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Background: Cytogenomic methods have gained space in the clinical investigation of patients with disorders/differences in sexual development (DSD). Here we evaluated the role of the SNP array in achieving a molecular diagnosis in Brazilian patients with syndromic DSD of unknown etiology. Methods: Twenty-two patients with DSD and syndromic features were included in the study and underwent SNP-array analysis. Results: In two patients, the diagnosis of 46,XX SRY + DSD was established. Additionally, two deletions were revealed (3q29 and Xp22.33), justifying the syndromic phenotype in these patients. Two pathogenic CNVs, a 10q25.3-q26.2 and a 13q33.1 deletion encompassing the FGFR2 and the EFNB2 gene, were associated with genital atypia and syndromic characteristics in two patients with 46,XY DSD. In a third 46,XY DSD patient, we identified a duplication in the 14q11.2-q12 region of 6.5 Mb associated with a deletion in the 21p11.2-q21.3 region of 12.7 Mb. In a 46,XY DSD patient with delayed neuropsychomotor development and congenital cataracts, a 12 Kb deletion on chromosome 10 was found, partially clarifying the syndromic phenotype, but not the genital atypia. Conclusions: The SNP array is a useful tool for DSD patients, identifying the molecular etiology in 40% (2/5) of patients with 46,XX DSD and 17.6% (3/17) of patients with 46,XY DSD.

Most infants born with very low birth weight (VLBW, birth weight 1500 g) show spontaneous catch-up growth in postnatal life. The reasons for the absence of catch-up growth are not entirely understood. We performed a comprehensive investigation of 52 children born with VLBW. Ten children had a history of an external cause that explained the VLBW and five refused genetic evaluation. Twenty-three cases were initially evaluated by a candidate gene approach. Patients with a negative result in the candidate gene approach (n = 14) or without clinical suspicion (n = 14) were assessed by chromosome microarray analysis (CMA) and/or whole-exome sequencing (WES). A genetic condition was identified in 19 of 37 (51.4%) patients without an external cause, nine by candidate gene approach, and 10 by a genomic approach (CMA/WES). Silver-Russell syndrome was the most frequent diagnosis (n = 5) and the remaining patients were diagnosed with other rare monogenic conditions. Almost all patients with a positive genetic diagnosis exhibited syndromic features (94.4%). However, microcephaly, neurodevelopmental disorders, major malformation, or facial dysmorphism were also frequently observed in children with an external cause. In conclusion, a significant proportion of children born with VLBW with persistent short stature have a genetic/epigenetic condition.

Wilms’ tumor suppressor gene 1 (WT1) plays an essential role in urogenital and kidney development. Heterozygous germline pathogenic allelic variants of WT1 have been classically associated with Denys–Drash syndrome (DDS) and Frasier syndrome (FS). Usually, exonic pathogenic missense variants in the zinc finger region are the cause of DDS, whereas pathogenic variants affecting the canonic donor lysine-threonine-serine splice site in intron 9 cause FS. Phenotypic overlap between WT1 disorders has been frequently observed. New WT1 variant-associated phenotypes, such as 46,XX testicular/ovarian-testicular disorders of sex development (DSD) and primary ovarian insufficiency, have been reported. In this report, we describe the phenotypes and genotypes of 7 Brazilian patients with pathogenic WT1 variants. The molecular study involved Sanger sequencing and massively parallel targeted sequencing using a DSD-associated gene panel. Six patients (5 with a 46,XY karyotype and 1 with a 46,XX karyotype) were initially evaluated for atypical genitalia, and a 46,XY patient with normal female genitalia sought medical attention for primary amenorrhea. Germ cell tumors were identified in 2 patients, both with variants affecting alternative splicing of WT1 between exons 9 and 10. Two pathogenic missense WT1 variants were identified in two 46,XY individuals with Wilms’ tumors; both patients were WT1 variant, c.1453_1456 (p.Arg485Glyfs*14), was identified in a 46,XX patient with testicular DSD. Nephrotic proteinuria was diagnosed in all patients, including 3 who underwent renal transplantation after progressing to end-stage kidney disease. The expanding phenotypic spectrum associated with WT1 variants in XY and XX individuals confirms their pivotal role in gonadal and renal development as well as in tumorigenesis, emphasizing the clinical implications of these variants in genetic diagnosis.

