity required for normal sexual maturation and for ovulation, Kiss1(-/-) and Gpr54(-/-) mice retain some degree of GnRH activity. This GnRH activity is sufficient to produce significant effects on vaginal cytology and uterine weights in female mice and on spermatogenesis and testicular weights in male mice.

CHD7 mutations in patients initially diagnosed with Kallmann syndrome--the clinical overlap with CHARGE syndrome.

Kallmann syndrome (KS) is the combination of hypogonadotropic hypogonadism and anosmia or hyposmia, two features that are also frequently present in CHARGE syndrome. CHARGE syndrome is caused by mutations in the CHD7 gene. We performed analysis of CHD7 in 36 patients with KS and 20 patients with normosmic idiopathic hypogonadotropic hypogonadism (nIHH) in whom mutations in KAL1, FGFR1, PROK2 and PROKR2 genes were excluded. Three of 56 KS/nIHH patients had de novo mutations in CHD7. In retrospect, these three CHD7-positive patients showed additional features that are seen in CHARGE syndrome. CHD7 mutations can be present in KS patients who have additional features that are part of the CHARGE syndrome phenotype. We did not find mutations in patients with isolated KS. These findings imply that patients diagnosed with hypogonadotropic hypogonadism and anosmia should be screened for clinical features consistent with CHARGE syndrome. If such features are present, particularly deafness, dysmorphic ears and/or hypoplasia or aplasia of the semicircular canals, CHD7 sequencing is recommended.

Kisspeptin and GPR54: discovery of a novel pathway in reproduction.

In order to find novel modulators of gonadotrophin-releasing hormone (GnRH) secretion, genetic tools were employed in patients with idiopathic hypogonadotrophic hypogonadism (IHH). Mutations in a G-protein coupled receptor, GPR54, were identified, making this receptor a genetic determinant and indisputable gatekeeper of normal reproductive function. This article places these investigations into historical context and reviews some of the new findings relevant to this pathway.

Peripheral and central hypomyelination with hypogonadotropic hypogonadism and hypodontia.

We identified four unrelated patients (three female, one male) aged 20 to 30 years with hypomyelination, pituitary hypogonadotropic hypogonadism, and hypodontia. Electron microscopy and myelin protein immunohistochemistry of sural nerves showed granular debris-lined clefts, expanded abaxonal space, outpocketing with vacuolar disruption, and loss of normal myelin periodicity. Reduced galactocerebroside, sphingomyelin, and GM1-N-acetylglucosamine and increased esterified cholesterol were found. This is a clinically homogeneous progressive hypomyelinating disorder. The term 4H syndrome is suggested.

Changes of thyroid function during long-term hGH therapy in GHD children. A possible relationship with catch-up growth?

BACKGROUND: Growth hormone (GH) treatment in patients with GH deficiency (GHD) can determine changes in the thyroid function. The clinical significance of these changes remains controversial, and all studies have so far covered rather a short period--usually no longer than one year. OBJECTIVE: To determine the effect of long-term recombinant hGH treatment in children with idiopathic GHD on the thyroid function. PATIENTS AND METHODS: Nineteen prepubertal children (12 boys and 7 girls, mean age 9.2 +/- 3.1 years) with idiopathic GHD were studied and followed for twenty-four months. None of the patients showed multiple pituitary hormone deficiencies. Nineteen healthy children matched for age and sex acted as controls. RESULTS: Patients with GHD showed a significant increase in TT (3) at twelve months and in FT (3) at six and twelve months after starting GH treatment, with a significant decrease at eighteen and twenty-four months. TT (4) level decreased significantly at twelve months and increased significantly at eighteen and twenty-four months. FT (4) also decreased, but only slightly, after twelve months of hGH treatment, and then increased significantly at twenty-four months. TSH levels did not vary significantly during the course of therapy. TT (3)/TT (4) and FT (3)/FT (4) ratios increased significantly after six and twelve months of therapy and significantly decreased later, approaching pre-therapy values. The SDS of Growth Velocity (SDS-GV) increased remarkably during the first year of therapy and then decreased significantly during the second year, although it remained significantly higher than the pre-therapy values. TT (3) and TT (3)/TT (4) ratio displayed a significant correlation with SDS-GV at twelve months of therapy. In a multiple regression analysis with age, bone age, parental height, GH dose, TT (3,) TT (3)/TT (4), and the SDS of IGF-I, only the TT (3)/TT (4) ratio at twelve months of therapy (p < 0.001) was identified as a significant predictor of SDS-GV. CONCLUSION: Our data confirm that changes in thyroid function are present in GHD children during long-term hGH therapy. These changes probably resulted from the effect of hGH on the peripheral metabolism of thyroid hormones and appear to be transitory, disappearing during the second year of hGH treatment. We speculate on the functional significance of these changes, and in particular, on their role in catch-up growth after hGH therapy.

