Context 46,XY Gonadal dysgenesis (GD) is a heterogeneous group of disorders with a wide phenotypic spectrum, including embryonic testicular regression syndrome (ETRS). Objective To report a gene for 46,XY GD etiology, especially for ETRS. Design Screening of familial cases of 46,XY GD using whole-exome sequencing and sporadic cases by target gene-panel sequencing. Setting Tertiary Referral Center for differences/disorders of sex development (DSD). Patients and Interventions We selected 87 patients with 46,XY DSD (17 familial cases from 8 unrelated families and 70 sporadic cases); 55 patients had GD (among them, 10 patients from 5 families and 8 sporadic cases had ETRS), and 32 patients had 46,XY DSD of unknown etiology. Results We identified four heterozygous missense rare variants, classified as pathogenic or likely pathogenic in the Asp-Glu-Ala-His-box (DHX) helicase 37 (DHX37) gene in five families (n = 11 patients) and in six sporadic cases. Two variants were recurrent: p.Arg308Gln (in two families and in three sporadic cases) and p.Arg674Trp (in two families and in two sporadic cases). The variants were specifically associated with ETRS (7/14 index cases; 50%). The frequency of rare, predicted-to-be-deleterious DHX37 variants in this cohort (14%) is significantly higher than that observed in the Genome Aggregation Database (0.4%; P < 0.001). Immunohistochemistry analysis in human testis showed that DHX37 is mainly expressed in germ cells at different stages of testis maturation, in Leydig cells, and rarely in Sertoli cells. Conclusion This strong genetic evidence identifies DHX37 as a player in the complex cascade of male gonadal differentiation and maintenance.

Background: Differences of sex development (DSD) is a term used for conditions in which the chromosomal, gonadal or phenotypical sex is atypical. 46,XY DSD patients frequently present undervirilized external genitalia. The expression of different miRNAs in many organs of the male genital system has been reported, and these miRNAs have been associated with testicular function and its disorders, but no description has been related to DSD conditions. This study aimed to evaluate the plasma expression of miR-210 in 46,XY DSD patients who presented atypical genitalia at birth. Methods:Eighteen 46,XY DSD patients who presented atypical genitalia (undescended testis and/or hypospadias, bifid scrotum or micropenis) at birth and 36 male control individuals were selected. Plasma levels of miR-210 and reference miR-23a were measured using RT-qPCR and the data were analysed by the 2 −ΔCt method.Results: MiR-210 plasma levels were significantly higher in 46,XY DSD patients with atypical genitalia than in male control subjects (p = 0.0024). A positive association between miR-210 levels and the presence of cryptorchidism and hypospadias (p = 0.0146 and p = 0.0223) was found in these patients. Significantly higher levels of miR-210 were observed in patients with 46,XY DSD and cryptorchidism than in control subjects (p = 0.0118). These results are in agreement with previous literature reports, in which increased levels of miR-210 expression were observed in human testicular tissue from adult males with undescended testes in comparison with samples of descended testes. Conclusion:Our study showed a positive association between the presence of atypical genitalia and plasma levels of miR-210 expression in the group of patients with 46,XY DSD of unknown aetiology studied. These findings contributeThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Introduction: Androgen Insensitivity Syndrome (AIS) is the most common cause of Differences of Sexual Development (DSD) in 46, XY individuals. It is a X-linked genetic disease caused by allelic variants in the Androgen Receptor Gene (Xq11-12), leading to 3 different phenotypes: Complete (CAIS), Partial (PAIS) or Mild (MAIS). Methods: Patients with clinical suspicion of AIS (familial history, atypical genitalia, primary amenorrhea and/or inguinal hernia) performed hormonal serum measurements (LH, FSH, estradiol, testosterone) and molecular sequencing of the ARgene, including all exonic regions (8 exons) and the 5’UTR region. Psychosexual variables (gender identity, gender role and sexual orientation) were evaluated through questionnaires. Gender identity was also evaluated through projective psychological test (HTP test). A histopathological study and immunostainining of CD240 and OCT3/4 were carried out for all individuals submitted to gonadectomy.Results: This cohort is made up of 64 individuals: CAIS (n=26) and PAIS (n=38), from 46 different families (24 PAIS; 22 CAIS). Inguinal hernia was the first clinical presentation in 35% of CAIS. Among the PAIS, 20 (52%) were assigned as female at birth, while 18 (48%) as male. Among In the group of PAIS, external genitalia virilization (Sinnecker score) influenced sex assignment (p&lt;0.01). Final height and weight were similar between PAIS and CAIS. Furthermore, gender identity at adulthood, gender role at childhood and sexual orientation were in agreement with sex assignment in virtually all cases of both PAIS and CAIS. No gender change was observed. Molecular diagnosis was obtained in 96% of CAIS and in 87% of PAIS. There were 10 novel AR allelic variants (4 in CAIS - 2 small deletions, 1 missenseand 1 at splicing site and PAIS - 5 missenseand 2 synonymous variants (both causing a new exonic splicing site leading to a short AR protein). LH ranged from 9 to 48 UI/L (mean 19), testosterone from 190 to 1500 ng/dL (mean 438), without phenotype differences. Seminoma was identified in 2 out 24 (8%) individuals with CAIS (at 19 and 20 years of age). This rate was higher taking into account only those who underwent gonadectomy after puberty (16 years old or later: 2 out 17 (12%). Among PAIS there were 2 cases of NICG (at 3 and 19 years of age) and none of seminoma. Conclusion: Hormonal levels did not enable us to differ PAIS and CAIS. Inguinal hernia is a common clinical presentation of AIS. External genitalia appearance in PAIS influenced sex assignment. The psychosexual development in AIS usually complies with sex assignment. No gender change was observed. There is a risk of seminoma in CAIS, especially after puberty, which is not low enough to be ignored.

