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The birth of a child with ambiguous genitalia is a challenging and distressing event for the family and physician and one with life-long consequences. Most disorders of sexual differentiation (DSD) associated with ambiguous genitalia are the result either of inappropriate virilization of girls or incomplete virilization of boys. It is important to establish a diagnosis as soon as possible, for psychological, social, and medical reasons, particularly for recognizing accompanying life-threatening disorders such as the salt-losing form of congenital adrenal hyperplasia. In most instances, there is sufficient follow-up data so that making the diagnosis also establishes the appropriate gender assignment (infants with congenital adrenal hyperplasia, those with androgen resistance syndromes), but some causes of DSD such as steroid 5α-reductase 2 deficiency and 17β-hydroxysteroid dehydrogenase deficiency are associated with frequent change in social sex later in life. In these instances, guidelines for sex assignment are less well established., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

A group of basic scientists, clinicians, clinical investigators, psychologists, patient advocacy groups, and representatives from professional societies and governmental agencies met at the National Institutes of Health in October, 2007 with the long-term goal of having the menstrual cycle accepted and understood as a marker of general health in adolescent girls. An equally important goal was to develop a research agenda for this area of investigation. This chapter comprises the highlights of discussions throughout that meeting, with an emphasis on ideas generated during a final session led by an internationally renowned physician-scientist, in which reports from four breakout groups were presented. The specific goal assigned to each group was to develop an agenda that would set the stage for how research should be conducted over the next 100 years, and to identify the pressing research questions that should be addressed related to the menstrual cycle and adolescent health. The four research areas represented in discussion groups included: emotional health; genetics; metabolism and reproduction; and the promotion of conduct of clinical research. Insights are also provided by five clinical investigators, including two outside experts, on topics of priority for a research agenda in the area of adolescent reproductive health, as well as how the research itself should be conducted.

A common cause of micropenis is congenital hypogonadotropic hypogonadism, whether isolated or associated with multiple pituitary hormone deficiencies. The postnatal surge in FSH, LH, and testosterone in the male infant as a consequence of the continued function of the fetal GnRH pulse generator provides a 6-month window of opportunity to establish the diagnosis of hypogonadotropic hypogonadism and alert the clinician to the possibility of its association with multiple pituitary hormone deficiencies. When ACTH or GH deficiency or both deficiencies are present, hypoglycemia and cortisol deficiency can lead to neonatal and infantile death or increased morbidity. Establishing the diagnosis of hypogonadotropic hypogonadism in infancy preempts the uncertainties and delays in distinguishing constitutional delay in puberty from hypogonadotropic hypogonadism. Accordingly, hormone replacement therapy can be initiated at the normal age of pubertal onset. The ontogenesis of infantile testicular function, including the possible significance of the infantile surge in gonadotropins and testosterone, is reviewed. The molecular basis for certain developmental disorders associated with hypogonadotropic hypogonadism and micropenis is considered and the management and treatment of congenital hypopituitarism discussed.

The discovery of males with mutations in CYP19, the gene that encodes aromatase, the enzyme catalyzing the final step in the conversion of androgen to estrogen, and of a man with a mutation in the estrogen receptor alpha gene has led to increasing recognition of the critical role of estrogen in the male. The diverse roles of estrogen in the male, both circulating and locally synthesized, is supported by extensive studies in male mice generated with disruption of the aromatase gene or the genes encoding the alpha- and beta-estrogen receptor. This presentation focuses on the crucial role of estradiol in the human male on the pubertal growth spurt, skeletal maturation and the cessation of linear growth, and the accrual of bone mass.

The fundamental aspects of the hypothalamic luteinizing hormone-releasing hormone (LHRH)(1) [1]pulse generator-pituitary gonadotrophin-gonadal apparatus in mammals have striking commonalities. There are, however, critical, substantive differences in the neuroendocrinology of puberty among species. The onset of puberty in the human is marked by an increase in the amplitude of LH pulses, an indirect indicator of the increase in amplitude of LHRH pulses. The hypothalamic LHRH-pituitary gonadotrophin complex is functional by at least 0.3 gestation in the human foetus; the sex difference in the fetal and neonatal pattern of LH and FSH secretion is an apparent consequence of imprinting of the fetal hypothalamus-pituitary-gonadotropin apparatus by fetal testosterone. Until about 6 months of age in boys and 12-24 months in girls, the testes and ovaries respond to the increased LH in boys and follicle-stimulating hormone (FSH) in girls by secreting testosterone and oestradiol, respectively, reaching levels that are not again achieved before the onset of puberty. Striking features of the ontogeny of the human hypothalamic pulse generator are: (1) its development and function in the foetus; (2) the continued function of the hypothalamic LHRH pulse generator-pituitary gonadotrophin-gonadal axis in infancy; (3) the gradual damping of hypothalamic LHRH oscillator activity during late infancy; (4) its quiescence during childhood - the so-called juvenile pause; (5) during late childhood the gradual disinhibition and reactivation of the LHRH pulse generator, mainly at night; (6) the increasing amplitude of the LHRH pulses, which are reflected in the progressively increased and changing pattern of circulating LH pulses, with the approach of and during puberty. The intrinsic central nervous system (CNS) mechanisms responsible for the inhibition of the LHRH pulse generator during childhood (the juvenile phase) involve the major role of an inhibitory neuronal system - the CNS inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and GABAergic neurons, as revealed by studies in the rhesus monkey by Terasawa and her associates. With the onset of puberty, the disinhibition and reactivation of the LHRH pulse generator is associated with a fall in GABAergic neurotransmission and a concomitant increase in the input of excitatory amino acid neurotransmitters (including glutamate) and possibly astroglial-derived growth factors. Despite remarkable progress over the past three decades, large gaps remain in our understanding of the neurobiological, genetic and environmental mechanisms involved in the control of the onset of puberty. The role of leptin in the control of the onset of puberty is reviewed. Severe leptin deficiency is associated with hypogonadotrophic hypogonadism; it appears that a critical level of leptin and a leptin signal is required to achieve puberty. The weight of evidence supports the hypothesis that leptin acts as one of several permissive factors and not a trigger in the onset of human puberty. The application of these advances provides a framework for the described classification of sexual precocity and delayed puberty.1 GnRH is synonymous with LHRH., (Copyright 2002 S. Karger AG, Basel)