A role for kisspeptins in the regulation of gonadotropin secretion in the mouse.

Kisspeptins are products of the KiSS-1 gene, which bind to a G protein-coupled receptor known as GPR54. Mutations or targeted disruptions in the GPR54 gene cause hypogonadotropic hypogonadism in humans and mice, suggesting that kisspeptin signaling may be important for the regulation of gonadotropin secretion. To examine the effects of kisspeptin-54 (metastin) and kisspeptin-10 (the biologically active C-terminal decapeptide) on gonadotropin secretion in the mouse, we administered the kisspeptins directly into the lateral cerebral ventricle of the brain and demonstrated that both peptides stimulate LH secretion. Further characterization of kisspeptin-54 demonstrated that it stimulated both LH and FSH secretion, at doses as low as 1 fmol; moreover, this effect was shown to be blocked by pretreatment with acyline, a potent GnRH antagonist. To learn more about the functional anatomy of kisspeptins, we mapped the distribution of KiSS-1 mRNA in the hypothalamus. We observed that KiSS-1 mRNA is expressed in areas of the hypothalamus implicated in the neuroendocrine regulation of gonadotropin secretion, including the anteroventral periventricular nucleus, the periventricular nucleus, and the arcuate nucleus. We conclude that kisspeptin-GPR54 signaling may be part of the hypothalamic circuitry that governs the hypothalamic secretion of GnRH.

The importance of autosomal genes in Kallmann syndrome: genotype-phenotype correlations and neuroendocrine characteristics.

Kallmann syndrome (KS) consists of congenital, isolated, idiopathic hypogonadotropic hypogonadism (IHH) and anosmia. The gene responsible for the X-linked form of KS, KAL, encodes a protein, anosmin, that plays a key role in the migration of GnRH neurons and olfactory nerves to the hypothalamus. In addition to X-linked pedigrees, autosomal dominant and recessive kindreds with KS have been reported. The relative importance of these autosomal vs. X-linked genes in producing KS, and the frequency of KAL mutations, are currently unknown because these are rare disorders and large series are unusual. We examined 101 individuals with IHH (+/- anosmia) and their families to determine their modes of inheritance, incidence of mutations in the coding sequence of KAL, genotype-phenotype correlations, and [in a subset (n = 38)] their neuroendocrine phenotype. Of the 101 patients, 59 had true KS (IHH + anosmia/hyposmia); whereas, in the remaining 42, no anosmia was evident in the patients or their families. Of the 59 KS patients, 21 were familial, whereas 38 were sporadic cases. Mutations in the coding sequence of KAL were identified in only 3 of 21 familial cases (14%) and 4 of 38 (11%) of the sporadic cases. Of the X-linked cases confirmed by mutational analysis, only 1 of 3 pedigrees appeared X-linked by inspection whereas the other 2 contained only affected brothers. Female members of known KAL mutation families (n = 3) exhibited no reproductive phenotype and were not anosmic, whereas families with anosmic women (n = 3) were not found to carry mutations in KAL. Mutations were uniformly absent in nonanosmic IHH probands (n = 42), as well as in families with both anosmic and nonanosmic members (n = 2). Overall, 4 novel mutations were identified (C172R, R191x, R457x, and delC@L600). With respect to neuroendocrine phenotype, KS men with documented KAL mutations (n = 8) had completely apulsatile LH secretion, whereas those with autosomal modes of inheritance demonstrated a more variable spectrum with evidence of enfeebled (but present) GnRH-induced LH pulses. Our conclusions are: 1) Confirmed mutations in the coding sequence of the KAL gene occur in the minority of KS cases, i.e. only 14% of familial and 11% of sporadic cases; 2) The majority of familial (and presumably sporadic) cases of KS are caused by defects in at least two autosomal genes that are currently unknown; 3) Obligate female carriers in families with KAL mutations have no discernible phenotype; 4) KAL mutations are uniformly absent in patients with either normosmic IHH or in families with both anosmic and nonanosmic individuals; and 5) Patients with KAL mutations have apulsatile LH secretion consistent with a complete absence of GnRH migration of GnRH cells into the hypothalamus, whereas evidence of present (but enfeebled) GnRH-induced LH pulses may be present in autosomal KS cases. Taken together, these findings suggest that autosomal genes account for the majority of familial cases of KS, and that unique neuroendocrine phenotypes consistent with some GnRH neuronal migration may exist in these patients.

