Masculinization Of External Genitalia Research Articles

RF02 | PMON189 Temporal and Social trends towards sex assignment and gender change in the 5α-reductase type 2 deficiency

Abstract 5α-reductase type 2 (5αR2) deficiency is an autosomal recessive inherited condition affecting 46,XY individuals resulting from the inability to convert testosterone into dihydrotestosterone. Affected individuals present a range of external genitalia undervirilization at birth and pronounced masculinization at puberty. Unlike most other 46,XY DSD, the frequency of gender change among 5αR2 deficiency is high. Objective to analyze the 451 individuals carrying pathogenic 5αR2 variants in the SRD5A2 gene in both alleles (in homozygous or compound heterozygous state) in the literature (Pubmed, EMBASE, Medline) and websites (ensemble, HGMD, ClinVar) regarding sex assignment, external genitalia virilization (1-5 Sinnecker score), and gender change accordingly country income Methods A total of 357 cases out of 451 had clinical information. All patients included were divided between those born before or after 2006 (the DSD consensus year). Country income was based on the 2018 World Bank classification, classifying income into four categories: low, lower-middle, upper-middle, and high (www.worldbank.org). Categorical variables were analyzed using the Chi-square test followed by the Cramer's V. Continuous variables were analyzed either by Student t-test or ANOVA one way. A binary logistic regression was built using sex assignment and gender change as categorical dependent variables. A p<.05 was considered significant. Results When we divided the individuals into those diagnosed after and before 2006, the percentage of female sex assignments dropped from 72% to 56% (p<.0001), followed by an odds ratio reduction (from 2.52 to 1.65) for female sex assignment. Country income did not impact sex assignment (p=.21). However, most cases (86%) were reported from countries with high and upper-middle economies. The absence of reports from low-income countries may be due to several reasons, such as barriers to molecular diagnosis, scientific access, and specialized medical assistance. Most cases were assigned as female (69%). The overall rate of gender change was 25% (89 out 357). All but one changed their gender from female to male. The rate of gender change was significantly different across all countries (V=0.46; p<.001), but it ranged from 16% to 70%, and gender change was lower among those diagnosed after 2006 (p=.01). The association between less external genitalia virilization and female sex assignment was significant (p<.001). On the other hand, the virilization score was similar between those who kept or changed gender (p=.37). Neither the type of SRD5A2 variant (missense, nonsense, indel) nor zygosity (homo/hetero) impacted sex assignment or gender change. Conclusion An apparent temporal trend indicates an increased likelihood of affected 5αR2 deficiency individuals being raised as boys, providing a gender change drop. The external virilization degree influenced sex assignment, but it does not affect gender change. There is no correlation between either both sex assignment and gender development with 5αR2 genetics. Presentation: Saturday, June 11, 2022 1:06 p.m. - 1:11 p.m., Saturday, June 11, 2022 1:06 p.m. - 1:11 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Ambulatory Blood Pressure Monitoring in Children and Adults Prenatally Exposed to Dexamethasone Treatment.

ContextThe clinical use of dexamethasone (DEX) prenatally to reduce virilization of external genitalia in female fetuses with congenital adrenal hyperplasia (CAH) is efficient but still controversial. It remains challenging to prevent the excessive exposure of DEX in unborn healthy babies during the first trimester of pregnancy.ObjectiveSince endogenous glucocorticoids contribute to the maintenance of blood pressure (BP) and since events during fetal life may program the fetus and affect future metabolic health, the aim of this study was to analyze ambulatory BP measurements in CAH-unaffected children and adults that were prenatally exposed to DEX treatment.MethodsAmbulatory BP measurements were analyzed in 33 (16 female) DEX-treated participants aged 5.1 to 26.3 years (19 participants aged ≤ 18 years) and in 54 (28 female) age- and sex-matched apparently healthy controls aged 5.5 to 25.3 years (27 participants aged ≤ 18 years) with ambulatory normotension.ResultsParticipants’ age, height, weight, and body mass index were similar between the DEX-treated group and the control group. Heart rate, 24-hour BP, pulse pressure, and nighttime dipping did not statistically significantly differ between DEX-treated participants and controls.ConclusionOur study suggests that prenatal DEX treatment in CAH-unaffected children and adults does not appear to adversely affect ambulatory BP later in life. Our observations need to be confirmed in larger studies.

