Therapeutic effects of melatonin in female mice with central precocious puberty by regulating the hypothalamic Kiss-1/Kiss1R system.

In recent years, central precocious puberty (CPP) in children is becoming more common, which seriously affects their physical and psychological health and requires finding a safe and effective treatment method. The aim of this study was to investigate the therapeutic effect of melatonin on CPP. A CPP model was established by subcutaneous injection of 300 micrograms of danazol into 5-day-old female mice, followed by treatment with melatonin and leuprolide. The vaginal opening was checked daily. Mice were weighed, gonads were weighed, gonadal index was calculated, and gonadal development was observed by hematoxylin and eosin (HE) staining. Serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) levels were measured by ELISA. By using RT-PCR and Western blotting, the mRNA and protein expression of the hypothalamus Kiss-1, Kiss-1 receptor (Kiss1R), gonadotropin-releasing hormone (GnRH), and pituitary GnRH receptor (GnRHR) were identified. The results showed that melatonin delayed vaginal opening time and reduced body weight, gonadal weight and indices in female CPP mice. Melatonin treatment prevents uterine wall thickening and ovarian luteinization in female CPP mice. Melatonin treatment reduces serum concentrations of FSH, LH, and E2 in female CPP mice. Melatonin suppressed the expressions of Kiss-1, Kiss1R and GnRH in the hypothalamus, and the expression of GnRHR in the pituitary of the female CPP mice. Our results suggest that melatonin can inhibit the hypothalamic-pituitary-gonadal (HPG) axis by down-regulating the Kiss-1/Kiss1R system, thereby treating CPP in female mice.

Relationship between Body Mass Index and Bone Turnover Markers in Girls with Idiopathic Central Precocious Puberty.

Background: This study aimed to determine the effect of body mass index (BMI) on bone turnover markers in girls with idiopathic central precocious puberty (ICPP) according to weight status at diagnosis. Methods: Two hundred and eleven girls with ICPP were divided according to their weight status at diagnosis into three groups: normal weight, overweight, and obese. The serum levels of total procollagen type 1 N-terminal propeptide (P1NP), N-terminal midfragment of osteocalcin, ß-C-terminal telopeptide of type 1 collagen, and some biochemical indicators were measured. Associations between variables were evaluated by multiple regression analysis. Results: Serum P1NP concentrations were significantly different among groups (p < 0.001). No other significant differences were noted in N-terminal midfragment of osteocalcin and ß-C-terminal telopeptide of type 1 collagen. BMI was associated with estradiol (r = 0.155, p < 0.05) and inversely associated with P1NP (r = -0.251, p < 0.01), luteinizing hormone peak (r = -0.334, p < 0.01), follicle-stimulating hormone peak (r = -0.215, p < 0.01), and luteinizing hormone/follicle-stimulating hormone peak (r = -0.284, p < 0.01). Multiple regression analysis of factors associated with BMI showed that it was correlated with P1NP, follicle-stimulating hormone base, and luteinizing hormone peak in the overweight group and the obese group. Conclusions: Our findings showed that BMI was associated with P1NP, revealing the reduction of bone formation in overweight and obese girls with ICPP. During the diagnosis and treatment of girls with ICPP, attention should be paid to body weight and bone metabolism.

MKRN3 inhibits puberty onset via interaction with IGF2BP1 and regulation of hypothalamic plasticity.

Makorin ring finger protein 3 (MKRN3) was identified as an inhibitor of puberty initiation with the report of loss-of-function mutations in association with central precocious puberty. Consistent with this inhibitory role, a prepubertal decrease in Mkrn3 expression was observed in the mouse hypothalamus. Here, we investigated the mechanisms of action of MKRN3 in the central regulation of puberty onset. We showed that MKRN3 deletion in hypothalamic neurons derived from human induced pluripotent stem cells was associated with significant changes in expression of genes controlling hypothalamic development and plasticity. Mkrn3 deletion in a mouse model led to early puberty onset in female mice. We found that Mkrn3 deletion increased the number of dendritic spines in the arcuate nucleus but did not alter the morphology of GnRH neurons during postnatal development. In addition, we identified neurokinin B (NKB) as an Mkrn3 target. Using proteomics, we identified insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) as another target of MKRN3. Interactome analysis revealed that IGF2BP1 interacted with MKRN3, along with several members of the polyadenylate-binding protein family. Our data show that one of the mechanisms by which MKRN3 inhibits pubertal initiation is through regulation of prepubertal hypothalamic development and plasticity, as well as through effects on NKB and IGF2BP1.