Prevalence, phenotypic spectrum, and modes of inheritance of gonadotropin-releasing hormone receptor mutations in idiopathic hypogonadotropic hypogonadism.

Mutations in the GnRH receptor (GNRHR) have been described as a cause of reproductive failure in a subset of patients with idiopathic hypogonadotropic hypogonadism (IHH). Given the apparent rarity of these mutations, we set out to determine the frequency and distribution of GNRHR mutations in a heterogeneous population of patients with IHH who were well characterized with respect to diagnosis, phenotype, and mode of inheritance and to define their distribution within the receptor protein. One hundred and eight probands with IHH were screened for mutations in the coding sequence of GNRHR. Forty-eight of the 108 patients had a normal sense of smell, whereas the remaining 60 had anosmia or hyposmia (Kallmann syndrome). Exon segments in the GNRHR were screened for mutations using temperature gradient gel electrophoresis, and all mutations were confirmed by direct sequencing. Five unrelated probands (3 men and 2 women), all normosmic, were documented to have changes in the coding sequence of the GNRHR. Two of these probands were from a subgroup of 5 kindreds consistent with a recessive mode of inheritance, establishing a GNRHR mutation frequency of 2 of 5 (40%) in patients with normosmic, autosomal recessive IHH. The remaining 3 probands with GNRHR mutations were from a subgroup of 18 patients without evidence of familial involvement, indicating a prevalence of 3 of 18 (16.7%) in patients with sporadic IHH and a normal sense of smell. Among the five individuals bearing GNRHR mutations, a broad spectrum of phenotypes was noted, including testicular sizes in the male that varied from prepubertal to the normal adult male range. Three probands had compound heterozygous mutations, and two had homozygous mutations. Of the eight DNA sequence changes identified, four were novel: Thr(32)Ile, Cys(200)Tyr, Leu(266)Arg, and Cys(279)TYR: COS-7 cells transiently transfected with complementary DNAs encoding the human GNRHR containing each of these four novel mutations failed to respond to GnRH agonist stimulation. We conclude that 1) the spectrum of phenotypes in patients with GNRHR mutations is much broader than originally anticipated; 2) the frequency of GNRHR mutations may be more common than previously appreciated in familial cases of normosmic IHH and infrequent in sporadic cases; and 3) functional mutations of the GNRHR are distributed widely throughout the protein.

Differential regulation of gonadotropin secretion by testosterone in the human male: absence of a negative feedback effect of testosterone on follicle-stimulating hormone secretion.