Treatment of Hypertension in a Child With 11Beta-Hydroxylase Deficiency: A Case Report

Background: 11beta-hydroxylase deficiency (11βOHD) is clinically presented with external genitalia virilization in girls and precocious puberty in boys. Low renin hypertension occurs in both sexes. Early diagnosis and treatment of hypertension can prevent complications. Objectives: To provide a practical management for controlling hypertension in patients with 11-beta-hydroxylase deficiency. Methods: We described a 4.5 years old girl of 46.XX, who presented with ambiguous genitalia at birth and hypertension later in follow-up. The patient had received the appropriate dosage of hydrocortisone and the level of 17-hydroxy progesterone was within the acceptable range but the hypokalemia persisted. Both hypertension and hypokalemia were normalized when spironolactone was added. Results: Blocking mineralocorticoid receptor treat hypertension in 11-beta-hydroxylase deficiency. Conclusions: Intermittent measurement of blood pressure is necessary for patients with 11βOHD. In these patients, spironolactone is effective in treating mineralocorticoid-mediated hypertension and hypokalemia by blocking mineralocorticoid receptors.

Hyponatremia, Metabolic Acidosis, and Abnormal Newborn Screen in a Preterm Neonate.

A 2,250-g male neonate is born at 34 weeks of gestation via spontaneous vaginal delivery to a 30-year-old gravida 1, para 0 woman after presentation to a community hospital with uterine contractions and spontaneous rupture of membranes. The mother receives 1 dose of ampicillin, 1 dose of betamethasone, and magnesium before delivery. At delivery, the neonate is vigorous and crying. His Apgar scores are 8 and 9 at 1 and 5 minutes after birth, respectively, with no abnormalities noted on physical examination. In the first couple of hours after birth, he develops persistent oxygen desaturations and is started on continuous positive airway pressure.He is admitted to the NICU for prematurity and low birthweight. Admission examination demonstrates nondysmorphic features, no respiratory distress with good air exchange, regular heart rate and rhythm without murmur, soft and flat abdomen, patent anus, normal external male genitalia, unremarkable neurologic findings, and pink skin color.Chest radiography shows mild diffuse ground-glass reticular densities. He is placed on a stock solution of parenteral nutrition via a peripheral intravenous catheter. Antibiotics are started because of the mother’s unknown group B Streptococcus status and preterm labor. Initial surveillance of electrolytes demonstrates sodium, potassium, calcium, phosphorus, and glucose levels within normal limits. On day 4 after birth, the neonate continues on nasal canula for tachypnea, receiving parenteral nutrition, and advancing gavage feeds of maternal breast milk or formula. A capillary blood gas measurement shows mild metabolic acidosis, with a pH of 7.28, Pco2 32 mm Hg (4.26 kPa), and bicarbonate 19 mEq/L (19 mmol/L).On day 6 after birth, repeat surveillance of electrolytes shows a sodium concentration of 126 mEq/L (126 mmol/L) and potassium of 6.7 mEq/L (6.7 mmol/L). Glucose concentration is 53 mg/dL (2.94 mmol/L). Blood gas continues to show mild metabolic acidosis with a pH of 7.27, Pco2 31 mm Hg (4.12 kPa), and bicarbonate 19 mEq/L (19 mmol/L). Results of the state newborn screening demonstrate an elevated 17-hydroxyprogesterone (17-OHP) of 221 nmol/L (normal <80 nmol/L) on dried blood spot testing, or 14,586 ng/dL (normal <5,280 ng/dL) as serum equivalent. A confirmatory serum 17-OHP level is sent that same day. The pediatric endocrinology service is consulted for the concerning laboratory results.The neonate is transferred to a tertiary care pediatric hospital for further management. He is started on hydrocortisone and fludrocortisone replacements along with sodium chloride supplementation. Repeat serum 17-OHP before treatment is greater than 50,000 ng/dL, and the actual diagnosis of classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is made.The neonate continues to appear well with normal vital signs, including blood pressure. He is breathing room air, receiving full enteral feeds, and working on oral feeds with the occupational therapist. Parenteral nutrition has been discontinued.The differential diagnosis for hyponatremia and hyperkalemia in a neonate includes CAH, pseudohypoaldosteronism, inappropriate parenteral nutrition composition, and exogenous diuretic use.CAH is a group of autosomal recessive disorders encompassing enzyme deficiencies in the adrenal steroidogenesis pathway and is the most common cause of primary adrenal insufficiency in children. The classic form of CAH affects 1 in 15,000 neonates and is commonly identified through newborn screening, (1) with screening mandated across the United States since 2008. (2) Most cases of CAH are caused by 21-OHD, which is marked by an elevated serum 17-OHP. In the steroidogenic pathway, 17-OHP is the precursor in the enzymatic reaction that is catalyzed by 21-OHD, and is the primary analyte tested in newborn screening for CAH. If treatment is not initiated early, neonates may present in acute salt-wasting adrenal crisis with vomiting, hypotension, and severe dehydration, electrolyte abnormalities, and hypoglycemia. (1)(3)(4)(5) Female neonates with classic CAH also present with virilization of external genitalia at birth.False-positive newborn screening results for CAH are common in preterm neonates, related to the fact that healthy preterm neonates have higher overall levels of 17-OHP compared with term neonates. (6) Thus, many screening programs have established reference ranges that are based on weight and gestational age.For neonates with classic CAH, therapy should be initiated soon after birth to reduce the potentially life-threatening sequelae of CAH. Classic CAH is treated with steroid hormone replacements including hydrocortisone (to replace cortisol) and fludrocortisone (to replace aldosterone). Supplemental sodium chloride (salt) is also recommended for neonates with classic CAH who could have salt-wasting.