Background: Molecular biology and cytogenomics methods have gained space in the clinical investigation for patients with disorders/differences in sexual development (DSD). Here we aimed to evaluate the role of SNP-array in achieving a molecular diagnosis in a sample of 22 Brazilian patients with syndromic DSD of unknown etiology, and to compare the relationship between the clinical diagnosis and the SNP-array results.Methods: 22 patients with DSD associated with syndromic features in several systems other than the genital tract were included in the study and underwent search for genomic copy number variations (CNVs) by SNP-array.Results: In two patients, the diagnosis of 46,XX SRY+ DSD was established. Additionally, two deletions were revealed (3q29 and Xp22.33), justifying the syndromic phenotype in these patients. Two pathogenic CNVs, a 10q25.3-q26.2 and 13q33.1 deletion encompassing the FGFR2 and the EFNB2 gene were associated with genital atypia and syndromic characteristics in two patients with 46, XY DSD. In a third 46,XY DSD patient, we identified a duplication in the 14q11.2-q12 region of 6.5 Mb associated with a deletion in the 21p11.2-q21.3 region of 12.7Mb. In a 46,XY DSD patient with delayed neuropsychomotor development and congenital cataract, a 12Kb deletion on chromosome 10 was found, justifying the congenital cataract.Conclusions: The SNP-array technique identified the molecular etiology in 40% (2/5) of patients with 46,XX DSD and 17.6% (3/17) of patients with 46,XY DSD. In one patient the array contributed to partially clarify the patient's syndromic phenotype, but not the genital atypia.

Reaching a firm diagnosis is vital for the long-term management of a patient with a difference or disorder of sex development (DSD). This is especially the case in XY DSD where the diagnostic yield is particularly low. Molecular genetic technology is playing an increasingly important role in the diagnostic process, and it is highly likely that it will be used more often at an earlier stage in the diagnostic process. In many cases of DSD, the clinical utility of molecular genetics is unequivocally clear, but in many other cases there is a need for careful exploration of the benefit of genetic diagnosis through long-term monitoring of these cases. Furthermore, the incorporation of molecular genetics into the diagnostic process requires a careful appreciation of the strengths and weaknesses of the evolving technology, and the interpretation of the results requires a clear understanding of the wide range of conditions that are associated with DSD.

Context Massively parallel sequencing (MPS) technologies have emerged as a first-tier approach for diagnosing several pediatric genetic syndromes. However, MPS has not been systematically integrated into the diagnostic workflow along with clinical/biochemical data for diagnosing 46,XY differences of sex development (DSD). Objective To analyze the contribution of phenotypic classification either alone or in association with genetic evaluations, mainly MPS, for diagnosing a large cohort of 46,XY DSD patients. Design/patients 209 nonsyndromic 46,XY DSD index cases from a Brazilian DSD center were included. Patients were initially classified into 3 subgroups according to clinical and biochemical data: gonadal dysgenesis (GD), disorders of androgen secretion/action, and DSD of unknown etiology. Molecular genetic studies were performed by Sanger sequencing and/or MPS. Results Clinical/biochemical classification into either GD or disorders of hormone secretion/action was obtained in 68.4% of the index cases. Among these, a molecular diagnosis was obtained in 36% and 96.5%, respectively. For the remainder 31.6% classified as DSD of clinically unknown etiology, a molecular diagnosis was achieved in 31.8%. Overall, the molecular diagnosis was achieved in 59.3% of the cohort. The combination of clinical/biochemical and molecular approaches diagnosed 78.9% of the patients. Clinical/biochemical classification matched with the genetic diagnosis in all except 1 case. DHX37 and NR5A1 variants were the most frequent genetic causes among patients with GD and DSD of clinical unknown etiology, respectively. Conclusions The combination of clinical/biochemical with genetic approaches significantly improved the diagnosis of 46,XY DSD. MPS potentially decreases the complexity of the diagnostic workup as a first-line approach for diagnosing 46,XY DSD.

Purpose: To analyze aspects of the sexual life, sexual orientation, and fertility desire among 46,XY DSD people, including those who changed their gender. Methods: It is a cross-sectional study including 127 adults (> 16 years of age) with 46,XY DSD (83 females; 44 males) from a single tertiary center. Results: Sexual fantasies and masturbation practice were more frequent in 46,XY DSD males, whereas orgasm and sexual life satisfaction were similar in both genders. More 46,XY DSD men than women had a long-term romantic relationship. 46,XY DSD women with prenatal androgen exposure reported more fear of being romantically rejected. External genitalia appearance at birth did not impact the sexuality of 46,XY DSD women after surgical genital treatment has been completed. Overall, the sexual life was similar between 46,XY men assigned as males and those who changed to the male gender. Regarding sexual orientation, most self-reported as heterosexual (91% and 92% of women and men, respectively). The desire for fertility had a similar prevalence in both genders, but more women than men considered infertility a barrier to a long-term romantic relationship. A total of 12 (7 males) had children; 10 out of 12 have adopted children. Conclusion: Fertility desire was common among 46,XY DSD people, regardless of gender. Prenatal androgen exposure reduced the desire for motherhood in 46,XY women. 46 XY DSD people who changed from female to male gender presented similar sexual parameters as those assigned as males. Among females, virilized genitalia at birth did not affect sexuality once the surgical treatment is completed.