Studies of sex steroid regulation of gonadotropin secretion in the human male have focused primarily on the respective site(s) of negative feedback of testosterone (T) and estradiol (E(2)). The use of pharmacological doses of sex steroids in these studies has precluded conclusions about the relative roles of T and E(2) in gonadotropin feedback. Thus, the aims of the present study were to 1) determine the relative contributions of T vs. E(2) to the sex steroid component of gonadotropin regulation, and 2) distinguish the feedback effects of T that that are direct (i.e. mediated by the androgen receptor) vs. indirect (mediated by aromatization to E(2)). Two experimental interventions were used: 1) inhibition of aromatization by a selective aromatase inhibitor to examine the impact of selective E(2) withdrawal; and 2) acute medical castration to examine the effect of ablating both T and E(2). Sixteen normal (NL) men (mean age, 30.5 +/- 2.2 yr) were studied. Nine NL subjects were treated with the aromatase inhibitor, anastrozole (10 mg, orally, daily, for 5 days). Twelve NL men underwent medical castration with ketoconazole (1-g loading dose followed by 400 mg, orally, four times a day for 5 days). Ketoconazole-treated subjects received concomitant treatment with dexamethasone (0.5 mg twice daily) to prevent the development of adrenal insufficiency. Single blood samples were drawn daily between 0800-1000 h. To ensure that dexamethasone was not altering the gonadotropin response to sex steroid ablation by a direct pituitary effect, five GnRH-deficient men (mean age, 37.6 +/- 3.9 yr) underwent GnRH dose-response studies at baseline and after treatment with dexamethasone (0.5 mg twice daily). Aromatase blockade caused significant lowering of E(2) (33 +/- 3 to 14 +/- 1 pg/mL; P: < 0.0005) with a corresponding increase in T levels (563 +/- 42 to 817 +/- 81 ng/dL; P: < 0.05). Treatment with ketoconazole resulted in equivalent suppression of E(2) (41 +/- 4 to 14 +/- 1 pg/mL; P: < 0.0005), but also induced castrate levels of T (491 +/- 28 to 40 +/- 3 ng/dL; P: < 0.0005). Both treatment regimens were associated with a significant increase in gonadotropin levels. For LH, the percent increase in serum levels after castration was almost 3-fold greater than that seen after selective E(2) withdrawal (275 +/- 23% with ketoconazole vs. 95.6 +/- 21% with anastrozole; P: < 0.005). Despite the divergent changes in T levels with these two maneuvers (a marked decrease after ketoconazole and a significant increase with anastrozole), the percent rise in FSH levels was similar in the two protocols (91 +/- 6% vs. 71 +/- 7%, respectively; P: = NS). Inhibin B levels were unchanged after selective E(2) withdrawal (156 +/- 23 vs. 176 +/- 19 pg/mL), but decreased slightly with ketoconazole (156 +/- 15 to 131 +/- 11 pg/mL; P: < 0.05). In contrast to the effects of glucocorticoid administration on gonadotropin secretion in women, no significant changes were observed in the GnRH-deficient men treated with dexamethasone in terms of mean LH levels (19.8 +/- 3.2 vs. 23.3 +/- 5.4 IU/L), mean LH pulse amplitude after GnRH (16.0 +/- 2.5 vs. 19.0 +/- 5.1 IU/L), or mean FSH levels (8.0 +/- 1.9 vs. 9.2 +/- 2.4 IU/L, pre vs. post). These studies provide evidence of differential regulation of gonadotropin secretion by T in the human male. T exerts both direct and indirect feedback on LH secretion, whereas its effects on FSH appear to be mediated largely by aromatization to E(2). From these data we conclude that in terms of sex steroid feedback, E(2) is the predominant regulator of FSH secretion in the human male.

Genetics of hypogonadotropic hypogonadism.

Determining the physiologic influences that modulate GnRH secretion, the prime initiator of reproductive function in the human, is fundamental not only to our understanding of the rare condition of congenital idiopathic hypogonadotropic hypogonadism (IHH), but also common disorders such as constitutional delay of puberty and hypothalamic amenorrhea. IHH is characterized by low levels of sex steroids and gonadotropins, normal findings on radiographic imaging of the hypothalamic-pituitary regions, and normal baseline and reserve testing of the remainder of the hypothalamic-pituitary axes. Failure of the normal pattern of episodic GnRH secretion results in delay of puberty and infertility. IHH is characterized by rich clinical and genetic heterogeneity, variable modes of inheritance, and association with other anomalies. To date, 4 genes have been identified as causes of IHH in the human; KAL [the gene for X-linked Kallmann syndrome (IHH and anosmia)], DAX1 [the gene for X-linked adrenal hypoplasia congenita (IHH and adrenal insufficiency)], GNRHR (the GnRH receptor), and PC1 (the gene for prohormone convertase 1, causing a syndrome of IHH and defects in prohormone processing). As these mutations account for less than 20% of all IHH cases, discovery of additional gene mutations will continue to advance our understanding of this intriguing syndrome.

Catch-up growth in short-at-birth NICU graduates.

