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Congenital adrenal hyperplasia is a group of autosomal recessive disorders second most often caused by deficiency of steroid 11-hydroxylase (CYP11B1) due to mutations in the CYP11B1 gene. We studied the functional and structural consequences of two novel missense mutations (W116C, L299P) and an in-frame 3-bp deletion (DeltaF438) in the CYP11B gene, detected in three unrelated families. All patients are suffering from classical CYP11B1 deficiency. In vitro expression studies in COS-7 cells revealed a decreased CYP11B1 activity in the W116C mutant to 2.9 +/- 0.9% (sd) for the conversion of 11-deoxycortisol to cortisol. The L299P mutant reduced the enzymatic activity to 1.2 +/- 0.9%, whereas the DeltaF438 mutation resulted in no measurable residual CYP11B1 activity. Introduction of these mutations in a three-dimensional model structure of the CYP11B1 protein provides a possible explanation for the in vitro measured effects. We hypothesize that the W116C mutation influences the conformational change of the 11-hydroxylase protein necessary for substrate access and product release. The L299P mutation causes a change in the position of the I helix relative to the heme group, whereas the DeltaF438 mutation results in a steric disarrangement of the heme group relative to the enzyme. Studying the enzyme function in vitro helps to understand the phenotypical expression and disease severity of 11-hydroxylase deficiency, which is the basis for accurate genetic counseling, prenatal diagnosis, and treatment. Moreover, the combination of in vitro enzyme function and molecular modeling provides new insights in cytochrome P450 structural-functional relationships.

Tpit is a T box transcription factor important for terminal differentiation of pituitary proopiomelanocortin-expressing cells. We demonstrated that human and mouse mutations of the TPIT gene cause a neonatal-onset form of congenital isolated ACTH deficiency (IAD). In the absence of glucocorticoid replacement, IAD can lead to neonatal death by acute adrenal insufficiency. This clinical entity was not previously well characterized because of the small number of published cases. Since identification of the first TPIT mutations, we have enlarged our series of neonatal IAD patients to 27 patients from 21 unrelated families. We found TPIT mutations in 17 of 27 patients. We identified 10 different TPIT mutations, with one mutation found in five unrelated families. All patients appeared to be homozygous or compound heterozygous for TPIT mutations, and their unaffected parents are heterozygous carriers, confirming a recessive mode of transmission. We compared the clinical and biological phenotype of the 17 IAD patients carrying a TPIT mutation with the 10 IAD patients with normal TPIT-coding sequences. This series of neonatal IAD patients revealed a highly homogeneous clinical presentation, suggesting that this disease may be an underestimated cause of neonatal death. Identification of TPIT gene mutations as the principal molecular cause of neonatal IAD permits prenatal diagnosis for families at risk for the purpose of early glucocorticoid replacement therapy.

Disruption of the P450 side-chain cleavage cytochrome (P450scc) enzyme due to deleterious mutations of the CYP11A1 gene is thought to be incompatible with fetal survival because of impaired progesterone production by the fetoplacental unit. We present a 46,XY patient with a homozygous disruption of CYP11A1. The child was born prematurely with complete sex reversal and severe adrenal insufficiency. Laboratory data showed diminished or absent steroidogenesis in all pathways. Molecular genetic analysis of the CYP11A1 gene revealed a homozygous single nucleotide deletion leading to a premature termination at codon position 288. This mutation will delete highly conserved regions of the P450scc enzyme and thus is predicted to lead to a nonfunctional protein. Both healthy parents were heterozygous for this mutation. Our report demonstrates that severe disruption of P450scc can be compatible with survival in rare instances. Furthermore, defects in this enzyme are inherited in an autosomal-recessive fashion, and heterozygote carriers can be healthy and fertile. The possibility of P450scc-independent pathways of steroid synthesis in addition to the current concept of luteoplacental shift of progesterone synthesis in humans has to be questioned.