Hypospadias is a common congenital disorder of male genital formation. Children born small for gestational age (SGA) present a high frequency of hypospadias of undetermined etiology. No previous study investigated the molecular etiology of hypospadias in boys born SGA using massively parallel sequencing. Our objective is to report the genetic findings of a cohort of patients born SGA with medium or proximal hypospadias. We identified 46 individuals with this phenotype from a large cohort of 46,XY DSD patients, including 5 individuals with syndromic features. DNA samples from subjects were studied by either whole exome sequencing or target gene panel approach. Three of the syndromic patients have 5 main clinical features of Silver-Russell syndrome (SRS) and were first studied by MLPA. Among the syndromic patients, loss of DNA methylation at the imprinting control region H19/IGF2 was identified in 2 individuals with SRS clinical diagnosis. Two novel pathogenic variants in compound heterozygous state were identified in the CUL7 gene establishing the diagnosis of 3M syndrome in one patient, and a novel homozygous variant in TRIM37 was identified in another boy with Mulibrey nanism phenotype. Among the non-syndromic subjects, 7 rare heterozygous variants were identified in 6 DSD-related genes. However, none of the variants found can explain the phenotype by themselves. In conclusion, a genetic defect that clarifies the etiology of hypospadias was not found in most of the non-syndromic SGA children, supporting the hypothesis that multifactorial causes, new genes, and/or unidentified epigenetic defects may have an influence in this condition.

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

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.

Background: It is recommended a multidisciplinary approach consisted of clinical, hormonal and genetic workups for diagnosing 46,XY DSD. However, no previous study has quantified how useful is this combined approach. Objectives: To retrospectively review the clinical and genetic findings for diagnosing a large cohort of patients with 46,XY DSD from a single Brazilian center. Methods: 247 non-syndromic 46,XY DSD individuals (159 sporadic and 88 familial cases from 39 families) were studied. Clinical and hormonal data were collected from medical files. Testosterone (T), androstenedione (A) were measured by immunoradiometric or immunofluorimetric assays and dihydrotestosterone (DHT) by RIA after celite chromatography or by liquid chromatography tandem mass spectrometry; T/DHT and T/A ratios were calculated. Analysis of sensitivity (SE), specificity (SP) of T/DHT was performed, being the molecular diagnosis considered the gold standard for diagnosing SRD5A2 deficiency. A T/A>0.8 was considered indicative of 17ß-HSDB3 deficiency. The patients were clinically classified into four subgroups: 1) androgen insensitivity syndrome (AIS), 2) gonadal dysgenesis (GD); 3) defects in androgen synthesis (DAS) and 4) DSD of unknown etiology. Molecular studies were performed by Sanger sequencing and/ or massively parallel sequencing (MPS). Results: The median age at first visit was 14 years (range 0.1 to 59 years). The molecular diagnosis was established in 96.5% of the cases with AIS (n=28/29), in 96% of the subjects with DAS (n=46/48), in 36% of the patients with GD (n=21/57) and in 26.7% (n=15/56) with DSD of unknown etiology. The best cut-off for T/DHT in basal state and hCG stimulated was 12.5 (SE=100%; SP=78.57%) and 24 (SE=87.5%; SP=95.7%) respectively. A T/A Conclusion: Considering the phenotype heterogeneity, pitfalls of the hormonal assessment and number of genes involved, it is reasonable to consider MPS as a first test for diagnosing patients with 46,XY DSD. However, the combination of clinical and molecular diagnosis is more accurate than either strategies alone in diagnosing 46,XY DSD.