Diagnostic Value of LH Peak Value of the GnRH Stimulation Test for Girls with Precocious Puberty and Its Correlation with Body Mass Index.

Objective: To analyze the diagnostic value of luteinizing hormone (LH) peak value of the gonadotropin-releasing hormone (GnRH) stimulation test for girls with precocious puberty and its correlation with body mass index (BMI). Methods: A total of 230 girls with precocious puberty who came to our hospital for testing from June 2019 to June 2021 were selected and divided into a true group (n = 130) and sham group (n = 100) according to the results of the GnRH stimulation test. According to the BMI, the true group was further divided into a normal group (48 cases), overweight group (43 cases), and obese group (39 cases). The GnRH stimulation test was performed on all subjects, and the basal value and peak value of LH and the basal value and peak value of follicle-stimulating hormone (FSH) were recorded. The general data and serological indexes of the true group and the sham group were compared. Indicators of the GnRH stimulation test, breast stage, bone age, BMI, uterine volume, ovarian volume, and serological indicators (leptin, sex hormone-binding protein (SHBG), and adiponectin (APN)) were compared among the normal group, the overweight group, and the obese group. Results: There were no significant differences in age and breast stage between the true group and the sham group (P > 0.05). There were statistically significant differences in bone age, BMI, uterine volume, and ovarian volume between the two groups (P < 0.05). The LH base value, LH peak value, FSH base value, and FSH peak value in the true group were higher than those in the sham group, and the differences were statistically significant (P < 0.05). ROC curve analysis showed that the AUC of LH peak value in diagnosing girls with precocious puberty was 0.973, which was higher than 0.895, 0.875, and 0.912 of LH base value, FSH base value, and FSH peak value, respectively. There were statistically significant differences in LH base value, LH peak value, FSH base value, breast development stage, bone age, BMI, SHBG, leptin, and APN among the normal group, overweight group, and obese group (P < 0.05), but there were no significant differences in FSH peak value, uterine volume, and ovarian volume among the three groups (P > 0.05). There was a negative correlation between BMI, LH peak value, and FSH base value (P < 0.05), but there was no significant correlation between BMI and FSH peak value (P > 0.05). Conclusion: The LH peak value of the GnRH stimulation test has high diagnostic value for girls with precocious puberty, and BMI is negatively correlated with the LH peak value of CPP children.

The m6A mRNA demethylase FTO regulates GnRH secretion in Mn-induced precocious puberty.

The GABAA receptor (GABAAR) plays important roles in the regulation of Mn-induced GnRH secretion in immature female rats. However, the underlying molecular mechanisms remain unknown. Here, we assessed whether FTO and its substrate m6A are correlated with GABAAR expression in GnRH neurons after treatment with Mn in vitro and in vivo. Our study indicated that Mn treatment increased the expression of GnRH mRNA and decreased the levels of GABAAR protein but had no effect on GABAAR mRNA. Moreover, Mn upregulated the levels of FTO and inhibited global cellular m6A levels and GABAAα2 mRNA m6A levels. Knockdown of FTO increased the expression of GABAAR protein and GABAAα2 mRNA m6A levels. Data from rat models further demonstrate that inhibition of FTO suppressed GABAAR protein expression in the hypothalamus, causing delayed puberty onset. Collectively, our findings suggest that FTO-dependent m6A demethylation plays a critical role in regulating GABAAR mRNA processing in GnRH neurons.

A Putative Adverse Outcome Pathway Network for Disrupted Female Pubertal Onset to Improve Testing and Regulation of Endocrine Disrupting Chemicals.