The statural catch-up growth, defined as reaching at least tenth length/height percentile (P10) for normal population standards (-1.28 SD score, SDS), was studied in 73 infants short at birth (length < P10 for gestational age) admitted to NICU. Mean gestational age at birth was 35.2 weeks (range 29-41) and mean birth length standard deviation score -2.31 (-4.52/-1.46). Infants were measured at birth, at 3, 6, 12, 18, and 24 months corrected age and then once a year until 6 years chronological age. Statural catch-up growth was studied, with reference both to normal population standards and to individual genetic target. With reference to normal population standards, 44% of infants had caught-up at 3 months of age, 51% at 3 years, 66% at 4 years and 73% at 6 years. In the case of individual genetic targets, a similar trend was present, but the absolute values were slightly higher from 4 to 6 years (73 vs. 66% and 78 vs. 73%, respectively). Statistically significant changes in mean standard deviations score for chronological age were present from birth to 3 months, 3 to 12 months, 3 to 4 years and 5 to 6 years (p<0.05). No differences were found in this trend of recovery when considering ponderal index (PI) at birth (symmetrical vs. asymmetrical), sex (male vs. female) or gestational age (p>0.05). In the majority of cases infants with short stature at birth admitted to a NICU had a statural catch-up growth within the first years of life. This is more evident when considered in relation to individual genetic target rather than to normal population standards.

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.

Successful use of pulsatile gonadotropin-releasing hormone (GnRH) for ovulation induction and pregnancy in a patient with GnRH receptor mutations.

GnRH receptor mutations have recently been identified in a small number of familial cases of nonanosmic hypogonadotropic hypogonadism. In the present report we studied a kindred in which two sisters with primary amenorrhea were affected with GnRH deficiency due to a compound heterozygote mutation (Gln(106)Arg, Arg(262)Gln) and performed extensive phenotyping studies. Baseline patterns of gonadotropin secretion and gonadotropin responsiveness to exogenous pulsatile GnRH were examined in the proband. Low amplitude pulses of both LH and free alpha-subunit (FAS) were detected during 24 h of every 10 min blood sampling. The proband then received exogenous pulsatile GnRH i.v. for ovulation induction, and daily blood samples for gonadotropins and sex steroids were monitored. At the conventional GnRH replacement dose for women with hypogonadotropic hypogonadism (75 ng/kg), no follicular development occurred. At a GnRH dose of 100 ng/kg, the level and pattern of gonadotropin secretion more closely mimicked the follicular phase of normal women; a single dominant follicle was recruited, and an endogenous LH surge was elicited. However, the luteal phase was inadequate, as assessed by progesterone levels. At a GnRH dose of 250 ng/kg, the gonadotropin and sex steroid dynamics reproduced those of normal ovulatory women in both the follicular and luteal phases, and the proband conceived. The FAS responses to both conventional and high dose GnRH were within the normal range. The following conclusions were made: 1) Increased doses of GnRH may be used effectively for ovulation induction in some patients with GnRH receptor mutations. 2) Higher doses of GnRH are required for normal luteal phase dynamics than for normal follicular phase function. 3) Hypersecretion of FAS in response to exogenous GnRH, which is a feature of congenital hypogonadotropic hypogonadism, was not seen in this patient with a GnRH receptor mutation.

Mutational analysis of DAX1 in patients with hypogonadotropic hypogonadism or pubertal delay.

Although delayed puberty is relatively common and often familial, its molecular and pathophysiologic basis is poorly understood. In contrast, the molecular mechanisms underlying some forms of hypogonadotropic hypogonadism (HH) are clearer, following the description of mutations in the genes KAL, GNRHR, and PROP1. Mutations in another gene, DAX1 (AHC), cause X-linked adrenal hypoplasia congenita and HH. Affected boys usually present with primary adrenal failure in infancy or childhood and HH at the expected time of puberty. DAX1 mutations have also been reported to occur with a wider spectrum of clinical presentations. These cases include female carriers of DAX1 mutations with marked pubertal delay and a male with incomplete HH and mild adrenal insufficiency in adulthood. Given this emerging phenotypic spectrum of clinical presentation in men and women with DAX1 mutations, we hypothesized that DAX1 might be a candidate gene for mutation in patients with idiopathic sporadic or familial HH or constitutional delay of puberty. Direct sequencing of DAX1 was performed in 106 patients, including 85 (80 men and 5 women) with sporadic HH or constitutional delay of puberty and patients from 21 kindreds with familial forms of these disorders. No DAX1 mutations were found in these groups of patients, although silent single nucleotide polymorphisms were identified (T114C, G498A). This study suggests that mutations in DAX1 are unlikely to be a common cause of HH or pubertal delay in the absence of a concomitant history of adrenal insufficiency.