Although low fertility rates have traditionally been reported among women with classic CAH and especially among women with the salt-wasting variant, more recent data suggest that fertility rates are significantly improved, largely owing to earlier treatment of CAH, improved compliance with therapy, and surgical advances in genital reconstruction. Furthermore, ovulation induction and assisted reproductive techniques are now available to women who remain infertile despite effective adrenal androgen suppression. Although the pregnancy experience in women with classic CAH remains limited, it is apparent that, once pregnant, these women have a high probability of successful outcome. Key issues should be emphasized in the management of CAH during gestation, including the need for assessing adrenal steroid replacement and adrenal androgen suppression, particularly in light of the interplay between maternal hyperandrogenism and the protective effect of placental aromatase activity, which provides a relatively large margin of safety for the female fetus. Maternal hormone levels should be evaluated in the context of laboratory-specific reference ranges for pregnancy. The infant should be examined for ambiguous genitalia and monitored for evidence of adrenal insufficiency. Although an affected female infant with classic CAH has not been reported as a pregnancy outcome of a mother with classic virilizing CAH, these concerns should be discussed during preconception counseling. Patients should also be aware of the importance of medication compliance and careful hormonal monitoring during the entire pregnancy. In most cases, successful gestational management requires the close coordination of care between the obstetrician and endocrinologist.

The discovery of a man with a homozygous mutation in the estrogen receptor alpha gene, which results in estrogen-receptor alpha resistance, and of males and females with autosomal recessive mutations in the CYP19 gene encoding aromatase, which leads to a failure to synthesize estrogens, has challenged conventional wisdom about the 'unimportant' role of estrogen in the male. For example, in the male, estrogen (not androgen) derived from direct testicular secretion (approximately 20%) and from extragonadal aromatization of testosterone and androstenedione (approximately 80%), is the critical sex hormone in the pubertal growth spurt, skeletal maturation, accrual of peak bone mass, and the maintenance of bone mass in the adult. Estrogen stimulates chondrogenesis in the epiphyseal growth plate increasing pubertal linear growth. At puberty, estrogen promotes skeletal maturation and the gradual, progressive closure of the epiphyseal growth plate, possibly as a consequence of both estrogen-induced vascular and osteoblastic invasion and the termination of chondrogenesis. In addition, during puberty and into the third decade, estrogen has an anabolic effect on the osteoblast and an apoptotic effect on the osteoclast, increasing bone mineral acquisition in axial and appendicular bone. In the adult, estrogen is important in maintaining the constancy of bone mass through its effects on remodeling and bone turnover. Establishing a role for estrogen does not exclude a direct action of testosterone on bone in the human male (especially on cortical bone), but this action is less characterized than thought in the past and is relatively minor in comparison with the major effect of estrogen in the male.

Recent developments have advanced our knowledge of the role of estrogen in the male. Studies of the mutations in CYP19, the gene encoding aromatase, in six females and two males and a mutant estrogen receptor alpha in a man are described. These observations provide illuminating new insights into the critical role of estrogen in the male (as well as female) in the pubertal growth spurt and skeletal maturation, and in the importance of estrogen sufficiency in the accrual and maintenance of bone mass. The weight of evidence supports an effect of androgens on the latter processes, but this effect has not been quantitated. There is a discordance in the estrogen-deficient male between skeletal growth and skeletal maturation and the accrual of bone mass and density. Estrogen synthesis by the testis is limited before puberty, and estrogen deficiency does not affect the age of pubertal onset. Estrogen deficiency in men leads to hypergonadotropism, macroorchidism, and increased testosterone levels. Estrogen lack has a significant effect on carbohydrate and lipid metabolism, and estrogen resistance was associated with evidence of premature coronary atherosclerosis in a man. These observations have highlighted the role of extraglandular estrogen synthesis and intracrine and paracrine actions. In the human, in contrast to nonprimate vertebrates, aromatase deficiency and estrogen resistance (alpha) does not seem to affect gender identity or psychosexual development. The clinical repercussions of mutations in CYP19 on the fetal-placental unit have highlighted the major role of placental aromatase in the protection of the female fetus from androgen excess, thus preventing androgen-induced pseudohermaphrodism and virilization of the mother. These features are compared with the virilization that occurs in utero in the female spotted hyena. The novel features of the aromatase deficiency syndrome in the affected female--in the fetus, during childhood, and at puberty--are discussed, including virilization at puberty and development of polycystic ovaries. The severity of the syndrome correlates with the severity of impairment of aromatase formation in expression systems. Finally, the structural consequences of missense mutations in CYP19 are described in accordance with a model of the structure of human aromatase.

Micropenis is commonly due to fetal testosterone deficiency. The clinical management of this form of micropenis has been contentious, with disagreement about the capacity of testosterone treatment to induce a functionally adequate adult penis. As a consequence, some clinicians recommend sex reversal of affected male infants. We studied 8 male subjects with micropenis secondary to congenital pituitary gonadotropin deficiency from infancy or childhood to maturity (ages 18 to 27 years). Four patients were treated with testosterone before 2 years of age (group I) and four between age 6 and 13 years (group II). At presentation, the mean penile length in group I was 1.1 cm (-4 SD; range, 0.5 to 1.5 cm) and in group II it was 2.7 cm (-3.4 SD; range, 1.5 to 3.5 cm). All patients received one or more courses of 3 intramuscular injections of testosterone enanthate (25 or 50 mg) at 4-week intervals in infancy or childhood. At the age of puberty the dose was gradually increased to 200 mg monthly and later to an adult replacement regimen. As adults, both group I and II had attained a mean final penile length of 10.3 cm 2.7 cm with a range of 8 to 14 cm (mean adult stretched penile length for Caucasians is 12.4 2.7 cm). Six of 8 men were sexually active, and all reported normal male gender identity and psychosocial behavior. We conclude that 1 or 2 short courses of testosterone therapy in infancy and childhood augment penile size into the normal range for age in boys with micropenis secondary to fetal testosterone deficiency; replacement therapy at the age of puberty results in an adult size penis within 2 SD of the mean. We found no clinical, psychologic, or physiologic indications to support conversion of affected male infants to girls. Further, the results of this study do not support the notion, derived from data in the rat, that testosterone treatment in infancy or childhood impairs penile growth in adolescence and compromises adult penile length.