The average age for pubertal onset in girls has declined over recent decades. Epidemiological studies in humans and experimental studies in animals suggest a causal role for endocrine disrupting chemicals (EDCs) that are present in our environment. Of concern, current testing and screening regimens are inadequate in identifying EDCs that may affect pubertal maturation, not least because they do not consider early-life exposure. Also, the causal relationship between EDC exposure and pubertal timing is still a matter of debate. To address this issue, we have used current knowledge to elaborate a network of putative adverse outcome pathways (pAOPs) to identify how chemicals can affect pubertal onset. By using the AOP framework, we highlight current gaps in mechanistic understanding that need to be addressed and simultaneously point towards events causative of pubertal disturbance that could be exploited for alternative test methods. We propose 6 pAOPs that could explain the disruption of pubertal timing by interfering with the central hypothalamic trigger of puberty, GnRH neurons, and by so doing highlight specific modes of action that could be targeted for alternative test method development.

The first central precocious puberty proteomic profiles revealed multiple metabolic networks and novel key disease-associated proteins.

Though central precocious puberty (CPP) as a disease that seriously affects the development of a child is increasing year by year, treatment options remain limited and is the same as the 1980s' method. These are mainly due to the complex pathogenesis of central precocious puberty. Therefore, systems biology approach to identify and explore the multiple factors related to the pathogenesis of central precocious puberty is necessary. Our data established the first proteome profile of CPP revealed 163 down-regulated and 129 were up-regulated differentially expressed proteins. These altered proteins were primarily enriched in three metabolic process including energy metabolism, amino acid metabolism and nitrogenous base metabolism. The identified altered members of the metabolic signaling are valuable and potential novel therapeutic targets of central precocious puberty.

Diet-Induced Hypothalamic Inflammation, Phoenixin, and Subsequent Precocious Puberty.

Recent studies have shown a rise in precocious puberty, especially in girls. At the same time, childhood obesity due to overnutrition and energy imbalance is rising too. Nutrition and fertility are currently facing major challenges in our societies, and are interconnected. Studies have shown that high-fat and/or high-glycaemic-index diet can cause hypothalamic inflammation and microglial activation. Molecular and animal studies reveal that microglial activation seems to produce and activate prostaglandins, neurotrophic factors activating GnRH (gonadotropin-releasing hormone expressing neurons), thus initiating precocious puberty. GnRH neurons' mechanisms of excitability are not well understood. In this review, we study the phenomenon of the rise of precocious puberty, we examine the physiology of GnRH neurons, and we review the recent literature regarding the pathophysiological mechanisms that connect diet-induced hypothalamic inflammation and diet-induced phoenixin regulation with precocious puberty.

Case Report: Lipoma of the Tuber Cinereum Mimicking a Pituitary Gland Abnormality in a Girl With Central Precocious Puberty.

Introduction: Magnetic Resonance Imaging (MRI) is the best approach to investigate the hypothalamic-pituitary region in children with central precocious puberty (CPP). Routine scanning is controversial in girls aged 6-8 year, due to the overwhelming prevalence of idiopathic forms and unrelated incidentalomas. Cerebral lipomas are rare and accidental findings, not usually expected in CPP. We report a girl with CPP and an unusually shaped posterior pituitary gland on SE-T1w sequences. Case Description: A 7.3-year-old female was referred for breast development started at age 7. Her past medical history and physical examination were unremarkable, apart from the Tanner stage 2 breast. X-ray of the left-hand revealed a bone age 2-years ahead of her chronological age, projecting her adult height prognosis below the mid parental height. LHRH test and pelvic ultrasound were suggestive for CPP. Routine brain MRI sequences, SE T1w and TSE T2w, showed the posterior pituitary bright spot increased in size and stretched upward. The finding was considered as an anatomical variant, in an otherwise normal brain imaging. Patient was started on treatment with GnRH analogue. At a thorough revaluation, imaging overlap with adipose tissue was suspected and a new MRI scan with 3D-fat-suppression T1w-VIBE sequences demonstrated a lipoma of the tuber cinereum, bordering a perfectly normal neurohypophysis. 3D-T2w-SPACE sequences, acquired at first MRI scan, would have provided a more correct interpretation if rightly considered. Conclusion: This is the first evidence, to our knowledge, of a cerebral lipoma mimicking pituitary gland abnormalities. Our experience highlights the importance of considering suprasellar lipomas in the MRI investigation of children with CPP, despite their rarity, should the T1w sequences show an unexpected pituitary shape. 3D-T2w SPACE sequences could be integrated into standard ones, especially when performing MRI routinely, to avoid potential misinterpretations.

