Background: Numerous studies have focused on the association between endocrine-disrupting chemicals (EDCs) and hypospadias. Phenotype variability, the absence of representative comparison groups and concomitant genetic testing prevent any definitive conclusions.Objective: To identify the role of occupational and environmental exposures to EDCs in nongenetic isolated hypospadias.Design, setting, and participants: A total of 408 consecutive children with isolated hypospadias and 302 normal boys were prospectively included (2009-2014) in a multi-institutional study in the south of France, the area of the country with the highest prevalence of hypospadias surgery.Outcome measurements and statistical analysis: In patients without AR, SRD5A2, and MAMLD1 mutations, parental occupational and professional exposures to EDCs were evaluated based on European questionnaire QLK4-1999-01422 and a validated job-exposure matrix for EDCs. Environmental exposure was estimated using the zip code, the type of surrounding hazards, and distance from these hazards. Multivariate analysis was performed.Results: Fetal exposure to EDCs around the window of genital differentiation was more frequent in the case of hypospadias (40.00% vs 17.55%, odds ratio 3.13, 95% confidence interval 2.11-4.65). The substances were paints/solvents/adhesives (16.0%), detergents (11.0%), pesticides (9.0%), cosmetics (5.6%), and industrial chemicals (4.0%). Jobs with exposure were more frequent in mothers of hypospadiac boys (19.73% vs 10.26%, p = 0.0019), especially cleaners, hairdressers, beauticians, and laboratory workers. Paternal job exposure was more frequent in the cases of hypospadias (40.13% vs 27.48%, p = 0.02). Industrial areas, incinerators, and waste areas were more frequent within a 3-km radius for mothers of hypospadiac boys (13.29% vs. 6.64%, p < 0.00005). Association of occupational and environmental exposures increases this risk.Conclusions: This multicenter prospective controlled study with a homogeneous cohort of hypospadiac boys without genetic defects strongly suggests that EDCs are a risk factor for hypospadias through occupational and environmental exposure during fetal life. The association of various types of exposures may increase this risk.Patient summary: Our multi-institutional study showed that parental professional, occupational, and environmental exposures to chemical products increase the risk of hypospadias in children. (C) 2015 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Background: Androgens are critical in male external genital development. Alterations in the androgen sensitivity pathway have been identified in severely undermasculinized boys, and mutations of the androgen receptor gene (AR) are usually found in partial or complete androgen insensitivity syndrome (AIS).Objective: The aim of this study was to determine whether even the most minor forms of isolated hypospadias are associated with AR mutations and thus whether all types of hypospadias warrant molecular analysis of the AR.Materials and Methods: Two hundred and ninety-two Caucasian children presenting with isolated hypospadias without micropenis or cryptorchidism and 345 controls were included prospectively. Mutational analysis of the AR through direct sequencing (exons 1-8) was performed. In silico and luciferase functional assays were performed for unreported variants.Results: Five missense mutations of the AR were identified in 9 patients with glandular or penile anterior (n = 5), penile midshaft (n = 2) and penile posterior (n = 2) hypospadias, i.e., 3%: p.Q58L (c.173A > T), 4 cases of p.P392S (c.1174C > T), 2 cases of p.A475V (c.1424C > T), p.D551H (c.1651G > C) and p.Q799E (c.2395C > G). None of these mutations was present in the control group. One mutation has never been reported to date (p.D551H). It was predicted to be damaging based on 6 in silico models, and in vitro functional studies confirmed the lowered transactivation function of the mutated protein. Three mutations have never been reported in patients with genital malformation but only in isolated infertility: p.Q58L, p.P392S, and p.A475V. It is notable that micropenis, a cardinal sign of AIS, was not present in any patient.Conclusion: AR mutations may play a role in the cause of isolated hypospadias, even in the most minor forms. Identification of this underlying genetic alteration may be important for proper diagnosis and longer follow-up is necessary to find out if the mutations cause differences in sexual function and fertility later in life.