Women with congenital adrenal hyperplasia due to 21-hydroxylase deficiency, especially those patients with the salt-losing form, have decreased fertility rates. Pregnancy experience in this population is limited. We report the pregnancy outcomes and serial measurements of maternal serum steroid levels in four women with classic 21-hydroxylase deficiency, three of whom were female pseudohermaphrodites with the salt-losing form. These glucocorticoid-treated women gave birth to four healthy female newborns with normal female external genitalia, none of whom were affected with 21-hydroxylase deficiency. In three women, circulating androgen levels increased during gestation, but remained within the normal range for pregnancy during glucocorticoid therapy. In the fourth patient, androgen levels were strikingly elevated during gestation despite increasing the dose of oral prednisone from 5 to 15 mg/day (two divided doses). Notwithstanding the high maternal serum concentration of androgens, however, placental aromatase activity was sufficient to prevent masculinization of the external genitalia of the female fetus and quite likely the fetal brain, consistent with the idea that placental aromatization of androgens to estrogens is the principal mechanism that protects the female fetus from the masculinizing effects of maternal hyperandrogenism. These four patients highlight key issues in the management of pregnancy in women with 21-hydroxylase deficiency, particularly the use of endocrine monitoring to assess adrenal androgen suppression in the mother, especially when the fetus is female. Recommendations for the management of pregnancy and delivery in these patients are discussed.

To assess whether fetal luteinizing hormone releasing hormone (LH-RH) neurosecretory neurons have the capacity to respond to an exogenous stimulus, a synthetic excitatory amino acid analogue, N-methyl-D-L-aspartate (NMDA; 15 mg/kg), was given rapidly intravenously to 8 chronically catheterized fetuses (130-142 days of gestation; term 147 +/- 3 days). All 8 fetuses exhibited a rise in plasma ovine luteinizing hormone (oLH) and ovine follicle-stimulating hormone (oFSH) within 5 min. The mean maximal increments of oLH (2.25 +/- 0.36 ng/ml) and oFSH (1.21 +/- 0.32 ng/ml) were significantly greater than in 6 normal saline-injected controls (oLH p < 0.0002; oFSH p < 0.03). The secretion of ovine prolactin (oPRL) and ovine growth hormone (oGH) was unaffected. LH-RH (5 microg) evoked a greater oLH response (p < 0.0009) and a greater oFSH response (p < 0.03) than NMDA (n = 6). Desensitization of the fetal gonadotrope by a potent LH-RH agonist, D-Trp6Pro9NEt-LH-RH (10 microg/day i.v. x 4 days), abolished the fetal oLH and the oFSH response to NMDA (n = 5). Moreover, D, L-2-amino-5-phosphonovalerate, a specific competitive antagonist for the NMDA receptor, completely inhibited the fetal oLH and oFSH response to NMDA, whereas D-L-2-amino-5-phosphonovalerate alone did not affect the plasma oLH or oFSH levels, the gonadotropin response to LH-RH, or the release of oGH or oPRL (n = 3). In primary ovine fetal pituitary cell cultures, NMDA (10(-10) to 10(-6) M) had no effect on oLH, oFSH, oGH, or oPRL secretion, whereas LH-RH stimulated oLH (10(-8) M; p < 0.0004) and oFSH (10(-8) M; p < 0. 0001) release, evidence that NMDA did not have a direct pituitary effect. The results suggest that NMDA induces oLH and oFSH secretion by stimulation of the fetal LH-RH pulse generator and is mediated by central NMDA receptors. Fetal LH and FSH secretion and the response to LH-RH decrease in late gestation in the ovine and human fetus. The relative importance of sex steroid dependent and sex steroid independent central nervous system inhibition in this developmental change is unclear. It appears that central neural inhibition in addition to sex steroid negative feedback contributes to the decrease in fetal gonadotropin concentrations in late gestation. NMDA did not affect fetal oGH or oPRL secretion.

Cushing's disease refers specifically to an ACTH-producing pituitary adenoma that stimulates excess cortisol production. Transsphenoidal surgery is the treatment of choice in children and adolescents, but disparate cure rates have been reported, ranging from 50-98%. The discrepancies in cure rate are due primarily to the technical success of the surgery and the length and method of follow-up. We studied 42 consecutive children and adolescents (age, < or = 18 yr) who underwent transsphenoidal exploration for the primary treatment of Cushing's disease at University of California-San Francisco from 1974-1993. Only 7 patients had persistent disease, defined as evidence of Cushing's disease within 6 months of surgery, yielding an initial remission rate of 83%. We comprehensively evaluated 26 of the 35 patients who experienced an initial remission, including testing of the ACTH-adrenocortical axis. The mean duration of follow-up is 7.2 yr (range, 1.5-13.6 yr). Seven experienced a relapse of Cushing's disease, yielding a net remission rate of 73%. Relapses occurred an average of 4.2 yr postoperatively (range, 0.75-6.2 yr). Five patients experienced relapse within 5 yr of surgery, whereas 2 relapsed more than 5 yr postoperatively. Repeat transsphenoidal surgery was performed in 8 patients with persistent or recurrent disease, and 6 of these remain in remission. Low serum or urinary cortisol measurements within the first post-operative week predicted remission of Cushing's disease, but were not necessarily predictive of long-term cure. Hypercortisolism had significant effects on bone metabolism, as reflected by both diminished bone density in the majority of patients examined and decreased growth rate. Both parameters improved after surgical care, although they did not fully normalize. We conclude that transsphenoidal surgery is a safe and effective treatment for pediatric Cushing's disease, but long-term surveillance is necessary to detect possible recurrences.