Congenital adrenal hyperplasia is a group of autosomal recessive disorders most often caused by deficiency of steroid 21-hydroxylase due to mutations in the CYP21 gene. We studied the functional and structural consequences of two novel missense mutations in the CYP21 gene, detected in two simple virilizing congenital adrenal hyperplasia patients. Both the male and female patient were compound heterozygous for the novel I77T and A434V point mutations, respectively. The in vitro expression analysis in COS-7 cells revealed a reduced 21-hydroxylase activity in the I77T mutant of 3 +/- 2% (sd) for the conversion of 17-hydroxyprogesterone to 11-deoxycortisol and of 5 +/- 3% for the conversion of progesterone to 11-deoxycorticosterone. The A434V mutant had a residual enzyme activity of 14 +/- 2% for 17-hydroxyprogesterone and 12 +/- 6% for progesterone. Substrate affinity was similar in the mutants as in the CYP21 wild-type protein, whereas reaction velocity was markedly decreased in both mutants. These effects could be readily explained by structural changes induced by the mutations, which were rationalized by a three-dimensional-model structure of the CYP21 protein. We hypothesize that the I77T mutation markedly decreases the reaction product release and/or substrate entrance to the enzyme's active site, whereas the A434V mutant reduces both the catalytic capacity and reaction velocity. Studying the enzyme function in vitro helps to understand the phenotypical expression and disease severity of 21-hydroxylase deficiency and also provides new insights into cytochrome P450 structure-function relationships.

Pseudohypoaldosteronism type 1 (PHA1) is a rare congenital disease inherited in either an autosomal-recessive or an autosomal-dominant trait. The autosomal-dominant form manifests with renal salt loss in infancy and a gradual improvement with advancing age. PHA1 presents with potential life-threatening salt wasting and failure to thrive in early infancy. Autosomal-dominant forms of PHA1 are often caused by heterozygous mutations of the MR gene coding for the mineralocorticoid receptor. Whether heterozygous mutations of the MR gene impair biological function as a result of haplo-insufficiency or due to a dominant-negative effect needs further clarification. We report a case of a renal form of PHA1 in a Turkish family. A heterozygous nonsense mutation c3055C>T (R947X) in exon 9 of the MR gene leading to a premature stop codon was identified in the index patient. The truncated receptor is free of aldosterone binding. The segregation analysis revealed the identical mutation in the patient's father, who never showed any symptoms of PHA. This shows the incomplete penetrance of the phenotype, although a mild salt loss might have been overlooked in the father's childhood.

The pathogenesis of central precocious puberty (PP) and/or gelastic seizures due to a hypothalamic hamartoma (HH) is still under debate. We evaluated the association of clinical symptoms with morphology and localization of the HH in 34 patients. The majority (86.4%) of HHs in patients with isolated PP (n = 22; 68.2% females) revealed a parahypothalamic position without affecting the third ventricle (91%). Half of them were pedunculated, and 40.9% showed a diameter less than 10 mm. In contrast, 11 of 12 patients with seizures, eight of whom were male, presented with a sessile intrahypothalamic hamartoma, 10 of which distorted the third ventricle. Logistic regression analysis revealed an increased relative risk (RR) for epilepsy in males (RR, 4.3; 95% confidence interval, 0.96-19). However, combination of the risk factor gender with intrahypothalamic position (RR, 19; 1.3-285) and distortion of the third ventricle (RR, 10; 0.6-164) reduced the risk associated with male gender to 1.1. The position of a HH and involvement of the third ventricle are likely to be more predictive for clinical characteristics than size and shape. Male gender was associated with an intrahypothalamic HH and epilepsy, suggesting a sexually dimorphic developmental pattern of this heterotopic mass.

Pseudohypoaldosteronism (PHA) type 1 presents in infancy with potential life-threatening salt wasting and failure to thrive. Plasma renin activity and aldosterone levels are markedly elevated. PHA1 is inherited in either an autosomal recessive or autosomal dominant trait. The autosomal dominant form manifests with renal salt loss in infancy and a gradual improvement with advancing age. We report the case of a renal form of PHA1 in a German family. PCR and direct sequencing revealed a heterozygous point mutation, c488C>G (S163X), leading to a premature stop codon in the index patient. The segregation analysis revealed the identical mutation in the patient's father, who showed no symptoms of PHA at the time of investigation or before. The family was screened for amino acid polymorphisms in the amiloride-sensitive epithelial sodium channel, which could be a cause for phenotypic differences within the family. PCR and direct sequencing revealed identical epithelial sodium channel haplotypes in the patient and his father. These polymorphisms can, therefore, not be responsible for the difference in clinical presentation. It remains to be elucidated whether other defects or polymorphisms in genes coding for regulatory proteins participating in sodium homeostasis are a cause of the heterogeneity of the clinical manifestations in autosomal dominant PHA1.