Background: Adrenal hypoplasia congenita (AHC) is a rare disease (1:70.000 men) characterized by reduction in all cortical adrenal hormones and also by hypogonadism. NR0B1 -related AHC includes both X-linked AHC and Xp21 deletion. AHC gene mutation was first described in 1994 and occurs in less than 1% of adrenal insufficiency (AI) cases and it is imperative that clinical endocrinologists increase their knowledge on this condition to recognize it as promptly as they were used to do so in other causes of pediatric AI.Clinical Cases: We describe here two sporadic cases and three familial cases from one family, all of them attending at our Endocrine Unit at HCPA, in the South of Brazil. The median age of AI first symptoms was 1 month old in two patients (acute infantile onset due to a salt wasting crises), and eleven years old in the remaining patients (childhood onset). Their exams confirmed the clinical suspicion of primary AI because of low plasma cortisol (from < 0.5 to 4 mcg/dl - reference range from 7-24 mcg/dl), as well as all adrenal steroids levels, high ACTH levels (from 672 to 2225 pg/ml - reference range from 7-63 pg/ml) and high plasmatic renin (from 92 to 448 mcUI/ml - reference range from 2.8-39.9 mcUI/ml). They were also extensively investigated searching for the AI pathophysiology. After some years of lost follow-up, one of the familial cases that had first been seen in 1993, returned to our hospital and was diagnosed with hypogonadism, leading to NR5OB1 gene evaluation.Regarding the genetic etiology, one of the sporadic cases developed a clinical picture including profound developmental delay, seizures and strabismus suggestive of a contiguous gene syndrome that involves Duchenne muscular dystrophy, glycerol kinase deficiency and AHC. A CGH array was performed and identified a Xp21.3-p21.1 deletion. The affected family had an extensive history suggestive of a genetic disease with X-linked inheritance pattern, as shown by the premature death of 3 uncles and a male cousin. A deleterious variant (c.131_212delinsTGAGACCTGTACCGT) in NR5OB1 gene was identified by Sanger sequencing in hemizygous state in the 46,XY affected patients.Conclusion: Albeit a rare disease, it is crucial that endocrinologist all around the World could be aware about clinical characteristics of this condition in order to properly diagnose it. And it is even possible, as it occurred with one of our patient, to make a late pathophysiologic diagnosis, making possible to treat the associated hypogonadism. Reference: Acherman JC, Vilain EJ. NR0B1- related adrenal hypoplasia congenita. GeneReviews 1993-2019.

Disorders (or differences) of sex development (DSD) are a heterogeneous group of congenital conditions with variations in chromosomal, gonadal, or anatomical sex. Impaired gonadal development is central to the pathogenesis of the majority of DSDs and therefore a clear understanding of gonadal development is essential to comprehend the impacts of these disorders on the individual, including impacts on future fertility. Gonadal development was traditionally considered to involve a primary ‘male’ pathway leading to testicular development as a result of expression of a small number of key testis-determining genes. However, it is increasingly recognized that there are several gene networks involved in the development of the bipotential gonad towards either a testicular or ovarian fate. This includes genes that act antagonistically to regulate gonadal development. This review will highlight some of the novel regulators of gonadal development and how the identification of these has enhanced understanding of gonadal development and the pathogenesis of DSD. We will also describe the impact of DSDs on fertility and options for fertility preservation in this context.

Background One of the defining moments of human life occurs early during embryonic development, when individuals sexually differentiate into either male or female. Perturbation of this process can lead to disorders/differences of sex development (DSD). Chromobox protein homolog 2 (CBX2) has two distinct isoforms, CBX2.1 and CBX2.2: the role of CBX2.1 in DSD has been previously established, yet to date the function of the smaller isoform CBX2.2 remains unknown. Methods The genomic DNA of two 46,XY DSD patients was analysed using whole exome sequencing. Furthermore, protein/DNA interaction studies were performed using DNA adenine methyltransferase identification (DamID) to identify putative binding partners of CBX2. Finally, in vitro functional studies were used to elucidate the effect of wild‐type and variant CBX2.2 on selected downstream targets. Results Here, we describe two patients with features of DSD i.e. atypical external genitalia, perineal hypospadias and no palpable gonads, each patient carrying a distinct CBX2.2 variant, p.Cys132Arg (c.394T>C) and p.Cys154fs (c.460delT). We show that both CBX2.2 variants fail to regulate the expression of genes essential for sexual development, leading to a severe 46,XY DSD defect, likely because of a defective expression of EMX2 in the developing gonad. Conclusion Our study indicates a distinct function of the shorter form of CBX2 and by identifying several of its unique targets, can advance our understanding of DSD pathogenesis and ultimately DSD diagnosis and management.

Context46,XY Gonadal dysgenesis (GD) is a heterogeneous group of disorders with a wide phenotypic spectrum, including embryonic testicular regression syndrome (ETRS).ObjectiveTo report a gene for 46,XY GD etiology, especially for ETRS.DesignScreening of familial cases of 46,XY GD using whole-exome sequencing and sporadic cases by target gene-panel sequencing.SettingTertiary Referral Center for differences/disorders of sex development (DSD).Patients and InterventionsWe 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.ResultsWe 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.ConclusionThis strong genetic evidence identifies DHX37 as a player in the complex cascade of male gonadal differentiation and maintenance.