Precocious puberty in Korean girls with and without exposure to endocrine-disrupting chemicals in toy slime: a comparative analysis.

BACKGROUND: Toy slime is popular in Korea, and in parallel, pre-pubertal girls visit hospitals for early pubertal signs. Thus far, numerous studies have investigated the association of endocrine-disrupting chemicals (EDCs) with precocious puberty (PP). However, there is a lack of studies on the clinical manifestations and sex hormones. We aimed to investigate early pubertal development in Korean girls with or without a history of toy slime exposure and determine changes in bone age, Tanner stage, and sex hormones. METHODS: In this study, 140 girls underwent stimulation tests at Kyungpook National University Children's Hospital Endocrinology Department, during January 2018 and December 2020. Patients were divided into two groups for gonadotropin-releasing hormone (GnRH) stimulation test and frequency of exposure to toy slime (EDCs). GnRH stimulation test was conducted after an intravenous injection of 100 µg of luteinizing hormone-releasing hormone. Slime exposure was defined as Slime ≥ 3 times/week for ≥ 3 months. RESULTS: History of slime exposure was found in 14 of 58 and 65 of 82 patients in the central PP (CPP) and non-CPP groups, respectively. Slime-exposed patients had advanced bone age, although their Tanner stage was low. Patients with a history of toy slime exposure were 5.5 times more likely to be diagnosed with non-CPP than patients without slime exposure (p < 0.05). CONCLUSIONS: Exposure to toy slime in prepubertal girls may be associated with rapid clinical advancement of pubertal development and bone age, and the patients appear more likely to be diagnosed with non-CPP.

The Diagnostic Utility of the Basal Luteinizing Hormone Level and Single 60-Minute Post GnRH Agonist Stimulation Test for Idiopathic Central Precocious Puberty in Girls.

Objective: The present study aimed to assess the diagnostic utility of the Luteinizing hormone (LH) levels and single 60-minute post gonadotropin-releasing hormone (GnRH) agonist stimulation test for idiopathic central precocious puberty (CPP) in girls. Methods: Data from 1,492 girls diagnosed with precocious puberty who underwent GnRH agonist stimulation testing between January 1, 2016, and October 8, 2020, were retrospectively reviewed. LH levels and LH/follicle-stimulating hormone (FSH) ratios were measured by immuno-chemiluminescence assay before and at several timepoints after GnRH analogue stimulation testing. Mann-Whitney U test, Spearman's correlation, χ2 test, and receiver operating characteristic (ROC) analyses were performed to determine the diagnostic utility of these hormone levels. Results: The 1,492 subjects were split into two groups: an idiopathic CPP group (n = 518) and a non-CPP group (n = 974). Basal LH levels and LH/FSH ratios were significantly different between the two groups at 30, 60, 90, and 120 minutes after GnRH analogue stimulation testing. Spearman's correlation analysis showed the strongest correlation between peak LH and LH levels at 60 minutes after GnRH agonist stimulation (r = 0.986, P < 0.001). ROC curve analysis revealed that the 60-minute LH/FSH ratio yielded the highest consistency, with an area under the ROC curve (AUC) of 0.988 (95% confidence interval [CI], 0.982-0.993) and a cut-off point of 0.603 mIU/L (sensitivity 97.3%, specificity 93.0%). The cut-off points of basal LH and LH/FSH were 0.255 mIU/L (sensitivity 68.9%, specificity 86.0%) and 0.07 (sensitivity 73.2%, specificity 89.5%), respectively, with AUCs of 0.823 (95% CI, 0.799-0.847) and 0.843 (95% CI, 0.819-0.867), respectively. Conclusions: A basal LH value greater than 0.535 mIU/L can be used to diagnose CPP without a GnRH agonist stimulation test. A single 60-minute post-stimulus gonadotropin result of LH and LH/FSH can be used instead of a GnRH agonist stimulation test, or samples can be taken only at 0, 30, and 60 minutes after a GnRH agonist stimulation test. This reduces the number of blood draws required compared with the traditional stimulation test, while still achieving a high level of diagnostic accuracy.