Pregnenolone (Preg) and 17-hydroxypregnenolone (17OH-Preg) are marker steroids that are elevated in the 3beta-hydroxysteroid-dehydrogenase-II deficiency form of congenital adrenal hyperplasia. The aim of this study was to establish normative data for both steroids in healthy preterm (28-33 and 34-37 wk gestation) and full-term infants, because reference values for the early neonatal period do not exist. At delivery, the main source of Preg is the placenta, because the highest levels were found in the retroplacental space (median, 141.31 nmol/liter), with no significant difference between preterm and full-term pregnancies. The fetal adrenals provide most of the circulating 17OH-Preg in full-term neonates, as demonstrated by a marked arteriovenous gradient in cord blood (40.96 nmol/liter vs. 10.77 nmol/liter). 17OH-Preg levels in the umbilical arteries were significantly lower in premature infants than in full-term infants (8.06 nmol/liter vs. 40.96 nmol/liter). During the first 2 postnatal weeks, Preg concentrations showed a rapid and significant fall in early preterm infants [95.78 nmol/liter (0 h) to 36.69 nmol/liter (d 14)] as well as in full-term infants [66.62 nmol/liter (0 h) to 14.81 nmol/liter (d 6)]. In addition, a significant fall in 17OH-Preg levels was found in full-term neonates [40.96 nmol/liter (0 h) to 11.32 nmol/liter (d 6)]. After 12 h, significantly higher levels for Preg and 17OH-Preg were found in early preterm infants (98.01 nmol/liter and 69.13 nmol/liter), compared with full-term neonates (36.29 nmol/liter and 28.55 nmol/liter, P < 0.05), reflecting the increased fetocortical activity as a response to the stress of delivery in the prematures. With these longitudinal data, it is now possible to confirm or exclude the diagnosis of 3beta-hydroxysteroid-dehydrogenase-II deficiency within the first postnatal week.

Estrogens have been shown to rapidly inhibit longitudinal growth in tall boys. To antagonize the initial growth accelerating effect of T, 41 boys with an initial height prediction in excess of 203 cm were treated prospectively with either T enanthate (TE) 250 mg/wk im in combination with ethinylestradiol (EE2) 0.1 mg/d taken orally for the first 5.8 +/- 0.47 wk (mean +/- SE) of treatment (group 1, n = 20) or with TE alone (group 2, n = 21). Patients were randomized to one or the other treatment regimen. Mean (+/-SE) predicted adult height was 206.8 +/- 0.7 cm in group 1 and 206.4 +/- 0.8 cm in group 2. Total duration of treatment was 16.1 +/- 0.8 months and 14.0 +/- 1.2 months in group 1 and 2, respectively (NS). EE2-induced side effects in group 1 (gynecomastia) were limited and fully reversible. No negative long-term sequelae were found at final height with respect to hypothalamic-pituitary-gonadal axis activity and to testis volumes. Although there was a tendency to a lower initial growth velocity measured by knemometry in group 1 (0.30 +/- 0.05 vs. 0.38 +/- 0.05 mm/wk, NS), final height did not differ in both study groups (195.0 +/- 0.8 cm in group 1, 194.6 +/- 0.8 cm in group 2). Similarly, height reduction (initial predicted adult height minus final height) was not significantly different between the two groups (12.0 +/- 0.9 cm in group 1, 11.7 +/- 0.9 cm in group 2). In conclusion, the addition of EE2 during the initial treatment phase to high-dose T in tall boys has no significant effect on height reduction. The results of this clinical trial suggest that the initial growth inhibiting effect of EE2 on the epiphyseal growth plates is overridden by the long-term administration of high dose TE.

Mutations of the PROP-1 gene cause combined pituitary hormone deficiency. Progressive ACTH/cortisol insufficiency is found in a few patients. Congenital hypoplasia of the anterior pituitary gland is the most common magnetic resonance imaging finding in patients with PROP-1 mutations. We present two brothers with compound heterozygosity for the two mutations 150delA and 301-302delAG of the PROP-1 gene. Both showed combined pituitary hormone deficiency of GH, TSH, PRL, and gonadotropins, as is typical for PROP-1 deficiency. We observed a developing insufficiency of ACTH and cortisol secretory capacity in both patients. Computed tomography revealed an enlarged pituitary in the older brother at 3.5 yr of age. Repeated magnetic resonance imaging after 12 yr showed a constant hypoplasia of the anterior pituitary lobe. Similarly, magnetic resonance imaging of the younger brother showed a constant enlargement of the anterior pituitary gland until 10 yr. At the age of 11 yr, the anterior pituitary was hypoplastic. The reason for pituitary enlargement in early childhood with subsequent decrease in pituitary size is not known. We speculate that altered expression of early transcription factors could be involved. Because both patients have the same PROP-1 mutations and an identical pattern of combined pituitary hormone deficiency, we suggest that early pituitary enlargement may be the typical course in such patients in whom pituitary surgery is not indicated.