The main complain of patients with partial androgen insensitivity patients (PAIS) is undervirilization signs and the small penile length. Supplemental androgen therapy has enhanced virilization in only a few patients with PAIS. Aromatase inhibitors as anastrozole and letrozole are nonsteroidal inhibitors able to bind reversibly to the heme group of cytochrome P450 reducing estrogen and estrone levels and increasing LH and FSH levels and consequently testosterone levels. We hypothesized that the combination of high-dose testosterone with anastrozole can maintain a sustained elevated testosterone levels and also deviated testosterone metabolization from estrogen to DHT, therefore increasing penile length. Case Report This experimental treatment was proposed and both patient and parents willing to participate. Patient was firstly evaluated at 7 yrs of age. The hemizygous c.2599G>C; p.V867L AR in the ligand-binding domain (LBD) was found in both affected siblings. He was previously submitted to three unsuccessful genital masculinizing surgeries and bilateral orchidopexia. The external genitalia presented a microphalus (penile length of 2 cm, -4.5 SDS), perineal hypospadias and bilateral topic testis. He received testosterone cypionate (50 mg IM injections monthly, for 3 times at age of 7 and 200 mg IM injections monthly, for 4 times at age of 8); topic DHT gel (5-10 g/daily for three months at age of 7 yrs). At age of 12.3 yrs, the genital masculinization surgeries were completed, penile length was 3.5 cm (-2.6 SDS), testicular size of 2.6x1.5 cm and pubic hair Tanner II stage. LH and FSH levels were (9.4 and 3.2 IU/L respectively), testosterone (304 ng/dL), DHT (26 ng/dL) and estradiol (

Abstract: The sexuality of 46,XY DSD individuals can be affected by several factors as external genitalia appearance, negative body image, traumas, social stigma, and previous genital surgery. Previous studies has been reported an impaired sexual quality of life (SexQoL) in 46,XY DSD individuals in comparison with controls. To observe aspects regarding sexual life, 144 individuals with 46,XY DSD (100 female; 44 male) were enrolled in this study. All of them were adults (>16 years old) and had completed all surgical treatments. Sexual life and fertility issues were assessed using self-developed questionnaire with likert-scale questions. Sexual orientation was assessed on the basis of orientation, sexual behavior and sexual attraction. For comparison, the cohort was divided into 3 subgroups (A: gender at adulthood; B: genitalia appearance at birth; and C: male individuals at adulthood who were assigned as male versus who changed from female to male). About fertility issues, 12 individuals (8%) had children (7 male; 5 female). 10 out of 12 were adopted children and 2 patients had children by Intravaginal insemination (IVI). Desire of fertility was similar in women and men (78.3%, 80.7% respectively; p = NS). Fear of rejection due to infertility was reported by 28% (34 out of 117). More women than men considered infertility a barrier to a steady relationship (p=0.04). Regarding sexual orientation, more than 90% referred themselves as heterosexual. Homosexuality was reported by 4% in both genders. Most 46,XY DSD women reported androphilic sexual attraction and female sexual behavior (have sex with male partners), regardless of the appearance of external genitalia. The opposite (gynephilic sexual attraction and male sexual behavior) was observed in most male patients either in individuals who were assigned male at birth and in individuals who changed from female to male. Regarding sexual life, there was significant difference between males and females on sexual fantasies, masturbation practice and fixed sexual partner, which were more frequent in males. Nevertheless, both gender reported a similar sexual life satisfaction (76% and 80%). All sexual parameters were similar comparing individuals who changed from female to male and who were assigned as male. In conclusion, sexual life satisfaction was reported by the majority of adult patients with 46,XY DSD in both gender. Among female patients, the appearance of external genitalia did not influence on sexuality, once the surgical correction of atypical genitalia have been performed. In male individuals, the sexual life was similar either if they were assigned as male or changed for that gender later. Fertility desire was common among 46,XY DSD people, regardless of gender or 46,XY etiology, which should be taken into consideration in the 46,XY DSD management.

17β-hydroxysteroid dehydrogenase 3 deficiency consists of a defect in the last phase of steroidogenesis, in which androstenedione is converted into testosterone and estrone into estradiol. External genitalia range from female-like to atypical genitalia and most affected males are raised as females. Virilization in subjects with 17β-HSD3 deficiency occurs at the time of puberty and several of them change to male social sex. In male social sex patients, testes can be safely maintained, as long as they are positioned inside the scrotum The phenotype of 46,XY DSD due to 17β-HSD3 deficiency is extremely variable and clinically indistinguishable from other causes of 46,XY DSD such as partial androgen insensitivity syndrome and 5α-reductase 2 deficiency. Laboratory diagnosis is based on a low testosterone/androstenedione ratio due to high serum levels of androstenedione and low levels of testosterone. The disorder is caused by a homozygous or compound heterozygous mutations in the HSD17B3 gene that encodes the 17β-HSD3 isoenzyme leading to an impairment of the conversion of 17-keto into 17-hydroxysteroids. Molecular genetic testing confirms the diagnosis and provides the orientation for genetic counseling. Our proposal in this article is to review the previously reported cases of 17β-HSD3 deficiency adding our own cases.