Effects of Pre and Post-Natal Androgen Exposure on Psychosexual Aspects and Gender Change in Congenital Adrenal Hyperplasia Due to 21-hydroxylase Deficiency

Introduction: Congenital Adrenal Hyperplasia (CAH) is defined as a group of autosomal recessive disorders characterized by a deficiency of the enzyme required to synthesize cortisol by the adrenal cortex. Defects in the 21-hydroxylase enzyme make up 90% of CAH. These defects impaired cortisol synthesis leading to an ACTH increase resulting in androgen excess in both salt-wasting (SW) or simple virilizing (SV) forms. As androgens play a role in the human psychosexual development favoring male psychosexuality, this study was designed to evaluate the impact of androgen exposure on the psychosexuality of individuals with CAH due 21-hydroxylase deficiency. Methods: This retrospective cohort includes 46,XX individuals (115 female-assigned; 8 male-assigned) with a molecular diagnosis of CAH due to CYP21A2 pathogenic variants in homozygous or compounds heterozygous state. External genitalia virilization was scored using Prader scale. Phenotype, time at diagnosis, sex assignment, and gender change were assessed. The gender role at childhood was assessed through the playmates and toys profile at childhood. Gender identity was assessed by a projective psychological test (HTP). Sexual orientation was assessed by self-report sexual identity. Compliance of glucocorticoid replacement was assessed by adequate testosterone and androstenedione serum levels for age. Results: CAH was diagnosed at the neonatal time in 73% (n=78). Fifth-nine (51%) had the SW form and 49% (n=56) had the SV form. While all cases of SW were diagnosed at the neonatal time (0.12 ± 0.14 months), the mean age at diagnosis among SV was 6.03 ± 8.45 years (p=<.001). The median of Prader score was 3 in both forms. Male sex assignment was associated with more virilized external genitalia (p=.002). Gender change occurred in 6 cases (female to male), all with SV form. The prader score was higher among those who changed gender (p=.01). All of those who changed their gender had poor treatment compliance. A total of 13% (n=15) of all groups defined themselves as homosexual. There was a strong association between male toys and preference for male playmates in childhood with homosexuality and male gender identity in adulthood with both gender change from female to male and homosexuality. Conclusion: Prenatal androgen exposure favors male psychosexuality in 46,XX CAH individuals as observed by the association between highest Prader scores and all assessed psychosexual outcomes. This influence is also substantiated by post-natal androgen exposure as observed by compliance issues and late diagnosis among those who changed from female to male gender.