OBJECTIVE:To determine whether NR5A1 (SF-1) variants are a cause of primary ovarian insufficiency (POI) in 26 young women with similar genetic background.DESIGN:Genetic and functional mutation study.SETTING:University hospitals.PATIENT(S):Genetic analysis of the NR5A1 gene in 26 XX girls with POI.INTERVENTION(S):None.MAIN OUTCOME MEASURE(S):NR5A1 molecular and functional analysis.RESULT(S):Genetic analysis revealed a new c.763C>T (p.Arg255Cys) mutation and a recurrent c.437G>C (p.Gly146Ala) variant. Functional analysis of the p.Arg255Cys mutant showed a marked decrease in transactivation on the Cyp11a1 and Amh promoters. The p.Gly146Ala variant was identified significantly more often in the patients (46.1%) than in ancestry-matched control subjects (10%).CONCLUSION(S):We identified one new NR5A1 mutation in a patient of our POI cohort (prevalence 3.8%). Moreover, although our study is limited in the number of cases, we report the high frequency of the p.Gly146Ala variant in this cohort compared with the ancestry-matched control subjects. This work highlights the important role of SF-1 in ovarian function.

Background: WNT4 and SF1 genes play an important role in ovarian development. They constitute coherent candidate genes associated with premature ovarian failure (POF) pathogenesis.Methods: We sequenced the coding region of WNT4 and SF1 in 55 Tunisian women with POF and 100 healthy controls.Results: We identified a synonymous variation in WNT4 (c.99G>A, p.Ser33Ser) and a substitution (c.G437C) in SF1 gene inducing G146 to Ala (GGG-GCG) missense mutation. WNT4 (c.99G>A, p.Ser33Ser) was not associated with POF pathology. However, a positive association of SF1 Gly146Ala polymorphism was noted. Gly146Ala minor allele frequency was significantly higher (p = 0.029) in POP patients versus controls and Ala allele containing genotypes (p = 0.005) were positively associated with POP pathology. The carriage of 146Ala allele was also associated with a significant reduction in estradiol plasma levels.Conclusions: SF1 Gly146Ala polymorphism seems to be associated with POF pathology in the Tunisian population likely by reducing estradiol levels. (C) 2012 Elsevier B.V. All rights reserved.

More than 50% of children with severe 46,XY disorders of sex development (DSD) do not have a definitive etiological diagnosis. Besides gonadal dysgenesis, defects in androgen biosynthesis, and abnormalities in androgen sensitivity, the Mastermind-like domain containing 1 (MAMLD1) gene, which was identified as critical for the development of male genitalia, may be implicated. The present study investigated whether MAMLD1 is implicated in cases of severe 46,XY DSD and whether routine sequencing of MAMLD1 should be performed in these patients. Seventy children with severe non-syndromic 46,XY DSD of unknown etiology were studied. One hundred and fifty healthy individuals were included as controls. Direct sequencing of the MAMLD1, AR, SRD5A2 and NR5A1 genes was performed. The transactivation function of the variant MAMLD1 proteins was quantified by the luciferase method. Two new mutations were identified: p.S143X (c.428C>A) in a patient with scrotal hypospadias with microphallus and p.P384L (c.1151C>T) in a patient with penile hypospadias with microphallus. The in vitro functional study confirmed no residual transactivating function of the p.S143X mutant and a significantly reduced transactivation function of the p.P384L protein (p = 0.0032). The p.P359S, p.N662S and p.H347Q variants are also reported with particularly high frequency of the p.359T- p.662G haplotype in the DSD patients. Severe undervirilization in XY newborns can reveal mutations of MAMLD1. MAMLD1 should be routinely sequenced in these patients with otherwise normal AR, SRD5A2 and NR5A1genes.