Both 17β-HSD3 and 5α-RD2 deficiencies are caused by homozygous or compound heterozygous mutations in the HSD17B3 and SRD5A2 genes, respectively. These molecular deficiencies result in androgen synthesis defects and cause DSD in 46,XY affected individuals. In both conditions, most patients have severe undervirilization of the external genitalia at birth and are usually assigned female. This review discuss the clinical, hormonal, psychosexual and long term outcomes, highlighting similarities and differences between these DSD 46,XY conditions.

46,XY gonadal dysgenesis is a heterogeneous disorder of sex development (DSD) that features abnormal gonadal development and varying degrees of undervirilization of the external genitalia, ranging from micropenis to female-like genitalia. Embryonic testicular regression syndrome (ETRS; MIM: 273250) is considered part of the clinical spectrum of 46,XY gonadal dysgenesis. Most ETRS patients present micropenis or atypical genitalia associated with a complete absence of gonadal tissue in one or both sides. In most patients with gonadal dysgenesis, the genetic diagnosis is unclear. We performed whole exome sequencing in ETRS patients and identified a rare variant, the p.Arg308Gln, in DEAH (Asp-Glu-Ala-His) box polypeptide 37 (DHX37) in 5 affected individuals from three unrelated families. We expanded the analysis of DHX37 coding region to additional 71 patients with 46,XY gonadal dysgenesis and identified the p.Arg308Gln and three other DHX37 missense variants (p.Arg151Trp, p.Thr304Met and p.Arg674Trp) in 11 affected members from eight distinct families (8 patients with ETRS, two with partial gonadal dysgenesis and one 46,XY DSD female patient previously gonadectomized). The p.Arg308Gln and p.Arg674Trp recurrent variants were identified in six and three families, respectively. Segregation analysis revealed sex-limited autosomal dominant inheritance in 4 families, autosomal dominant with incomplete penetrance in one family and autosomal recessive in another family. Immunohistochemical analysis of normal testes revealed that DHX37 is expressed in germ cells at different stages of maturation.This study demonstrates an expressive frequency of rare predicted to be deleterious DHX37 variants in 46,XY gonadal dysgenesis group, particularly those individuals exhibiting the ETRS phenotype (25% and 50%, respectively).Our findings indicate that DHX37 is a new player in the complex cascade of male gonadal differentiation and maintenance, thus establishing a novel and frequent molecular etiology for 46,XY gonadal dysgenesis spectrum, mainly for embryonic testicular regression syndrome.

Missense mutations in the gene, MAP3K1, are a common cause of 46,XY gonadal dysgenesis, accounting for 15-20% of cases [Ostrer, 2014, Disorders of sex development (DSDs): an update. J. Clin. Endocrinol. Metab., 99, 1503-1509]. Functional studies demonstrated that all of these mutations cause a protein gain-of-function that alters co-factor binding and increases phosphorylation of the downstream MAP kinase pathway targets, MAPK11, MAP3K and MAPK1. This dysregulation of the MAP kinase pathway results in increased CTNNB1, increased expression of WNT4 and FOXL2 and decreased expression of SRY and SOX9. Unique and recurrent pathogenic mutations cluster in three semi-contiguous domains outside the kinase region of the protein, a newly identified N-terminal domain that shares homology with the Guanine Exchange Factor (residues Met164 to Glu231), a Plant HomeoDomain (residues Met442 to Trp495) and an ARMadillo repeat domain (residues Met566 to Glu862). Despite the presence of the mutation clusters and clinical data, there exists a dearth of mechanistic insights behind the development imbalance. In this paper, we use structural modeling and functional data of these mutations to understand alterations of the MAP3K1 protein and the effects on protein folding, binding and downstream target phosphorylation. We show that these mutations have differential effects on protein binding depending on the domains in which they occur. These mutations increase the binding of the RHOA, MAP3K4 and FRAT1 proteins and generally decrease the binding of RAC1. Thus, pathologies in MAP3K1 disrupt the balance between the pro-kinase activities of the RHOA and MAP3K4 binding partners and the inhibitory activity of RAC1.

