A conserved NR5A1-responsive enhancer regulates SRY in testis-determination.

The Y-linked SRY gene initiates mammalian testis-determination. However, how the expression of SRY is regulated remains elusive. Here, we demonstrate that a conserved steroidogenic factor-1 (SF-1)/NR5A1 binding enhancer is required for appropriate SRY expression to initiate testis-determination in humans. Comparative sequence analysis of SRY 5' regions in mammals identified an evolutionary conserved SF-1/NR5A1-binding motif within a 250 bp region of open chromatin located 5 kilobases upstream of the SRY transcription start site. Genomic analysis of 46,XY individuals with disrupted testis-determination, including a large multigenerational family, identified unique single-base substitutions of highly conserved residues within the SF-1/NR5A1-binding element. In silico modelling and in vitro assays demonstrate the enhancer properties of the NR5A1 motif. Deletion of this hemizygous element by genome-editing, in a novel in vitro cellular model recapitulating human Sertoli cell formation, resulted in a significant reduction in expression of SRY. Therefore, human NR5A1 acts as a regulatory switch between testis and ovary development by upregulating SRY expression, a role that may predate the eutherian radiation. We show that disruption of an enhancer can phenocopy variants in the coding regions of SRY that cause human testis dysgenesis. Since disease causing variants in enhancers are currently rare, the regulation of gene expression in testis-determination offers a paradigm to define enhancer activity in a key developmental process.

Peak serum luteinising hormone cut-off during gonadotropin-releasing hormone analogue test for diagnosing central precocious puberty was lower in girls with obesity as compared with girls with normal weight.

OBJECTIVE: Serum luteinising hormone (LH) concentration has been reported to be lower in girls with overweight and obesity (OW/OB) as compared with girls with normal weight (NW). This study aimed to evaluate peak serum LH concentration during gonadotropin-releasing hormone analogue (GnRHa) test in girls with OW/OB and NW who had central precocious puberty (CPP) and to determine peak serum LH cut-off for diagnosing CPP in girls with OW/OB. DESIGN, PATIENTS AND MEASUREMENTS: Medical records of 971 girls with premature breast development who underwent subcutaneous GnRHa (100 µg of triptorelin acetate) test were reviewed. All girls were classified as either CPP or premature thelarche. All of them were further classified into two groups according to their body mass index as NW and OW/OB groups for each Tanner stage. RESULTS: There were 634 and 337 girls in NW and OW/OB groups, respectively. CPP was diagnosed in 600 girls (249 had Tanner stage II and 351 had Tanner stage III). There were no differences in peak serum LH concentrations between CPP girls with NW and OW/OB. Peak serum LH cut-off of 5 IU/L (the current widely used cut-off) had a sensitivity and a specificity of 75% and 90%, respectively in NW group. Peak serum LH cut-off for CPP diagnosis was lower at 4 IU/L in the OW/OB group with greater sensitivity and specificity of 86% and 93%, respectively. The results were reproducible for each Tanner stage of breasts. CONCLUSION: Lower peak serum LH cut-off to 4 IU/L for diagnosing CPP in girls with OW/OB should be considered to avoid underdiagnosis of the condition.

In vitro cellular reprogramming to model gonad development and its disorders.

During embryonic development, mutually antagonistic signaling cascades determine gonadal fate toward a testicular or ovarian identity. Errors in this process result in disorders of sex development (DSDs), characterized by discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells toward gonadal progenitors. Transcriptomic analysis reveals that the in vitro-derived murine gonadal cells are equivalent to embryonic day 11.5 in vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete anti-Müllerian hormone, migrate, and form tubular structures. Cells derived from 46,XY DSD female hiPSCs, carrying an NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR-Cas9-mediated variant correction rescued the phenotype. This is a robust tool to understand mechanisms of sex determination and model DSDs.

Central Precocious Puberty in a Boy with Pseudohypoparathyroidism Type 1A due to a Novel GNAS Variant, with Congenital Hypothyroidism as the First Manifestation

Pseudohypoparathyroidism (PHP) type 1A (PHP1A) is a disorder of multiple hormone resistance, mainly parathyroid hormone. It is associated with Albright hereditary osteodystrophy phenotypes. Patients with PHP1A may initially present with hypothyroidism during infancy and later develop typical PHP1A characteristics during their childhood. Central precocious puberty (CPP) is extremely rare among PHP1A patients in whom gonadotropin resistance is more usual. This is a case report of a 9.5-year-old boy with congenital hypothyroidism who developed hypocalcemia secondary to PHP. He had relatively short stature with height standard deviation score of -0.9. Obesity had been noted since the age of two years. At the presentation of PHP, pubertal-sized testes of 10 mL were observed, and CPP was documented with serum testosterone concentration of 298 ng/dL (normal for Tanner stage III, 100-320), luteinizing hormone of 3.9 IU/L (normal, 0.2-5.0), and follicle stimulating hormone of 4.8 IU/L (normal, 1.2-5.8). Pituitary magnetic resonance imaging was unremarkable. Genetic analysis confirmed the diagnosis of PHP1A with a novel heterozygous missense variant of GNAS gene in exon 13, c.1103A>G (p.Asp368Gly). Awareness of PHP1A diagnosis in patients with congenital hypothyroidism and early childhood-onset obesity is important for early diagnosis. Apart from multiple hormone resistance, CPP may manifest in patients with PHP1A.