We report nine consecutive children and adolescents [five females and four males; aged 2 yr 8 months (m) to 18 yr 1 m] studied over the last 5 yr with idiopathic central diabetes insipidus. In addition to vasopressin deficiency, anterior pituitary hormone deficiencies were detected, either on evaluation at presentation or during follow-up studies over the following 3 yr. Four patients had an increased concentration of plasma PRL. One patient had multiple pituitary hormone deficiencies at diagnosis, and two others developed the same by 21 m of follow-up. Brain magnestic resonance imaging scans, performed at presentation, were originally interpreted as normal in four of nine patients, except for absence of the bright posterior pituitary signal; after retrospective review, two of nine were considered normal. All of the brain magnetic resonance imaging (MRI) scans showed positive findings by 14 m of follow-up. The first abnormal finding in all patients was isolated pituitary stalk thickening. Evaluation of cerebrospinal fluid (CSF) for hCG was positive in three of eight evaluated patients; the three positive CSF values were found at presentation and 3 and 9 m after presentation. All eight patients assessed were negative for CSF alpha-fetoprotein and cytology, and no patient had serum tumor markers. Transsphenoidal biopsy of the lesion in seven of nine patients showed a germinoma in six patients and inflammatory cells in one. The six patients with documented germinoma comprise 31% of the intracranial germinomas diagnosed in this age group at the University of California-San Francisco during the last 5 yr. The patient with mononuclear inflammatory cells on biopsy along with one other patient have had spontaneous resolution of their stalk thickening. So-called "idiopathic" central diabetes insipidus warrants close follow-up to determine the etiology, especially if anterior pituitary hormone deficiencies are detected. Normal brain MRI scans or scans that show isolated pituitary stalk thickening merit follow-up with serial contrast enhanced brain MRI for the early detection of an evolving occult hypothalamic-stalk lesion. CSF evaluation is recommended at presentation because elevated CSF hCG may precede MRI abnormalities.

Purpose: Recent studies in the rat suggest that early exposure to exogenous testosterone accelerates the loss of androgen receptors and compromises eventual penile length. To determine whether this is true in men we measured adult penile length of patients treated in childhood for sexual precocity., Materials and Methods: We examined 21 men with sexual precocity due to true precocious puberty (12) or congenital adrenal hyperplasia (9) who had been followed at our institution since childhood. Penile lengths were compared with data from normal men., Results: Mean stretched penile length plus or minus standard deviation was 12.7 +/- 2.6 cm. in all patients, 12.1 +/- 2.6 cm. in those with true precocious puberty and 13.6 +/- 1.6 cm. in those with congenital adrenal hyperplasia. These lengths were not significantly different from those of normal men (12.4 +/- 2.7 cm.)., Conclusions: In contrast to findings in rats, exposure to endogenous testosterone during gestation and/or childhood does not reduce adult penile length in men. Thus, the use of testosterone to treat childhood genitourinary anomalies would likely not compromise mature penile size.

Autosomal recessive mutations in the 17 beta-hydroxysteroid dehydrogenase 3 gene impair the formation of testosterone in the fetal testis and give rise to genetic males with female external genitalia. Such individuals are usually raised as females, but virilize at the time of expected puberty as the result of increases in serum testosterone. Here we describe mutations in 12 additional subjects/families with this disorder. The 14 mutations characterized to date include 10 missense mutations, 3 splice junction abnormalities, and 1 small deletion that results in a frame shift. Three of these mutations have occurred in more than 1 family. Complementary DNAs incorporating 9 of the 10 missense mutations have been constructed and expressed in reporter cells; 8 of the 9 missense mutations cause almost complete loss of enzymatic activity. In 2 subjects with loss of function, missense mutations testosterone levels in testicular venous blood were very low. Considered together, these findings strongly suggest that the common mechanism for testosterone formation in postpubertal subjects with this disorder is the conversion of circulating androstenedione to testosterone by one or more of the unaffected 17 beta-hydroxysteroid dehydrogenase isoenzymes.

The aromatase enzyme complex catalyzes the conversion of androgens to estrogens in a wide variety of tissues, including the ovary, testis, placenta, brain, and adipose tissue. Only a single human gene encoding aromatase P450 (CYP19) has been isolated; tissue-specific regulation is controlled in part by alternative promoters in a tissue-specific manner. We report a novel mutation in the CYP19 gene in a sister and brother. The 28-yr-old XX proband, followed since infancy, exhibited the cardinal features of the aromatase deficiency syndrome as recently defined. She had nonadrenal female pseudohermaphrodism at birth and underwent repair of the external genitalia, including a clitorectomy. At the age of puberty, she developed progressive signs of virilization, pubertal failure with no signs of estrogen action, hypergonadotropic hypogonadism, polycystic ovaries on pelvic sonography, and tall stature. The basal concentrations of plasma testosterone, androstenedione, and 17-hydroxyprogesterone were elevated, whereas plasma estradiol was low. Cyst fluid from the polycystic ovaries had a strikingly abnormal ratio of androstenedione and testosterone to estradiol and estrone. Hormone replacement therapy led to breast development, menses, resolution of ovarian cysts, and suppression of the elevated FSH and LH values. Her adult height is 177.6 cm (+2.5 SD). Her only sibling, an XY male, was studied at 24 yr of age. During both pregnancies, the mother exhibited signs of progressive virilization that regressed postpartum. The height of the brother was 204 cm (+3.7 SD) with eunuchoid skeletal proportions, and the weight was 135.1 kg (+2.1 SD). He was sexually fully mature and had macroorchidism. The plasma concentrations of testosterone (2015 ng/dL), 5 alpha-dihydrotestosterone (125 ng/dL), and androstenedione (335 ng/dL) were elevated; estradiol and estrone levels were less than 7 pg/mL. Plasma FSH and LH concentrations were more than 3 times the mean value. Plasma PRL was low; serum insulin-like growth factor I and GH-binding protein were normal. The bone age was 14 yr at a chronological age of 24 3/12 yr. Striking osteopenia was noted at the wrist. Bone mineral densitometric indexes of the lumbar spine (cancellous bone) and distal radius (cortical bone) were consistent with osteoporosis; the distal radius was -4.7 SD below the mean value for age- and sex-matched normal men; indexes of bone turnover were increased. Hyperinsulinemia, increased serum total and low density lipoprotein cholesterol, and triglycerides and decreased high density lipoprotein cholesterol were detected.(ABSTRACT TRUNCATED AT 400 WORDS)