MINI REVIEW: Role of Kisspeptin-GPR54 system in regulation of reproductive functions in human and other mammals.

Kisspeptin is a 54- amino acid peptide that acts as a ligand of a receptor called GPR54 which is basically a transmembrane receptor that spins seven times across the cell membrane and coupled with G-protein. Kisspeptin regulates the development of reproductive functions and the onset of puberty in human and other mammals by acting at the brain, hypothalamus, pituitary and gonad levels of reproductive axis. Kisspeptin is also involved in regulation of trophoblastic invasion during pregnancy, ovulation, and sperm hyperactivation. Inactivating mutations in human kisspeptin gene (KISS1) cause idiopathic hypogonadotropic hypogonadism. Some mutations in human kisspeptin receptor gene (KISS1R) make the receptor inactive which result in idiopathic hypogonadotropic hypogonadism. Some mutations in human KISS1R gene make the receptor prematurely activated and result in the development of central precocious puberty. Central precocious puberty is also caused by some mutations in human KISS1 gene that make the kisspeptin resistant to degradation. This leads to an increased basal kisspeptin level and subsequently the development of central precocious puberty. Higher kisspeptin level has been detected in the serum and plasma of central precocious puberty patients, which suggest that serum or plasma kisspeptin level can be used as a marker for diagnosis of central precocious puberty.

MKRN3-mediated ubiquitination of Poly(A)-binding proteins modulates the stability and translation of GNRH1 mRNA in mammalian puberty.

The family of Poly(A)-binding proteins (PABPs) regulates the stability and translation of messenger RNAs (mRNAs). Here we reported that the three members of PABPs, including PABPC1, PABPC3 and PABPC4, were identified as novel substrates for MKRN3, whose deletion or loss-of-function mutations were genetically associated with human central precocious puberty (CPP). MKRN3-mediated ubiquitination was found to attenuate the binding of PABPs to the poly(A) tails of mRNA, which led to shortened poly(A) tail-length of GNRH1 mRNA and compromised the formation of translation initiation complex (TIC). Recently, we have shown that MKRN3 epigenetically regulates the transcription of GNRH1 through conjugating poly-Ub chains onto methyl-DNA bind protein 3 (MBD3). Therefore, MKRN3-mediated ubiquitin signalling could control both transcriptional and post-transcriptional switches of mammalian puberty initiation. While identifying MKRN3 as a novel tissue-specific translational regulator, our work also provided new mechanistic insights into the etiology of MKRN3 dysfunction-associated human CPP.

Short-term effects of gonadotropin-releasing hormone analogue treatment on leptin, ghrelin and peptide YY in girls with central precocious puberty.

OBJECTIVES: To determine appetite-regulating hormone levels in girls with central precocious puberty (CPP) before and after 20 weeks of gonadotropin-releasing hormone analogue (GnRH-A) treatment. METHODS: Eighteen newly diagnosed CPP girls were enrolled. Body composition measured by bioelectrical impedance analysis and GnRH-A test were performed with fasting serum leptin, ghrelin and peptide YY (PYY) measurements at baseline (before) and after 20 weeks of GnRH-A treatment. RESULTS: Following GnRH-A treatment, all patients had prepubertal gonadotropin and estradiol levels. Mean (SD) fat mass index (FMI) was significantly increased from 4.5 (1.7) to 5.0 (1.8) kg/m2 after treatment. Also, median (IQR) serum leptin level was significantly increased from 6.9 (4.2-8.6) to 7.4 (5.3-13.1) ng/mL. FMI had a positive correlation with serum leptin level (r=0.64, p=0.004). In contrast, no significant changes of serum ghrelin and PYY levels were observed. CONCLUSIONS: Decreased estrogen following short-term GnRH-A treatment in CPP girls may cause an increase in appetite and consequently an elevation of FMI. Increased serum leptin may be a result of having increased FMI secondary to an increase in appetite.