The etiology of short stature (SST) in Turner syndrome (TS) is still a subject of speculation. A variety of hypotheses have been put forward, from SST as a result of increased intrauterine tissue pressure after fetal lymphedema to haploinsufficiency of a specific growth gene(s). These hypotheses have various statistical-auxological implications on the growth distribution in TS. Empirical research has provided no clear evidence for any of these theories, but the well known correlation between patients' and midparental height (MPH) could be established. The influence of undetected mosaic status has often been cited as a major problem in the investigation of growth in TS. However, an assessment of mosaic status (simultaneous analysis of karyotype and phenotype) and its effect on growth with inclusion of MPH has been not yet carried out for a large sample. The aim of this study was to evaluate growth and its complex relationship to mosaic status and MPH in TS. In a mixed cross-sectional and longitudinal study we retrospectively analyzed the auxological and clinical data of 447 patients with a pure loss of X-chromosomal material (n = 381 with 45,X0; n = 66 mosaics). The 447 patients were selected from a series of 609 consecutive patients with TS. To assess the effect of mosaic status on growth, we computed a bifactorial analysis of variance (phenotype, karyotype), including MPH as a covariate. In line with the mosaic hypothesis, we found a correlation between individual loss of X-chromosomal material and phenotypical expressivity. In contrast, no correlation was found with respect to growth. With respect to MPH, we found growth retardation (GR) even in those patients with "normal" height above the third percentile (-2 or more SD score). The interindividual variance of GR in TS (comparable to growth variance in the normal population) seems to be unrelated to other TS-specific factors (e.g. mosaic status or single gene loss). Instead, both interindividual variance and the global growth shift distribution are best explained by the presence of an unspecific aneuploidic effect. Furthermore, consideration of patient height in relation to MPH should lead to a better understanding of the nature of GR in TS than the commonly used, strictly qualitative definition of SST.

A considerable number of patients with central precocious puberty (CPP) treated with depot GnRH agonists have reached final height (FH). The aim of this prospective, multicentric study was the evaluation of the benefits, side-effects, and long term outcome of depot GnRH agonist therapy. We investigated 50 young women (mean +/- SD age, 16.7+/-2.6 yr; range, 12.9-23.4 yr) at FH. They received depot triptorelin over a period of 4.4+/-2.1 yr (range, 1.0-9.7 yr). Target height (TH) and predicted adult height (PAH) at the start of treatment were 163.6+/-6.2 and 154.9+/-9.6 cm, respectively (P < 0.05). FH was 160.6+/-8.0 cm (FH vs. TH, P = NS; FH vs. PAH, P < 0.05). Young patients showed the highest height gain (FH minus initial PAH). Seventy-eight percent of all patients reached a FH within their TH range. Even in young patients and those with an unfavorable initial PAH below the TH range, 60% reached a FH within their individual TH range. Standardized bone mineral density and standardized bone mineral density SD score investigated by dual energy x-ray absorptiometry of the lumbar spine (L1-L4) were 1040.9+/-124.2 mg/cm2 and 0.0+/-1.0; those of the femoral neck were 902.2+/-115.4 mg/cm2 and 0.2+/-1.0, respectively. The SD score of the ratio of sitting height over lower leg length was normal (0.3+/-1.2). Body mass index SD scores at pretreatment, at the end of treatment, and at FH were not significantly different (2.0+/-2.0, 2.0+/-2.0, and 1.7+/-2.2, respectively). Menarche or remenarche started at age 12.3+/-1.4 yr (range, 9.3-15.8 yr) in all patients. In conclusion, long term depot GnRH agonist treatment of CPP girls preserved genetic height potential and improved FH significantly combined with normal body proportions. No negative effect on bone mineral density and reproductive function was seen. Treatment neither caused nor aggravated obesity.