We report a long term study on the effectiveness of chronic GnRH agonist treatment on final or near-final height in 26 patients (20 females and six males) with true precocious puberty (TPP). This study differs from other treatment studies in that the median age at onset of therapy was 4.7 yr for females and 6.2 yr for males, the youngest cohort of treated patients reported to date. We compared patients treated with GnRH agonists who attained final or near-final height with a historical control group of untreated children with TPP (n = 116) matched for mean age of pubertal onset, etiology of TPP (idiopathic or neurogenic), rate of progression, and sex ratio. The current mean height of GnRH agonist-treated females who began therapy at more than 5 yr of age (157.6 +/- 6.6 cm) is already significantly greater than the mean final height of untreated females (152.7 +/- 8.6 cm). The current mean predicted height of the treated females is 164.6 +/- 9.7 cm. The current mean height of females whose treatment was started before 5 yr of age is greater (164.1 +/- 7.7 cm) than that of females whose treatment began after 5 yr of age (157.6 +/- 6.6 cm). The final height of untreated children whose age of sexual precocity was less than 5 yr at diagnosis is significantly less than that of treated patients who were less than 5 yr when they developed TPP (P = 0.0006). The current mean height of GnRH agonist-treated males is 166.3 +/- 12.2 cm, and the current mean predicted height is 170.8 +/- 11.3 cm. This is in sharp contrast to the mean final height of untreated males (155.6 +/- 7.7 cm). The current predicted height correlates negatively with the age at initiation of treatment and the initial bone age and positively with height SD for bone age in the agonist-treated children. The current mean height deviation from target height is significantly less in the 20 treated females (-1 SD) than in 93 untreated females (-2.4 SD; P = 0.006). The mean final height deviation from target height in 23 untreated males (-3.7 SD) is significantly greater than the current height deviation from target height in 6 treated males (-1.7 SD; P = 0.03). The salutary effects of long term GnRH agonist therapy on stature are more clear-cut in the younger treated children. Young untreated children may have the worst outcome with respect to final height.(ABSTRACT TRUNCATED AT 400 WORDS)

N-Methyl-D-aspartate (NMDA) directly stimulates gonadotropin-releasing hormone (GnRH) neurons to secrete GnRH. It is not known if this stimulatory effect of NMDA is mediated by NO. Northern blot analysis of the immortalized hypothalamic GnRH neuronal cells (GT1-1) mRNA with a neuronal NOS cDNA revealed this clonal cell line expressed neuronal NOS transcripts as a single 10.5-kb band. Immunoblot analysis of GT1-1 proteins with anti-neuronal NOS serum showed that the GT1-1 cells contain neuronal NOS. GT1-1 cells were used to study the effects of NO and NMDA on GnRH release. L-Arginine (10(-2) M), a precursor of NO enhances basal GnRH secretion. Both oxyhemoglobin (Hb)(10(-6)-10(-4) M), a NO scavenger and N omega-nitro-L-arginine (NNA)(10(-3),10(-2) M), a NOS inhibitor and inactivator block basal as well as NMDA-induced GnRH release. Sodium nitroprusside (SNP) (10(-4), 10(-3) M), a NO donor stimulates GnRH release, an effect inhibited by Hb. Incubation of GT1-1 cells in Ca(2+)-free medium abolished the stimulatory effect of NMDA on GnRH release. In contrast, incubation in medium with increasing concentrations of Ca2+ enhances basal GnRH release as well as augments NMDA-mediated GnRH release. The results demonstrate that L-arginine-NO pathway is functional in the GT1-1 cells and an increase in intracellular Ca2+ [Ca2+]i following NMDA receptor activation activates NOS to generate NO. We conclude that endogenous NO mediates, at least in part, basal as well as NMDA-stimulated GnRH release and may play a role as an intercellular messenger in synchronizing pulsatile GnRH release.

We report the features of a new syndrome of aromatase deficiency due to molecular defects in the CYP19 (P450arom) gene in a 46,XX female. At birth, the patient presented with a nonadrenal form of female pseudohermaphrodism. At 17 months of age, laparotomy revealed normal female internal genital structures; the histological appearance of the ovaries was normal. FSH concentrations were markedly elevated at 9.4 ng/mL LER 869, and estrone and estradiol levels were undetectable (< 37 pmol/L). By 14 yr of age, she had failed to exhibit breast development. The clitoris had enlarged to 4 x 2 cm, and pubic hair was Tanner stage IV. The plasma concentration of testosterone was elevated at 3294 pmol/L, as was androstenedione at 9951 pmol/L. Plasma estradiol levels were below 37 pmol/L. ACTH and dexamethasone tests indicated a nonadrenal source of testosterone and androstenedione. Plasma gonadotropin levels were in the castrate range. Pelvic sonography and magnetic resonance imaging showed multiple 4- to 6-cm ovarian cysts bilaterally. Despite increased circulating androgens and clitoral growth, the bone age was 10 yr at chronologic age 14 2/12 yr. Estrogen replacement therapy resulted in a growth spurt, breast development, menarche, suppression of gonadotropin levels, and resolution of the cysts. The clinical findings suggested the diagnosis of P450arom deficiency. Analyses of genomic DNA from ovarian fibroblasts demonstrated two single base changes in the coding region of the P450arom gene, one at 1303 basepairs (C-T), R435C, and the other at 1310 basepairs (G-A), C437Y, in exon 10. The molecular genetic studies indicate that the patient is a compound heterozygote for these mutations. Expression of these mutations showed that the R435C mutation had 1.1% the activity of the wild-type P450arom enzyme, whereas the C437Y mutation demonstrated no activity. The cardinal features of this syndrome are a consequence of P450arom deficiency: 1) the fetal masculinization in this syndrome can be ascribed to defective placental conversion of C19 steroids to estrogens, leading to exposure of the female fetus to excessive amounts of testosterone; 2) the pubertal failure, mild virilization, multicystic ovaries, and hyperstimulation of the ovaries by FSH and LH are the result of the inability of the ovary to aromatize testosterone and androstenedione to estrogens; and 3) the striking delay in bone age at 14 2/12 yr supports the notion that estrogens, in contrast to androgens, are the major sex steroid driving skeletal maturation during puberty. Familial P450arom deficiency, although rare, may be more common than previously suspected.(ABSTRACT TRUNCATED AT 400 WORDS)