Introduction: Despite the use of robust techniques for diagnosis, such as arrays and large-scale sequencing of patients with differences of sex development (DSD), the etiology of a great number of DSD patients remains unclear. Investigation of alternative signaling pathways and epigenetic factors is scarce in 46,XY DSD patients. The ZEB proteins have been related to the occurrence of hypospadias in humans, a feature often observed in the atypical genitalia of patients with 46,XY DSD. Additionally, miR-200c has been reported to regulate ZEB. Objective: To evaluate the expression of miR-200c in plasma samples of 46,XY DSD patients with unknown etiology. Methods: Plasma miR-200c of six adult 46,XY DSD patients with unknown etiology (age 18-33, mean 19±8) and 15 adult male controls (age 18-55, mean 29±10 yo) were analyzed. External Masculinization Score (EMS) was used to describe the undervirilization degree of patients’ external genitalia and to classify them in two groups with low EMS (LEMS: 0-4.9 points) and high EMS (HEMS: 5-10 points). All patients presented atypical genitalia with hypospadias. miR-200c was selected based on its targeting to ZEB1 and in silico analysis; miR-23a was used as internal normalization control. RNA was extracted from plasma samples with Magmax Mirvana Total RNA isolation kit. cDNA was synthesized using TaqMan Advanced miRNA cDNA Synthesis Kit and qPCR was performed using TaqMan Advanced miRNA. The data analysis of qPCR results of patients, of each individualized patient and also the EMS groups were compared with the control group by statistical test. Results: LEMS group presented lower expression values of miR-200c when compared to HEMS group (P=0.0001) and control group (p=0.0009), but no difference was observed when comparing HEMS group and controls, the two patients with lower miR-200c expression presented the lowest EMS (EMS- 3 and 3.5). Altogether, patients presented lower values of miR-200c, although not significantly (p=0.09). Discussion: These findings corroborate with previous literature data correlating miR200-c, ZEB1 and hypospadias. The regulatory loop of miR-200c/Zeb1 was previously demonstrated in rats with hypospadias, confirming that low expression of miR-200c induce a higher Zeb1 expression. The ZEB1 upregulation in penile tissue is positively correlated with the severity of hypospadias in animal models and humans. In the present study, 46,XY DSD patients with severe genital undervirilization had lower miR-200c expression in plasma. Conclusion: Plasma miRNA expression patterns may be a new strategy research in 46,XY DSD, contributing for understanding the processes involved in the external genitalia development.

Esta dissertação de qualificação está de acordo com as seguintes normas, em vigor no momento desta publicação: Referências: ABNT, Autor (data).