X-linked congenital adrenal hypoplasia (AHC) is a rare developmental disorder of the human adrenal cortex and is caused by deletion or mutation of the DAX-1 gene, a recently discovered member of the nuclear hormone receptor superfamily. Hypogonadotropic hypogonadism is frequently associated with AHC. AHC occurs as part of a contiguous gene syndrome together with glycerol kinase deficiency (GKD) and Duchenne's muscular dystrophy. The present series, collected over the past 2 decades, includes 18 AHC boys from 16 families: 4 with AHC, GKD, and Duchenne's muscular dystrophy; 2 with AHC and GKD; and 12 with AHC (5 young adults with hypogonadotropic hypogonadism). Most of the boys presented with salt wasting and hyperpigmentation during the neonatal period. Plasma steroid determinations performed in the first weeks of life often showed confusing results, probably caused by steroids produced in the neonates' persisting fetocortex. Aldosterone deficiency usually preceded cortisol deficiency, which explains why the patients more often presented with salt-wasting rather than with hypoglycemic symptoms. An ACTH test was often necessary to detect cortisol deficiency in the very young infants. In some patients, serial testing was necessary to establish the correct diagnosis. In 4 boys studied during the first 3 months after birth, we found pubertal LH, FSH, and testosterone plasma levels indicating postnatal transient activation of the hypothalamic-pituitary-gonadal axis as in normal boys. Previous studies have shown that the DAX-1 gene is deleted in the AHC patients with a contiguous gene syndrome and is mutated in nondeletion patients. Most of the point mutations identified in AHC patients were frameshift mutations and stop mutations. In the 15 patients available for molecular analysis of the DAX-1 gene, there were large deletions in 6 patients and point mutations in another 7 patients. All of the point mutations identified in the present study resulted in a nonfunctional truncated DAX-1 protein. Two brothers with primary adrenal insufficiency and a medical history that strongly suggested AHC had no mutation in the DAX-1 gene. Thus, additional, as yet unknown genes must play a part in normal adrenal cortical development.

We describe a patient with onset of puberty at the age of 5 yr. characterized by accelerated growth, enlargement of genitalia, pubarche, and serum hormone levels compatible with noncentral precocious puberty. Exon 11 of the LH receptor gene was amplified from genomic DNA by PCR and directly sequenced. We identified a heterozygous C to T base change at nucleotide position 1126, exchanging codon 373 from Ala to Val in the first transmembrane domain. The LH receptor sequence of the parents was normal. The mutated receptor displayed an up to 7.5-fold increase in basal cAMP production compared to that of the wild-type receptor in transiently transfected COS-7 cells. Treatment of the patient with ketoconazole resulted in inconsistent suppression of serum testosterone levels. At the age of 9.1 yr, central activation of the hypothalamic-pituitary-gonadal axis occurred. Additional treatment with a GnRH agonist led to complete suppression of testosterone secretion. This is the first description of constitutive activation of the LH receptor in the first transmembrane segment. It suggests the involvement of the first transmembrane helix in signal transduction and provides further insight into the structural organization of the seven transmembrane domains of the glycoprotein hormone receptor proteins.

Aldosterone (Aldo), the most potent mineralocorticoid, is synthesized in the adrenal zona glomerulosa, requiring 11 beta-hydroxylation of 11-deoxycorticosterone (DOC) to form corticosterone (B), hydroxylation at position C18 to form 18-hydroxycorticosterone (18-OHB), and finally oxidation at position C18. There is a single cytochrome P450 enzyme (P450aldo) catalyzing all three reactions in the zona glomerulosa. The gene encoding for this enzyme is termed CYP11B2. There are two inborn errors of terminal aldosterone biosynthesis characterized by overproduction of B and deficient synthesis of Aldo. Corticosterone methyl oxidase deficiency type I (CMO-I) is characterized by decreased production of 18-OHB, whereas CMO-II is characterized by overproduction of 18-OHB and an elevated plasma ratio of 18-OHB to Aldo. Both disorders have an autosomal recessive inheritance and are rare causes of salt-wasting and failure to thrive in early infancy. In the last 10 yr, we diagnosed 16 infants with CMO deficiencies by simultaneous multisteroid analysis in a small plasma sample (RIA after extraction and automated high performance gel chromatography). All patients presented with severe failure to thrive in the first 3 months of life, associated with severe hyponatremia, hyperkalemia, and increased PRA. Basal Aldo levels were decreased (range, 0.055-0.11 nmol/L), whereas B was elevated (range, 19-154 nmol/L). Plasma 18-OHB, ranging from 0.063-0.44 nmol/L, was decreased or in the lower normal range in seven patients, whereas the other seven patients had elevated 18-OHB levels (range, 12.1-57.7 nmol/L). 18-OH-DOC (range, 0.81-7.8 nmol/L) and DOC (range, 0.7-9.53 nmol/L) levels were elevated in all patients. In seven patients, we found an elevated ratio of 18-OHB/Aldo (range, 286-900) and a low ratio of B/18-OHB (range, 1.1-5.8), whereas seven other patients had a low 18-OHB/Aldo ratio (range, 1.1-6.95) and a high B/18-OHB ratio (range, 41-1360). These findings confirmed the diagnosis of CMO-I in seven patients (low 18-OHB, 18-OHB/Aldo ratio < 10, and B/18-OHB ratio > 40) and the diagnosis of CMO-II in seven other patients (high 18-OHB, 18-OHB/Aldo ratio > 100, and B/18-OHB ratio < 10), whereas 18-OHB could not be determined in two patients. The B/18-OHB ratio appears particularly useful in CMO cases with undetectably low Aldo plasma levels and uncalculable 18-OHB/Aldo ratios. In conclusion, the simultaneous multisteroid determination method allows the precise differentiation of CMO-I and CMO-II in a small plasma sample during early infancy.