Previous studies demonstrated that an excitatory amino acid analog, N-methyl-D-aspartate (NMDA), stimulates GnRH secretion in the rat, prepubertal primate, and ovine fetus at the hypothalamic level. It is not known if this stimulatory effect of NMDA is mediated directly on the GnRH neurosecretory neuron. A hypothalamic GnRH neuronal cell line (GT1-1) provided a useful model system to study the effect of NMDA on GnRH release by both superfusion and static incubation techniques. Studies with GT1-1 cells indicate that GnRH neurons exhibit spontaneous autorhythmicity and function intrinsically as a neuronal oscillator for the synchronous discharge of GnRH pulses. In static incubation studies, 10(-4) and 10(-3) M NMDA increased GnRH release, whereas 10(-6), 10(-5), and 10(-2) M NMDA had no effect. A competitive NMDA receptor antagonist, AP-5 (10(-4)-10(-2) M), and a noncompetitive NMDA receptor antagonist, MK-801 (10(-12)-10(-5) M), inhibited the action of NMDA. Superfusion of GT1-1 cells after a 90-min control period followed by either continuous NMDA or intermittent NMDA (10(-4) and 10(-3) M) for 90 min increased GnRH pulse amplitude by 100-400%, but had no effect on the interpulse interval (17 min by Cluster); 10(-6), 10(-5), and 10(-2) M NMDA had no effect on either pulse amplitude or interpulse interval. MK-801 (10(-5) M) attenuated the stimulatory effect of NMDA on GnRH pulse amplitude. Incubation in glycine-free and high magnesium medium abolished the action of NMDA on GnRH release. Hybridization analysis of GT1-1 mRNA with an NMDA R1 receptor cDNA showed that this pure neuronal cell line expressed NMDA receptor transcripts observed as a 4.2-kilobase band. The results demonstrate that NMDA stimulates GnRH neurons directly to secrete GnRH through their NMDA receptors by increasing pulse amplitude without affecting pulse frequency.

Diagnostic accuracy of magnetic resonance imaging (MRI) interpretation was assessed prospectively in patients with ambiguous genitalia or intersex problems. MRI depiction of the uterus was possible in 93%, the vagina in 95%, the penis in 100%, the testis in 88%, and the ovary in 74% of patients. The strength of MRI lies in the multiplanar capability and tissue characterization by means of T1- and T2-weighted sequences. MRI contributes to accurate morphologic evaluation of müllerian duct structures, the gonads, and the development of the phallus, all of which are essential for appropriate gender assignment and planning of surgical reconstruction.

We identified two mutations in the CYP19 gene responsible for aromatase deficiency in an 18-year-old 46,XX female with ambiguous external genitalia at birth, primary amenorrhea and sexual infantilism, and polycystic ovaries. The coding exons, namely exons II-X, of the CYP19 gene were amplified by PCR from genomic DNA and sequenced directly. Direct sequencing of the amplified DNA from the patient revealed two single-base changes, at bp 1303 (C-->T) and bp 1310 (G-->A) in exon X, which were newly found missense mutations and resulted in codon changes of R435C and C437Y, respectively. Subcloning followed by sequencing confirmed that the patient is a compound heterozygote. The results of restriction fragment length polymorphism analysis and direct sequencing of the amplified exon X DNA from the patient's mother indicate maternal inheritance of the R435C mutation. Transient expression experiments showed that the R435C mutant protein had approximately 1.1% of the activity of the wild type, whereas C437Y was totally inactive. Cysteine-437 is the conserved cysteine in the heme-binding region believed to serve as the fifth coordinating ligand of the heme iron. To our knowledge, this patient is the first adult to have described the cardinal features of a syndrome of aromatase deficiency. Recognition that such defects exist will lead to a better understanding of the role of this enzyme in human development and disease.

Purpose: We report the MR findings in nine patients with clinical and laboratory evidence of Kallmann syndrome (KS), a genetic disorder of olfactory and gonadal development. In patients with KS, cells that normally express luteinizing hormone-releasing hormone fail to migrate from the medial olfactory placode along the terminalis nerves into the forebrain. In addition, failed neuronal migration from the lateral olfactory placode along the olfactory fila to the forebrain results in aplasia or hypoplasia of the olfactory bulbs and tracts. Patients with KS, therefore, suffer both reproductive and olfactory dysfunction., Methods: Nine patients with KS underwent direct coronal MR of their olfactory regions in order to assess the olfactory sulci, bulbs, and tracts. A 10th patient had MR findings of KS, although the diagnosis is not yet confirmed by laboratory tests., Results: Abnormalities of the olfactory system were identified in all patients. In particular, the anterior portions of the olfactory sulci were uniformly hypoplastic. The olfactory bulbs and tracts appeared hypoplastic or aplastic in all patients in whom the bulb/tract region was satisfactorily imaged. In two (possibly three) patients, prominent soft tissue in the region of the bulbs suggests radiographic evidence of neurons that have been arrested before migration., Conclusions: Previous investigators of patients with KS used axial MR images to demonstrate hypoplasia of the olfactory sulci but offered no assessment of the olfactory bulbs. In the present study we used coronal images to show hypoplasia of both olfactory sulci and bulbs. In addition, we found what we believe to be the radiologic correlate of arrested neuronal migration in KS.

The LHRH-secreting hypothalamic hamartoma (HH), a congenital malformation consisting of a heterotopic mass of nervous tissue that contains LHRH neurosecretory neurons attached to the tuber cinereum or the floor of the third ventricle, can cause true or central precocious puberty (TPP). We have suggested that it functions as an ectopic LHRH pulse generator independent of the central nervous system inhibitory mechanism that normally restrains the hypothalamic LHRH pulse generator. TPP associated with a hamartoma has all of the hormonal hallmarks of puberty, including a pubertal pattern of pulsatile LH and a pubertal plasma LH response to LHRH administration. Little is known about the natural history of HH. We present long term data on 10 children (5 females and 5 males) with TPP due to HH. Physical signs of puberty were observed at a mean age of 2.2 +/- 1.6 yr (range, 0.5-5.1). Two of 10 had a pedunculated mass, and 8 of 10 had a sessile mass. The hamartoma varied in diameter from 4-25 mm and did not change with time (3.5-8.7 yr). Four patients have a seizure disorder, 3 with gelastic seizures (1 with mental retardation) and 1 with tonic-clonic seizures. The shape of the hamartoma, sessile or pedunculated, did not correlate with the occurrence of seizures. At presentation with sexual precocity, the mean height SD for chronological age was +3.5 +/- 0.4, the mean height SD for bone age was -1.9 +/- 0.4, and the mean bone age SD for chronological age was +6.8 +/- 0.7. Baseline data were comparable to those of 10 females with idiopathic TPP. Nine of 10 HH patients and all idiopathic TPP patients were treated with a LHRH agonist. The response to therapy was excellent in both groups and indistinguishable. Nine of 10 HH children attend school regularly and, aside from those with seizures, have no neurological handicap. While surgical resection of the hamartoma has been recommended, it carries an increased risk of morbidity and mortality and, if removal is incomplete, does not arrest the sexual precocity. In our experience, LHRH agonist therapy for TPP due to HH is the preferable approach.