The Worldwide Landscape of Variants in the Androgen Receptor Gene Related to Androgen Insensitivity Syndrome: An Integrative Analysis

Androgen Insensitivity Syndrome (AIS) is an X-linked genetic disease and it is the most common cause of 46,XY DSD. It is divided into 3 phenotypes: complete (CAIS), partial (PAIS), and mild (MAIS). To analyse the landscape of AR variants in AIS we collected all AR variants reported among AIS in the literature (Pubmed, EMBASE, Medline) and websites (ensemble, HGMD, ClinVar). They were analyzed according to phenotype, exon location, domain, amino acid (aa) conservation, sex assignment, external genitalia virilization (EMS score), molecular and functional studies. Conservation analysis of the AR were performed using CONSURF plataform. To test our hypothesis that non-synonymous AR variants could also impact on splicing, we used both ESEfinder and Human Splicing Finder 3.1. We founded 901 individuals with AIS: CAIS = 565 (62.7%); PAIS = 282 (31.3%); and MAIS = 54 (6%). They had 465 different AR variants: CAIS = 290 (62.3%); PAIS = 135 (29.1%); and MAIS = 40 (8.6%). Among MAIS and PAIS, most variants were at LDB domain (22 out 40 = 55% and 84 out 135 = 62.2%, respectively) whereas they were at NTD domain among CAIS (129 out 290 = 44.5%). Most were missense (81%). However, small indels (11%), nonsense (3%), splicing sites (4%) and large deletions (1%) were all reported. Non-synonymous AR variants accounting for 60%, 96%, and 100% of CAIS, PAIS, and MAIS, respectively. Synonymous AR variants were rarely found (n=3). In 81% only the AR sequencing was performed. The remaining was detected by WES (18%) or WGS (1%). Deep intronic variant was detected in PAIS (n=1) while variants in the 5’UTR of the AR gene in both PAIS and CAIS (n=2). Most AR variants were located at conserved aa (78%), but AR variants at non-conserved aa were more frequently indels (p<.01). Functional studies were found in 38%, mostly showing reduced AR expression. Among PAIS, 48% (n=134) were assigned as male at birth. The median EMS was 5 (95% CI, 5-7) in those assigned as male while it was 3.2 (95% CI, 2-6) in those assigned as female (p<.01). The median of EMS score was lower in variants at NTD domain (2.8, 95% CI, 0-7). We identified 34 AR variants causing more than one AIS phenotype (mostly CAIS and PAIS) and 6 AR variants causing all of three AIS phenotypes. In silico analysis suggests potential to disrupt normal AR splicing in 18 (53%) by creating new acceptor or donor splicing sites (n=11) or exonic splicing signals (n=7). More severe AR variants are related to CAIS. Most AR variants were reported only based on AR sequencing. Therefore, the functional pathogenicity of these variants remains unclear. Further studies including WGS could help to expand the molecular diagnosis of AIS. There is phenotype variability in AIS. So, sex assignment of patients with PAIS cannot be based on a specific identified AR gene mutation. There is potential to alter splicing among non-synonymous AR variants, which could be an explanation for phenotype variability in AIS.

Genotypic Sex and Severity of the Disease Determine the Time of Clinical Presentation in Steroid 17α-Hydroxylase/17,20-Lyase Deficiency

Context: Steroid 17α-hydroxylase/17,20-lyase deficiency (17OHD) is characterized by decreased sex steroids and cortisol, and excessive mineralocorticoid action. The clinical symptoms of hypocortisolemia are subtle. Aim: The clinical, biochemical, and molecular characteristics of patients with 17OHD were evaluated to determine the factors influencing the time of diagnosis and the management. Patients and Methods: Clinical data, steroid profiles by liquid chromatography-tandem mass spectrometry, and Sanger sequencing of the CYP17A1 gene was evaluated in 12 patients with 17OHD diagnosed between 2004 and 2020. Results: Median age of diagnosis was 13.9 (range: 0.04–29.5) years. Ten of 12 patients had 46,XY karyotype. Except for one boy with partial 17OHD, all patients had female external genitalia hence raised as females. The clinical presentation of 17OHD was earlier (median age: 7 years) in patients, who presented with severe hypertension, atypical genitalia, or positive family history (n = 6, 50%) than those without (median age: 15.3 years; p = 0.0005). The latter group presented with amenorrhea (n = 6, 50%). Steroid profile of patients uniformly showed a typical pattern of 17OHD regardless of the age at diagnosis. Serum gonadotropin concentrations were elevated in patients >12 years (n = 7), normal in pre-adolescents (n = 4), and low in a patient, who had a digenic inheritance of homozygous CYP17A1 and KISS1R mutations. Conclusions: Early clinical presentation and diagnosis in 17OHD are associated with symptomatic hypertension in both 46,XX and 46,XY patients or inadequate virilization of external genitalia in 46,XY partial 17OHD. In the absence of these, the clinical presentation is at late pubertal ages at which time amenorrhea and elevated gonadotropins are the hints for diagnosis.