The interrelations of steroid hormone levels in plasma and amniotic fluid from mothers and their undisturbed fetuses at early midgestation of human pregnancy have not been defined previously. We, therefore, studied 12 healthy mothers and their fetuses undergoing termination of pregnancy for social reasons at 16-20 weeks gestation. Fetal arterial and venous blood was obtained by direct vessel puncture through a fetoscope in the conscious sedated mothers immediately before termination of pregnancy. Simultaneously, maternal peripheral venous blood and amniotic fluid were collected. Aldosterone (Aldo), corticosterone (B), 11-deoxycorticosterone, progesterone (P), 17-hydroxyprogesterone (17OHP), 11-deoxycortisol, cortisol (F), and cortisone were simultaneously determined by specific RIA after extraction and chromatography. Positive fetal arterio-venous differences were found for F, B, and Aldo, whereas arteriovenous differences were negative for P and 17OHP. In amniotic fluid, six of the eight corticosteroids showed significantly lower levels during fetoscopy than during routine amniocentesis, as reported previously using the same analytical methods. The present study demonstrates that the undisturbed human fetus at 16-20 weeks gestation actively secretes the most important gluco- and mineralocorticoids, F, B, and Aldo, independent of the mother. P and 17OHP were shown to be primarily derived from placental production and supplied to the fetus as a source of F and Aldo biosynthesis. The fetoscopy procedure with premedication seemed to give rise to less stress to the fetus than routine amniocentesis without sedation. Fetoscopy is, therefore, an ideal method to study feto-maternal steroid interrelations in human pregnancy.

It is not possible to differentiate reliably between male idiopathic hypothalamic hypogonadism (HH) and severe constitutional delay of puberty (CD) on the basis of a standard GnRH bolus test (GBT) or other known endocrine or clinical parameters. Therefore, we studied the response of 17 hypogonadal men, 8 with a diagnosis of HH (age, 15.5-41; bone age, 12.5-19 yr; testes, 1-4 ml) and 9 with CD (age, 14.5-20; bone age, 11-15 yr, testes, 2-10 ml) to pulsatile GnRH stimulation. Basal and peak LH and FSH levels after a single dose of GnRH greatly overlapped between the two groups. In each patient, a spontaneous nocturnal plasma profile of LH and FSH, sampled every 20 min, was followed by a pulsatile GnRH stimulation (5 micrograms iv every 90 min) via a portable minipump for 36 h. Before and after this pulsatile GnRH stimulation, a GBT (60 micrograms/m2 iv) was performed and plasma LH, FSH, testosterone, androstenedione, and dehydroepiandrosterone sulfate were measured. Pulse analysis revealed 0-5 spontaneous nocturnal LH peaks in the CD patients but only one in all of the HH patients. During the 36 h of pulsatile GnRH, mean LH and FSH levels were significantly higher (P less than 0.0001) than during the spontaneous nocturnal profile in all patients (except 1 from each group for LH). The GBT after pulsatile stimulation caused significantly higher (P less than 0.001) LH increments in CD than in HH patients, with no overlap between the two groups (range, 4.1-15.6 in CD vs. 0.8-2.4 mIU/ml in HH). Plasma testosterone rose significantly (P less than 0.01) during pulsatile GnRH from 67 to 155 ng/dl (median) in the CD men, but did not change in the HH group (21 to 22.5 ng/dl). Plasma androstenedione and dehydroepiandrosterone sulfate did not rise in either group. We conclude that, in contrast to other parameters investigated so far, the LH increment in the second GBT after 36 h of pulsatile GnRH allows clear-cut differentiation between CD and HH. These results indicate significantly lower pituitary LH reserve in patients with permanent HH after short term priming of the pituitary by pulsatile GnRH administration.