To analyze the secretion patterns of LH, FSH, GH, and PRL in the late gestational sheep fetus in vivo, we measured simultaneous plasma levels of these hormones during a period of frequent sampling under basal conditions (samples every 15 min for 5 h) in 17 chronically catheterized sheep fetuses. To calculate mean plasma levels and areas under the curve, we analyzed hormone pulses and coincident pulse patterns to assess interactions between the release of these pituitary hormones. Mean plasma levels for all fetuses were: LH, 0.8 +/- 0.2 ng/ml (mean +/- SEM); FSH, 4.6 +/- 0.7 ng/ml; GH, 136.6 +/- 16.5 ng/ml; and PRL, 40.5 +/- 10.3 ng/ml. Pulse analysis detected 20 LH pulses during 5100 min of total sampling time, which gave a mean interpulse interval of 255.0 min. For GH, 37 pulses were detected; the mean interpulse interval was 129.7 min. Twenty PRL pulses yielded a mean interpulse interval of 225.0 min. FSH pulses could not be analyzed due to the long half-life of this hormone, but hormone level fluctuations were screened for maxima. A new method was developed to detect an interaction between hormone pulses. The probability of the simultaneous occurrence of hormone pulses was calculated and compared with the rate of coincidences found in the experiments. Analysis of copulsatile release of LH, GH, and PRL revealed 11 GH pulses coinciding with the LH pulses (P = 0.0020). An interaction between the pulsatile release of LH and GH can, therefore, be assumed. There was also a significant interaction between GH and PRL. Seven PRL pulses preceded the GH pulses by 15 min (P = 0.0014). In contrast, no significant copulsatile release could be observed between LH and PRL; 95.5% of LH pulses were accompanied by a maximum FSH level, suggesting an interaction between LH and FSH secretion. In summary, we show that LH, GH, and PRL (and possibly FSH) are secreted in a pulsatile fashion in the ovine fetus. Furthermore, the pulsatile releases of LH, FSH, and GH as well as GH and PRL are temporarily coupled, as demonstrated by a significant number of coincident pulses between LH/GH and GH/PRL and a high number of FSH hormone maxima concomitant with LH pulses.

The detection of pulsatile ovine LH (oLH) secretion in the sheep fetus by 81 days gestation (term 147 days), the suppression of fetal gonadotropin secretion by chronic administration of an LH-releasing factor agonist or antagonist, and the capacity of N-methyl; D-aspartate (a neuroexcitatory amino acid analog) to evoke a fetal oLH pulse strongly support a functional LH-releasing factor pulse-generator in the ovine fetus. In light of the sex difference in fetal gonadal function and gonadotropin secretion before day 114, we postulated that fetal castration would have a discordant effect on the pattern of gonadotropin secretion in males and females. Fetal sheep were either castrated (male = 11; female = 9) or sham operated (male = 9; female = 6) at 110-115 days gestation. Chronic indwelling arterial and venous catheters were implanted, and animals were studied for up to 30 days. During each study period fetal arterial blood samples were drawn every 15 min for 5 h and the plasma assayed for oFSH and oLH by specific RIAs. Multiple studies were performed on each fetus. In all fetuses (both intact and castrated) a decrease in oLH pulse frequency occurred after day 130. In female fetuses before day 130, castration had no effect on mean oLH pulse frequency (sham, 0.72 +/- 0.19 pulses/5 h; castrate, 0.50 +/- 0.13 pulses/5 h; P greater than 0.05). After day 130, pulsatile oLH secretion decreased in both intact and castrated female fetuses to undetectable levels during the sampling period. In contrast, castration significantly (P less than 0.001) increased mean oLH pulsatility in males before and after day 130 (less than 130 days, sham, 1.06 +/- 0.24 pulse/5 h; castrate, 2.70 +/- 0.22 pulse/5 h; greater than 130 days, sham, 0.18 +/- 0.12 pulses/5 h; castrate, 1.65 +/- 0.26 pulses/5 h). Mean oLH pulse amplitude was increased by castration only in the male fetuses (sham, 3.89 +/- 0.87 ng/ml; castrate, 6.02 +/- 0.39 ng/ml; P less than 0.05). oFSH pulses were infrequent in both sexes and not influenced by castration. The mean plasma concentration of oFSH was greater in intact female fetuses than in intact males (female, 5.65 +/- 1.15 ng/ml vs. male, 2.07 +/- 0.45 ng/ml; P less than 0.01). Castration increased the mean value for plasma oFSH in males (4.40 +/- 0.43 ng/ml; P less than 0.001) but had no effect in females (3.83 +/- 0.64 ng/ml; P greater than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

Plasma ovine growth hormone (oGH) concentrations are strikingly elevated in the ovine fetus and decline at birth towards the low levels observed in the newborn lamb. We postulated that developmental changes in somatotrope function secondary to GH-releasing factor (GRF) desensitization and GH feedback play a role in the developmental pattern of oGH secretion and tested this hypothesis in vito in chronically catheterized ovine fetuses (123-145 days gestation; term 147 days) and newborn lambs (1-18 days). In the first set of studies, two consecutive intravenous GRF(1-44 amide) boluses (1 microgram/kg) were administered. When the GRF boluses were given 90 min apart, they elicited similar oGH responses, both in fetuses and in newborn lambs. In contrast, when the GRF boluses were given 20 min apart, a significant oGH response was evoked by the first GRF but an oGH response was not detected after the second GRF, either in fetuses or in newborn lambs. When the oGH response GRF(1-44 amide; 1 microgram/kg i.v.) was evaluated 40 min after the start of a human GH infusion (25 micrograms/kg hGH bolus followed by 0.5 microgram/kg/min hGH for 80 min, resulting in mean hGH plasma concentrations of 80 ng/ml), the exogenous hGH did not after the oGH response to GRF, either in fetuses or in newborn lambs. The present in vivo results demonstrate that the fetal and the neonatal somatotrope can be desensitized to GRF and suggest that a short-term latency, ultrashortloop GH feedback mechanism is not operative, either in the ovine fetus or in the newborn lamb.(ABSTRACT TRUNCATED AT 250 WORDS)