Gonadotropin releasing hormone analogue therapy in girls with idiopathic precocious puberty/early-fast puberty: dynamics in adiposity indices, eating habits and quality of life.

BACKGROUND: The impact of gonadotropin-releasing-hormone-analogue (GnRHa) treatment on weight and body composition is controversial. Exploring the nutritional, psychological patterns of this population may aid to clarify this propensity to gain weight. This prospective observational study aimed to evaluate longitudinal changes in adiposity, nutrition and quality of life in girls with central precocious/early-fast puberty (CPP/EFP) during GnRHa treatment. METHODS: Thirty-two GnRHa-treated girls with CPP/EFP and 27 prepubertal girls (7-10 years) were included in the analysis. Outcome measures assessed at baseline for CPP/EFP and the control groups and during up to two years of GnRHa treatment for the CPP/EFP group, included anthropometrics, body-composition, basal-metabolic-rate (BMR), 3-day food-diaries, child eating-behavior questionnaire, and pediatric quality-of-life questionnaire (PedsQL). RESULTS: Girls with CPP/EFP had higher pretreatment BMI-SDS, fat percentages, waist circumference and waist-per-height (p<0.01 for all), and lower psychosocial functioning than controls (p<0.05). Changes in anthropometric and body composition measurements indicated a gradual increase in adiposity and a decrease in muscle mass (p<0.001 for all). Dynamics in body composition could not be explained by the participants' self-reported dietary patterns and physical activity levels or by the measured BMR, which revealed an adequate and relatively low energy intake as compared to energy requirements. A gradual decline in physical functioning (PedsQL) after one and two years of GnRHa treatment was observed (p<0.001). CONCLUSIONS: Our findings highlight the need for comprehensive surveillance in girls with CPP/EFP. Dynamics in weight status and body composition during GnRHa treatment indicate the need for tailored nutritional and physical activity counseling aimed at preventing obesity.

DIAGNOSIS OF ENDOCRINE DISEASE: Sex steroid action in adolescence: too much, too little; too early, too late.

ABSTRACT: This review aims to cover the subject of sex steroid action in adolescence. It will include situations with too little sex steroid action, as seen in for example, Turners syndrome and androgen insensitivity issues, too much sex steroid action as seen in adolescent PCOS, CAH and gynecomastia, too late sex steroid action as seen in constitutional delay of growth and puberty and too early sex steroid action as seen in precocious puberty. This review will cover the etiology, the signs and symptoms which the clinician should be attentive to, important differential diagnoses to know and be able to distinguish, long-term health and social consequences of these hormonal disorders and the course of action with regards to medical treatment in the pediatric endocrinological department and for the general practitioner. This review also covers situations with exogenous sex steroid application for therapeutic purposes in the adolescent and young adult. This includes gender-affirming therapy in the transgender child and hormone treatment of tall statured children. It gives some background information of the cause of treatment, the patient's motivation for medicating (or self-medicating), long-term consequences of exogenous sex steroid treatment and clinical outcome of this treatment.

Central Ceramide Signaling Mediates Obesity-Induced Precocious Puberty.

Childhood obesity, especially in girls, is frequently bound to earlier puberty, which is linked to higher disease burden later in life. The mechanisms underlying this association remain elusive. Here we show that brain ceramides participate in the control of female puberty and contribute to its alteration in early-onset obesity in rats. Postnatal overweight caused earlier puberty and increased hypothalamic ceramide content, while pharmacological activation of ceramide synthesis mimicked the pubertal advancement caused by obesity, specifically in females. Conversely, central blockade of de novo ceramide synthesis delayed puberty and prevented the effects of the puberty-activating signal, kisspeptin. This phenomenon seemingly involves a circuit encompassing the paraventricular nucleus (PVN) and ovarian sympathetic innervation. Early-onset obesity enhanced PVN expression of SPTLC1, a key enzyme for ceramide synthesis, and advanced the maturation of the ovarian noradrenergic system. In turn, obesity-induced pubertal precocity was reversed by virogenetic suppression of SPTLC1 in the PVN. Our data unveil a pathway, linking kisspeptin, PVN ceramides, and sympathetic ovarian innervation, as key for obesity-induced pubertal precocity.