Molecular diagnosis is rarely established in 46,XX testicular (T) disorder of sex development (DSD) individuals with atypical genitalia. The Wilms' tumour factor-1 (WT1) gene is involved in early gonadal development in both sexes. Classically, WT1 deleterious variants are associated with 46,XY disorders of sex development (DSD) because of gonadal dysgenesis. We report a novel frameshift WT1 variant identified in an SRY-negative 46,XX testicular DSD girl born with atypical genitalia. Target massively parallel sequencing involving DSD-related genes identified a novel heterozygous WT1 c.1453_1456del; p.Arg485Glyfs*14 variant located in the fourth zinc finger of the protein which is absent in the population databases. Segregation analysis and microsatellite analysis confirmed the de novo status of the variant that is predicted to be deleterious by in silico tools and to increase WT1 target activation in crystallographic model. This novel and predicted activating frameshift WT1 variant leading to the 46,XX testicular DSD phenotype includes the fourth zinc-finger DNA-binding domain defects in the genetic aetiology of 46,XX DSD.

ContextIn 46,XY disorders of sexual development (DSD) patients, several factors may affect psychosexual development, leading to gender identity discrepancy and gender change later in life. Prenatal sexual steroid exposure and external genital virilization are considered to influence human psychosexual development, but their roles not completely understood yet.DesignA total of 144 individuals (18 to 60 years of age) with a clinical/molecular diagnosis of 46,XY DSD from a single tertiary center were enrolled. Psychosexual outcomes (gender role, gender identity, and sexual orientation) were assessed using questionnaires and psychological test. The Sinnecker score was used for genital virilization measurement. Prenatal androgen exposure was estimated according to 46,XY DSD etiology.ResultsWe found a positive association between prenatal androgen exposure and male psychosexual outcomes. Alternatively, prenatal estrogen exposure, age of gonadectomy, and the degree of external genital virilization did not influence any psychosexual outcome. There were 19% (n = 27) with gender change, which was associated with prenatal androgen exposure (P < 0.001) but not with the external genital virilization. The median age of gender change was 15 years, but most of the patients reported the desire for gender change earlier.ConclusionsPrenatal androgen exposure influenced psychosexual development in 46,XY DSD favoring male psychosexuality in all psychosexual outcomes, whereas the degree of external genital virilization did not influence these outcomes. The organizational effect of sexual steroids on psychosexuality at puberty appears to be weak in comparison with the prenatal effects. Prenatal androgen exposure also influenced female-to-male gender change frequency. All 46,XY DSD conditions with prenatal androgen exposure must be followed for gender issues in their management.

Background Follow-up data on patients with 46,XY partial gonadal dysgenesis (PGD) until adulthood are scarce, making information on prognosis difficult. Objective To analyse the long-term outcomes of patients with 46,XY PGD regarding testosterone production, germ cell tumour risk, genotype and psychosexual adaptation. Methods A retrospective longitudinal study of 33 patients (20 assigned male and 13 patients assigned female at birth). Molecular diagnosis was performed by Sanger sequencing or by targeted massively parallel sequencing of 63 genes related to disorders of sex development (DSDs). Results Age at first and last visit ranged from 0.1 to 43 and from 17 to 53 years, respectively. Spontaneous puberty was observed in 57% of the patients. During follow-up, six of them had a gonadectomy (four due to female gender, and two because of a gonadal tumour). At last evaluation, five of six patients had adult male testosterone levels (median 16.7 nmol/L, range 15.3-21.7 nmol/L) and elevated LH and FSH levels. Germ cell tumours were found in two postpubertal patients (one with an abdominal gonad and one patient with Frasier syndrome). Molecular diagnosis was possible in 11 patients (33%). NR5A1 variants were the most prevalent molecular defects (n = 6), and four of five patients harbouring them developed spontaneous puberty. Gender change was observed in four patients, two from each sex assignment group; all patients reported satisfaction with their gender at final evaluation. Sexual intercourse was reported by 81% of both gender and 82% of them reported satisfaction with their sexual lives. Conclusion Spontaneous puberty was observed in 57% of the patients with 46,XY PGD, being NR5A1 defects the most prevalent ones among all the patients and in those with spontaneous puberty. Gender change due to gender dysphoria was reported by 12% of the patients. All the patients reported satisfaction with their final gender, and most of them with their sexual life.

There are only 2 patients with 47,XXY karyotype and androgen receptor (AR) gene mutation reported in the literature, and both are diagnosed as complete androgen insensitivity syndrome (CAIS). We report a 22-year-old female with 47,XXY karyotype and atypical external genitalia. Sequencing of AR revealed the heterozygous p.Asn849Lys*32 mutation, and extensive X chromosome microsatellite analysis showed homozygosity for Xp and heterozygosity for Xq, suggesting partial X maternal isodisomy. Partial androgen insensitivity syndrome (PAIS) developed in this case, probably because of the presence of the heterozygous AR mutation and random X- inactivation of the healthy allele. This is the first report of a female patient with 47,XXY karyotype and PAIS phenotype.