The relative contributions of GH, insulin-like growth factor-I (IGF-I), estradiol, and testosterone to the pubertal growth spurt are incompletely understood. We studied 8 patients (5 girls and 3 boys) with true precocious puberty and GH deficiency due to CNS lesions to assess the role of sex steroids in pubertal growth independent of an increase in circulating GH. Included is 1 patient with an unusual hypothalamic lesion due to head trauma. A control group of 17 GH-sufficient patients with true precocious puberty (13 girls and 4 boys) was matched for chronological age. The GH-deficient girls grew at a mean velocity of 9.2 cm/yr (range, 7.2-14.4), and the boy's mean height velocity was 7.9 cm/yr (6.1-9.9). Mean bone age was advanced in the GH-deficient group (girls, +2.7 SD; boys, +2.6 SD), but not as much as the GH-sufficient controls (girls, +5.4 SD; boys, +4.3 SD). The mean concentration of plasma IGF-I was lower in the GH-deficient group than in the control group, but was greater than the mean concentration in age-matched prepubertal GH-deficient patients. Four GH-deficient patients were treated with a potent agonist of LRF. This caused suppression of gonadal sex steroid concentrations and a fall in mean height velocity from 9.1 to 4.3 cm/yr after 1 yr of therapy; however, circulating GH and IGF-I values were not uniformly altered. We conclude that a substantial pubertal growth spurt can occur in patients with true precocious puberty and GH deficiency that is dependent on gonadal sex steroids yet unaccompanied by normal pubertal levels of circulating GH or IGF-I. Reversal of this growth acceleration is possible with sex steroid suppression. The results, in light of previous in vivo and in vitro studies, suggest that the normal pubertal growth spurt is mediated in part by direct effects of sex steroids at the growth plate.

We previously reported that GH secretion evoked by GHRH is inhibited after 5 days of treatment with im GH. This impaired pituitary response was associated with a significant increase in the serum concentration of insulin-like growth factor I (IGF-I). To dissociate the possible effects of circulating IGF-I from other effects of GH on the pituitary response to GHRH, we carried out the following study in eight normal men. A bolus injection of GHRH (1 microgram/kg, iv) was administered 2 h after the start of a 4-h continuous iv infusion of GH (180-micrograms bolus dose, then 3 micrograms/min in 150 mmol/L NaCl) or placebo (150 mmol/L NaCl). In addition, a similar injection of GHRH was given 4 h after the start of a 6-h continuous iv GH infusion (180-micrograms bolus dose, then 3 micrograms/min). During the GH infusions, plasma GH levels reached steady state concentrations in the 9-13 micrograms/L range. The mean GHRH-induced GH response was not significantly blunted during the 4-h infusions of GH [724 +/- 163 (+/- SE) vs. 1184 +/- 373 micrograms.min/L during placebo; P = 0.29], but was significantly inhibited during the 6-h GH infusions (226 +/- 105 micrograms.min/L; P = 0.04 vs. control). Serum IGF-I or plasma glucose did not change significantly throughout the GH infusions. During the 6-h GH infusions, plasma FFA increased to levels significantly above basal values during the last 3 h of the 6-h infusion. These results indicate that short term GH infusion inhibits the plasma GH response to GHRH in a time-dependent manner. The inhibition is not due to changes in circulating IGF-I and glucose concentrations. Fluctuations in hypothalamic somatostatin secretion, changes in lipid or other GH-dependent metabolites, paracrine effects of IGF-I, or a direct effect of GH itself may cause the impaired pituitary responsiveness during short term iv GH infusion.

The administration of a dopamine antagonist, haloperidol, to the ovine fetus in late gestation elevates plasma concentrations of PRL, suggesting tonic dopaminergic inhibition of fetal PRL secretion. The source of this dopaminergic inhibition was investigated in chronically catheterized ovine fetuses (104-135 days of gestation) after hypophysial stalk section (SS; n = 4) and in sham-operated controls (CON; n = 7). Basal PRL levels were similar in the two groups of fetuses. After the administration of TRF (250 micrograms, iv), PRL levels rose comparably in both the SS and CON fetuses. The only difference was a higher mean incremental response (P less than 0.02) in the SS fetuses. The dopamine agonist apomorphine (100 micrograms/kg, iv) induced a similar suppression of fetal PRL concentrations in CON (n = 4) and SS (n = 2) fetuses. After the administration of haloperidol (1 mg, iv) to the CON fetuses (n = 7), the concentration of fetal PRL rose (P less than 0.01). In the SS fetus (n = 4), haloperidol induced a rise in PRL concentrations (P less than 0.01); however, the response to haloperidol was less (P less than 0.01) in SS than in CON fetuses. These data suggest that there is persistent dopaminergic inhibition of PRL secretion in the fetus after complete stalk section, and that the source of this dopamine is extrahypothalamic. The greater incremental PRL response to TRH and the lesser response to haloperidol in the SS fetus than in CON are evidence for a hypothalamic component to the dopaminergic inhibition in the intact fetus. Basal FSH concentrations and the gonadotropin response to LRF were not affected by stalk section in fetuses studied 5-8 days after surgery. Both the PRL and the GH responses to 5-hydroxytryptophan were abolished by stalk section. After stalk section GH levels fell, however, significant concentrations of GH were measurable in fetal plasma in late gestation, which suggests that the fetal pituitary can secrete GH in the absence of hypothalamic stimulation at this stage in gestation.

Fifteen short but otherwise normal children, 4.3 to 15.5 years old, with heights greater than 3 S.D. below the mean value for age, growth rates less than or equal to 5.0 cm per year, and normal serum levels of immunoreactive growth hormone in response to provocative stimuli (peak greater than or equal to 10 ng per milliliter) were treated with intramuscular injections of pituitary growth hormone (0.1 U per kilogram) three times weekly for six months, as were 14 children with documented growth hormone deficiency. In all the latter children growth rate increased by more tan 2.0 cm per year during treatment. In 6 of the 14 short normal children who remained prepubertal, growth rate also increased, by 2.2 to 4.2 cm per year during treatment; four of these children had normal base-line serum somatomedin C concentrations. In both short normal children and children with growth hormone deficiency, the increment in serum somatomedin C concentrations after 4 or 10 daily injections of growth hormone correlated with bone age but not with later growth or growth hormone levels. Among the short normal children, those who responded to growth hormone were younger and had a greater delay in bone age and a slower pretreatment growth rate than the nonresponders. These observations suggest that a dose of growth hormone comparable to that used for the treatment of hypopituitarism increases growth rate in some short normal children.