Clinical and Molecular Analysis of Four Patients With 11β-Hydroxylase Deficiency.

Objective: 11β-hydroxylase deficiency (11βOHD) is a rare autosomal recessive disorder caused by mutations in the CYP11B1 gene. It is characterized by virilization, hypertension, and significant final height impairment. In this study, we aim to investigate the clinical and molecular characteristics of four unrelated Chinese patients with 11βOHD disorder.Methods: The clinical information of four 11βOHD patients were carefully reviewed. Genetic analysis was performed using next-generation sequencing (NGS) based panel analysis. NGS coverage depth was analyzed to detect exonic copy-number variants (CNVs) on patient 1. Quantitative PCR (qPCR) was subsequently performed to confirm the CNVs detected from the NGS coverage depth analysis.Results: The mean age of the patients at diagnosis was 4.7 years (range, 2.0–9.3 years). Two genetically female patients (patients 1 and 2) with 11βOHD presented severe virilization of external genitalia and were raised as males. Two genetically male patients (patients 3 and 4) presented precocious puberty. Additionally, patients 1, 3, and 4 presented with hypertension. In patient 4, unilateral adrenal mass was detected and removed at the age of 9 years. Interestingly, the height of patient 4 (174.4 cm, +6.7 SD) wasn't impaired and reached his mid-parental height (173 cm). Three novel variants in the CYP11B1 gene (c.1150_1153del, c.217C>T, and c.400G>C) were identified by NGS. Various bioinformatics tools revealed potential pathogenic effects for the novel variants, and evolutionary-conservation revealed that the novel missense variant affected an amino acid that is highly conserved among species. Furthermore, NGS coverage depth analysis and qPCR identified a novel heterozygous deletion of exons 1–6 in patient 1.Conclusion: Our study expands the spectrum of mutations of the CYP11B1 gene in Chinese population. In addition, We reported the first case of a patient with classical 11βOHD disorder, whose final height wasn't compromised.

OR15-06 Integrative and Analytical Review of the 5 Alpha Reductase Type 2 Deficiency Worldwide

Introduction: The conversion of testosterone into dyhidrotestosterone is catalyzed by the 5 α reductase type 2 enzyme which plays a crucial role in the external genitalia virilization. It is encoded by the SRD5A2 gene. Allelic variants (AV) in this gene cause a 46,XY DSD with no genotype-phenotype relationship. It was firstly reported at early 70’s from isolated clusters. Since then, several cases have been reported. Putting together, it will expand the knowledge about the molecular bases of androgen regulation. Methods: We searched for SRD5A2 AV in the literature (Pubmed, EMBASE, Medline) and websites (ensemble, HGMD, ClinVar). Only cases with AV in both alleles, either in homozygous (HM) or compound heterozygous state (CH) and 5ARD2 phenotype were included. The AV were analyzed according to ethnicity, exon, domain, aminoacid (aa) conservation, age at diagnosis, sex assignment, gender change, external genitalia virilization and functional studies. External genitalia virilization was scored using Sinnecker scale. Conservation analysis was carried out using CONSURF platform. For categorical variables we used X2 test and Cramer’s V. Continuous variables were analyzed by t test or ANOVA. Concordance was estimated by Kappa. Results: We identified 434 cases of 5ARD2 deficiencies from 40 countries. Most came from Turkey (23%), China (17%), Italy (9%), and Brazil (7%). 69% were assigned as female. There were 70% of AV in HM and 30% in CH state. Most were missense variants (76%). However, small indels (11%), splicing (5%) and large deletions (4%) were all reported. They were distributed along all exons with exon 1 (33%) and exon 4 (25%) predominance. AV in the exon 4 (NADPH-binding domain) resulted in lower virilization (F=10.5; p<.0001). The positions 55, 65, 196, 235 and 246 are hotspots making up, 25% of all AV. Most AV (76%) were located at conserved aa. However, AV at non-conserved aa were more frequently indels (28% vs 6%; p<.01). The overall rate of gender change from female to male ranged from 16% to 70%. The lowest rate of gender change occurred in Turkey and the highest in Brazil. Virilization was similar between those who changed and kept their gender. The gender change rate was significantly different across the countries (V=0.44; p<.001) even with similar virilization scores. There was no concordance between genotype and phenotype in all recurrent AV (196, 235 and 246; k 0.6, 0.12 and 0.19). Conclusion: 5ARD2 deficiency has a worldwide distribution. AV at NADPH-ligand region cause lower virilization. Genitalia virilization influenced sex assignment but not gender change which was influenced by cultural aspects across the countries.