Premature Adrenarche in Girls Characterized By Enhanced 17,20-Lyase and 17ß-Hydroxysteroid Dehydrogenase Activities.

CONTEXT: Girls with premature adrenarche (PA) may have a higher risk of developing polycystic ovary syndrome (PCOS) and metabolic syndrome. The biological purpose of adrenarche is unknown and the role of novel biosynthetic pathways remains unclear. OBJECTIVE: To compare the urinary steroid metabolome and enzyme activities of girls with PA to age-matched control girls and to published steroid values of girls with normal adrenarche and of women with PCOS and their newborn daughters. DESIGN: Prospective observational study from 2009 to 2014. SETTING: Academic pediatric endocrinology referral center. PARTICIPANTS: Twenty-three girls with PA and 22 healthy, age-matched girls. MAIN OUTCOME MEASURES: Steroid metabolites in 24-hour urine samples, including 4 progesterones, 5 corticosterones, aldosterone, 13 androgens, 2 estrogens, 14 glucocorticoids, and enzyme activities represented by metabolite ratios. RESULTS: Girls with PA had a higher body mass index (mean standard deviation scores 0.9 vs -0.3, P = 0.013). Androgen excretion was higher in PA girls than in control girls (median 3257 nmol/24 hours vs 1627 nmol/24 hours, P < 0.001), in particular metabolites from alternate androgen pathways. The amount of progesterone, corticosterone, aldosterone, estrogen, and cortisol metabolites were similar between groups. Activities of 17ß-hydroxysteroid-dehydrogenase and of 17,20-lyase were higher in girls with PA. Activities of 3ß-hydroxysteroid-dehydrogenase, 21-hydroxylase, and 5α-reductase activity were not different between groups, in contrast to published results on girls with normal adrenarche or PCOS females. CONCLUSIONS: Metabolites and enzymes involved in alternate androgen pathways appear to be markers of PA. Prospective studies should assess whether steroid production in PA also differs from adrenarche at normal timing and persists into adulthood.

Novel Genetic and Biochemical Findings of DLK1 in Children with Central Precocious Puberty: A Brazilian-Spanish Study.

BACKGROUND: Central precocious puberty (CPP) has been associated with loss-of-function mutations in 2 paternally expressed genes (MKRN3 and DLK1). Rare defects in the DLk1 were also associated with poor metabolic phenotype at adulthood. OBJECTIVE: Our aim was to investigate genetic and biochemical aspects of DLK1 in a Spanish cohort of children with CPP without MKRN3 mutations. PATIENTS: A large cohort of children with idiopathic CPP (Spanish PUBERE Registry) was studied. Genomic deoxyribonucleic acid was obtained from 444 individuals (168 index cases) with CPP and their close relatives. Automatic sequencing of MKRN3 and DLK1 genes were performed. RESULTS: Five rare heterozygous mutations of MKRN3 were initially excluded in girls with familial CPP. A rare allelic deletion (c.401_404 + 8del) in the splice site junction of DLK1 was identified in a Spanish girl with sporadic CPP. Pubertal signs started at 5.7 years. Her metabolic profile was normal. Familial segregation analysis showed that the DLK1 deletion was de novo in the affected child. Serum DLK1 levels were undetectable (<0.4 ng/mL), indicating that the deletion led to complete lack of DLK1 production. Three others rare allelic variants of DLK1 were also identified (p.Asn134=; g.-222 C>A and g.-223 G>A) in 2 girls with CPP. However, both had normal DLK1 serum levels. CONCLUSION: Loss-of-function mutations of DLK1 represent a rare cause of CPP, reinforcing a significant role of this factor in human pubertal timing.