Serum Kisspeptin and Its Relation to Metabolic Parameters and Glucose Metabolism in Prepubertal and Pubertal Obese Children.

OBJECTIVE: Kisspeptin, a puberty control neuropeptide, has been discovered to have an additional role in metabolism and glucose homeostasis regulation. This study aimed to determine the association of serum kisspeptin with metabolic parameters and glucose metabolism in obese children. Design, Patients and Measurements. A cross-sectional study of 270 obese children was conducted. All children underwent an oral glucose tolerance test and had serum kisspeptin, glycated hemoglobin (HbA1c), and lipid profile measurements. Body fat mass was assessed by bioelectrical impedance analysis. Serum kisspeptin levels of both prepubertal and pubertal children with two HbA1c ranges, <5.7% (normal range) and 5.7-6.4% (prediabetes range), were analyzed and correlated with metabolic parameters and glucose metabolism status. RESULTS: The median (IQR) serum kisspeptin level of only pubertal (not prepubertal) children with prediabetes HbA1c was higher than those with normal HbA1c (53.2 (33.9, 69.8) and 37.8 (29.6, 67.5) pg/mL; p = 0.015, respectively). There were no differences in serum kisspeptin levels among children with different glucose metabolism status. During pubertal progression, serum kisspeptin reached the highest level at Tanner stage II only in obese boys. Additionally, there was a positive correlation between serum kisspeptin and HbA1c after adjusting for puberty (ß = 12.87; p = 0.001). No correlations between serum kisspeptin and insulin sensitivity indices, insulin secretion indices, lipid profile, blood glucose, as well as percentage of body fat were demonstrated. CONCLUSIONS: Serum kisspeptin levels in pubertal obese children with prediabetes HbA1c were greater than those with normal HbA1c. Serum kisspeptin was positively associated with HbA1c, but not with glucose metabolism status.

Basal serum luteinising hormone cut-off, and its utility and cost-effectiveness for aiding the diagnosis of the onset of puberty in girls with early stages of breast development.

OBJECTIVE: To determine basal and gonadotrophin-releasing hormone analogue (GnRHa)-stimulated peak luteinising hormone (LH) cut-offs to diagnose onset of early or normal puberty in girls with each Tanner stage of breast (II and III). DESIGN, PATIENTS AND MEASUREMENTS: A retrospective study of 601 girls with breast onset before 8 years of age who underwent GnRHa test was conducted. Patients were categorized as CPP and premature thelarche. Each group was divided into two subgroups; Tanner II and III. Cost-effectiveness analysis was performed. RESULTS: In comparison with basal LH cut-off of 0.3 IU/L, basal LH cut-off of 0.2 IU/L had comparable specificity (Tanner II: 98.0% vs 94.8%, Tanner III: 98.8% vs 93.8%), but greater sensitivity (Tanner II: 28.3% vs 41.7%, Tanner III: 45.2% vs 59.3%). Specificity of basal LH cut-off of 0.2 IU/L was not inferior to that of the traditionally used peak LH of 5 IU/L. Using basal LH cut-off of 0.2 IU/L followed by GnRHa test in girls with negative basal LH was more cost-saving when compared with using the cut-off of 0.3 IU/L. Moreover, using basal LH cut-off of 0.2 IU/L followed by GnRHa test provided a cost reduction when compared with performing GnRHa test in all patients. CONCLUSIONS: Basal serum LH cut-off of 0.2 IU/L could be a simple and cost-saving tool for initial diagnosis of onset of early or normal puberty in girls with Tanner II and III before proceeding to GnRH testing.

Acute Effects of Blood Transfusion on Insulin Sensitivity and Pancreatic ß-Cell Function in Children with ß-Thalassemia/Hemoglobin E Disease.

OBJECTIVE: To assess the acute effects of blood transfusion on insulin sensitivity and pancreatic ß-cell function in thalassemia patients. METHODS: Fifty children and adolescents with ß-thalassemia/HbE disease were enrolled in a prospective cohort study. Hemoglobin, serum ferritin and oral glucose tolerance test (OGTT) were performed prior to, and one week after blood transfusion. Insulin sensitivity indices [homeostatic model assessment (HOMA) of insulin resistance (HOMA-IR), whole body insulin sensitivity index (WBISI)] and ß-cell function indices [HOMA of ß-cell function (HOMA-ß), insulinogenic index (IGI), and disposition index (DI)] were calculated from glucose and insulin levels obtained during the OGTT. RESULTS: Following blood transfusion, hemoglobin and serum ferritin increased significantly; 8.5 to 10.1 g/dL (p<0.001) and 1764 to 2160 ng/mL (p<0.001), respectively. ß-Cell function indices also increased significantly [median HOMA-ß: 74.3 vs. 82.7 (p=0.033); median IGI: 59.6 vs. 79.3 (p=0.003); median DI: 658 vs. 794 (p=0.01)]. However, the insulin sensitivity index (WBISI) tended to decrease and the insulin resistance index (HOMA-IR) tended to increase although this did not reach significance. Multivariate analysis showed that pre-transfusion serum ferritin was the major factor negatively associated with WBISI and positively associated with HOMA-IR, but pre-transfusion hemoglobin had no significant association with insulin sensitivity indices post-transfusion. CONCLUSION: This study demonstrated that acute increases in serum ferritin and hemoglobin following blood transfusion in patients with thalassemia might contribute to an increase in insulin secretion and to a trend towards increased insulin resistance.