CONTEXT:In 46,XY disorders of sex development, 5α-reductase deficiency is rare and is not usually the first-intention diagnosis in newborn ambiguous genitalia, contrary to partial androgen insensitivity syndrome. Yet the cause of ambiguous genitalia may guide sex assignment, and rapid, precise diagnosis of 5α-reductase deficiency is essential.OBJECTIVE:The aim of the study was to describe relevant data for clinical diagnosis, biological investigation, and molecular determination from 55 patients with srd5A2 mutations identified in our laboratory over 20 yr to improve early diagnosis.SETTING:The study was performed at Montpellier University Hospital.PATIENTS:We studied a cohort of 55 patients with srd5A2 gene mutations.MAIN OUTCOME MEASURE(S):Genetic analysis of srd5A2 was conducted.RESULTS:Clitoromegaly (49.1%) and microphallus with various degrees of hypospadias (32.7%) were frequent phenotypes. Female external genitalia (7.3%) and isolated micropenis (3.6%) were rare. Seventy-two percent of patients were initially assigned to female gender; five of them (12.5%) switched to male sex in peripuberty. Over 72% of patients were considered for 5α-reductase deficiency diagnosis when the testosterone/dihydrotestosterone cutoff was 10. In 55 patients (with 20 having a history of consanguinity), we identified 33 different mutations. Five have never been reported: p.G32S, p.Y91H, p.G104E, p.F223S, and c.461delT. Homozygous mutations were present in 69.1% of cases, compound heterozygous mutations in 25.5%, and compound heterozygous mutations alone with the V89L polymorphism in 5.4%. Exons 1 and 4 were most affected, with 35.8 and 21.7% mutant alleles per exon, respectively.CONCLUSIONS:In the largest cohort to date, we demonstrate a wide spectrum of phenotypes and biological profiles in patients with 5α-reductase deficiency, whatever their geographical or ethnic origins.

Objective 46,XY disorders of sex differentiation (46,XY DSD) can be due to a testis determination defect, an androgen biosynthesis defect, or androgen resistance (complete or partial androgen insensitivity syndrome (PAIS), or 5α reductase deficiency). We aimed to evaluate the impact of a prenatal contamination by environmental xenoestrogens in ‘idiopathic’ PAIS-like phenotype. Subjects We investigated 28 newborn/infant males with 46,XY DSD, normal androgen production, and no androgen receptor or steroid-5αR type II enzyme ( SRD5A2 ) gene mutations. Methods To exclude other genetic defects, we sequenced the steroidogenic factor 1 (SF1) and mastermind-like domain-containing 1 ( MAMLD1 ) genes, which were recently found to be associated with the PAIS-like phenotype. Parents were interviewed about their environmental/occupational exposure to endocrine disrupting chemicals (EDCs) before/during the patients' fetal life. Total estrogenic bioactivity of patient serum was analyzed by ultrasensitive bioassay. Results All the patients had normal SF1 sequence and one patient showed a double polymorphism of MAMLD1. Eleven (39.3%) of the 28 patients had reported parental fetal exposure to EDCs. The mean estrogenic bioactivity in these 11 patients with fetal EDC exposure (6.65±8.07 pg/ml) versus 17 cases without contamination (1.27±0.34 pg/ml) and controls (1.06±0.44 pg/ml; P <0.05) was elevated. Conclusions Our results indicate that the ‘idiopathic’ PAIS-like phenotype may in some cases be related to EDC contamination during fetal life.

Knockout of the steroidogenic factor 1 (Sf1) gene in 46,XY mice causes complete adrenal failure associated with gonadal dysgenesis, undervirilization, and persistence of mullerian ducts [1]. SF1 (NR5A1, SF1/Ad4BP/FTZF1) is a member of the nuclear receptor superfamily, and it plays a crucial role in the fetal development of the adrenal gland [2] and testes [3]. Sf1 is considered to be the master regulator of the reproductive system because it regulates the expression of a wide array of genes required for testis and adrenal development and hormone synthesis [4].