Androgen insensitivity syndrome (AIS) is the most common cause of 46,XY disorders of sex development (46,XY DSD). This syndrome is an X-linked inheritance disease and it is caused by mutations in the human androgen receptor (AR) gene. Non-synonymous point AR mutations are frequently described in this disease, including in the complete phenotype. We present a novel synonymous mutation in the human AR gene (c.1530C > T) in four 46,XY patients from two unrelated families associated with complete androgen insensitivity syndrome (CAIS). The analysis of mRNA from testis showed that synonymous AR mutation changed the natural exon 1 donor splice site, with deletion of the last 92 nucleotides of the AR exon 1 leading to a premature stop codon 12 positions ahead resulting in a truncate AR protein. Linkage analyses suggested a probable founder effect for this mutation. In conclusion, we described the first synonymous AR mutation associated with CAIS phenotype, reinforcing the disease-causing role of synonymous mutations.

Steroidogenic factor 1 (NR5A1, SF‐1, Ad4BP) is a transcriptional regulator of genes involved in adrenal and gonadal development and function. Mutations in NR5A1 have been among the most frequently identified genetic causes of gonadal development disorders and are associated with a wide phenotypic spectrum. In 46,XY individuals, NR5A1‐related phenotypes may range from disorders of sex development (DSD) to oligo/azoospermia, and in 46,XX individuals, from 46,XX ovotesticular and testicular DSD to primary ovarian insufficiency (POI). The most common 46,XY phenotype is atypical or female external genitalia with clitoromegaly, palpable gonads, and absence of Müllerian derivatives. Notably, an undervirilized external genitalia is frequently seen at birth, while spontaneous virilization may occur later, at puberty. In 46,XX individuals, NR5A1 mutations are a rare genetic cause of POI, manifesting as primary or secondary amenorrhea, infertility, hypoestrogenism, and elevated gonadotropin levels. Mothers and sisters of 46,XY DSD patients carrying heterozygous NR5A1 mutations may develop POI, and therefore require appropriate counseling. Moreover, the recurrent heterozygous p.Arg92Trp NR5A1 mutation is associated with variable degrees of testis development in 46,XX patients. A clear genotype‐phenotype correlation is not seen in patients bearing NR5A1 mutations, suggesting that genetic modifiers, such as pathogenic variants in other testis/ovarian‐determining genes, may contribute to the phenotypic expression. Here, we review the published literature on NR5A1‐related disease, and discuss our findings at a single tertiary center in Brazil, including ten novel NR5A1 mutations identified in 46,XY DSD patients. The ever‐expanding phenotypic range associated with NR5A1 variants in XY and XX individuals confirms its pivotal role in reproductive biology, and should alert clinicians to the possibility of NR5A1 defects in a variety of phenotypes presenting with gonadal dysfunction. Birth Defects Research (Part C) 108:309–320, 2016. © 2016 The Authors Birth Defects Research Part C: Embryo Today: Reviews Published by Wiley Periodicals, Inc.

Purpose: Disorders or differences of sex development (DSDs) are rare congenital conditions characterized by atypical sex development. Despite advances in genomic technologies, the molecular cause remains unknown in 50% of cases. Methods: Homozygosity mapping and whole-exome sequencing revealed an ESR2 variant in an individual with syndromic 46, XY DSD. Additional cases with 46, XY DSD underwent whole-exome sequencing and targeted next-generation sequencing of ESR2. Functional characterization of the identified variants included luciferase assays and protein structure analysis. Gonadal ESR2 expression was assessed in human embryonic data sets and immunostaining of estrogen receptor-beta (ER-beta) was performed in an 8-week-old human male embryo. Results: We identified a homozygous ESR2 variant, c.541_543del p. (Asn181del), located in the highly conserved DNA-binding domain of ER-beta, in an individual with syndromic 46, XY DSD. Two additional heterozygous missense variants, c.251G>T p.(Gly84Val) and c.1277T>G p.(Leu426Arg), located in the N-terminus and the ligand-binding domain of ER-beta, were found in unrelated, nonsyndromic 46, XY DSD cases. Significantly increased transcriptional activation and an impact on protein conformation were shown for the p.(Asn181del) and p.(Leu426Arg) variants. Testicular ESR2 expression was previously documented and ER-beta immunostaining was positive in the developing intestine and eyes. Conclusion: Our study supports a role for ESR2 as a novel candidate gene for 46, XY DSD.