Objective: To further investigate the molecular mechanism by which NR5A1/SF-1 mutation led to gonadal dysgenesis with predominant Sertoli cell defect.Design: Genetic and functional mutation study.Setting: University hospital.Patient(s): Genetic analysis of an XY newborn with hypospadias and micropenis. Puberty developed spontaneously with a rise in T levels and normal LH contrasting with high FSH and low inhibin B concentrations, revealing a Sertoli cell defect.Intervention(s): None.Main Outcome Measure(s): NR5A1/SF-1 gene molecular analysis.Result(s): Genetic analysis identified a new NR5A1/SF-1 mutation, c. 842G>C (p. Arg281Pro). In vitro functional studies showed that the p. Arg281Pro mutant mainly altered Sertoli cell function, as observed in vivo with a high FSH level and low inhibin B concentration contrasting with normal LH concentration. The mutation was found in the father's DNA at a low copy number through direct sequencing and high-resolution melting assay, suggesting mosaicism.Conclusion(s): We describe a new heterozygous NR5A1/SF-1 mutation that mainly altered Sertoli cell function. However, this 46, XY disorders of sex development (DSD) boy had no Mullerian derivatives, suggesting normal Sertoli cell function during fetal life. During puberty, Sertoli cell deficiency became more apparent. This is the first report of a progressive and predominant Sertoli cell defect in an XY patient with testicular dysgenesis owing to NR5A1/SF-1 mutation. (Fertil Steril (R) 2011; 95: 1788. e5-e9. (C) 2011 by American Society for Reproductive Medicine.)

Partial androgen insensitivity syndrome (PAIS) is the milder variant of androgen receptor (AR) defects. The subtle effects of AR mutations present in a patient with micropenis, peno-scrotal hypospadias, infertility, clitoromegaly and posterior labial fusion. We studied the association of isolated micropenis with the genetic defects resulting in androgen resistance, that is, AR gene defects and 5-alpha reductase type 2 (SRD5A2) deficiency. We describe two cases of isolated micropenis: one in a 14-year-old boy and the other in a 3-year-old boy who was followed until he was 10 years old. There were no findings of hypospadias, cryptorchidism or gynecomastia in either of these patients. Serum gonadotrophin and androgen levels were obtained and karyotyping was done. Human chorionic gonadotropin (hCG) stimulation testing assessed the functional capacity of the testes. DNA was extracted from peripheral leukocytes, and all exons of the SRD5A2 and AR genes were amplified by polymerase chain reaction and sequenced. In both patients, baseline testosterone (T) level was low and the values were elevated after hCG testing. The sequence of the SRD5A2 gene was normal in patient 1, and a heterozygous polymorphism, V89L, was found in patient 2. Two known mutations, P390S and A870V, were identified in patients 1 and 2, respectively. Mutations in the AR gene can be associated with isolated micropenis without other features of PAIS, such as hypospadias or gynecomastia. This underlines the importance of including AR gene analysis in the evaluation of isolated micropenis with normal plasma T to ensure proper management of the patient and appropriate genetic counseling for the family.

Objective: To confirm the clinical diagnosis of complete androgen insensitivity syndrome (CAIS) by molecular genetic analysis and to determine the prevalence of exon 1 mutations in the androgen receptor (AR) transactivation defects of a large series of CAIS patients.Design: International retrospective study.Setting: University Hospital of Montpellier, Department of Hormonology.Patient(s): 105 patients with normal female external genitalia, bilateral intra-abdominal or inguinal testis, normal breast development, absent or sparse pubic hair, normal or high endogenous testosterone production, hypoplastic or absent wolffian structures, and 46,XY karyotype.Intervention(s): Sequencing of the AR gene.Main Outcome Measure(s): Prevalence of AR exon 1 mutations.Result(s): Over a 10-year period (1997 to 2007), we identified 78 AR gene mutations in 105 patients with CAIS; 21 of them were located in exon 1, and 13 of these were new mutations. We report 13 new mutations in the AR gene. All but one were stop codons, and the last was a splicing abnormality.Conclusion(s): The finding that 27% of the mutations in our cohort were localized in exon 1 versus 15% in previous works justifies the sequencing of this exon in patients with CAIS. (Fertil Steril(R) 2010;94:472-6. (C) 2010 by American Society for Reproductive Medicine.)