SUN-071 Prenatal and Post-Natal Influence of Androgens in the Psychosexual Development in Individuals with 21-hydroxylase Congenital Adrenal Hyperplasia

Introduction: Congenital Adrenal Hyperplasia (CAH) is defined as a group of autosomal recessive disorders characterized by a deficiency of the enzyme required to synthesize cortisol by the adrenal cortex. Defects in the 21-hydroxylase enzyme make up of 90% of CAH. It is caused by several mutations in the CYP21A2 gene. These defects impaired cortisol synthesis leading to an ACTH increase resulting in androgen excess, either with salt-wasting or simple virilizing forms. Androgens play a crucial role in the human psychosexual development favoring male psychosexuality. This study was designed to evaluate the impact of androgens on psychosexuality of a cohort of individuals with CAH due 21-hydroxylase deficiency. Methods: This retrospective cohort is made up of 94 four individuals with proved molecular defect of the CYP21A2 gene. External genitalia virilization was scored using Prader scale. Clinical phenotype, age at diagnosis, sex assignment, gender reassignment and gender change were all assessed. The gender role at childhood was assessed through the playmates and favorite toys at childhood. Gender identity was assessed by a projective psychological test (HTP). Sexual orientation was assessed by self-report and sexual attractiveness. Compliance of glucocorticoid replacement was assessed by serum testosterone and 17OHP levels. Results: CAH was diagnosed at neonatal time in 67% (61 out 94). 51% (n=48) had the salt-wasting form (SW) and 49% (n=46) had the simple virilizing form (SV). While all cases of SW were diagnosed at neonatal time (0.12 ± 0.14 months), the mean age at diagnosis among SV was 6.03 ± 8.45 years (F 38.9; p=<.001). A total of 84.5% were assigned as female at birth. The median of Prader score was 3 in both phenotypes. Male sex assignment was associated with more virilized external genitalia (Prader 4 and 5) while the opposite (Prader 1 and 2) was associated with female sex assignment (X2 16, p=.002, V=0.44). Gender change from female to male occurred in 7 cases (7.4%). All cases of gender change had SV form (p=.006). The mean age at diagnosis of those who changed gender was 7.92 yr and 2.67 yr for those who kept the assigned gender, p=.04). There was a strong association between male toys at childhood and male gender identity at adulthood (X2 28, p=.0001, V=0.59). A total of 11% defined themselves as homosexual. External genitalia virilization was associated with gynophilic sexual attractiveness (X2 82, p=0.001, V=0.59). Compliance issues were identified in 60% of individuals who changed their gender. Inappropriate hormonal control between 2 and 11 years of age was also associated with gender change (p=.04). Conclusion: Prenatal androgen exposure favors male psychosexuality in CAH. This influence is substantiated by post-natal androgen exposure since gender changed from female to male occurred in individuals with late diagnosis and bad compliance.

SUN-078 Clinical, Hormonal, Psychosexual Aspects, Gonadal Tumors and Genetic Background of an Androgen Insensitivity Syndrome Cohort

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<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.

SUN-204 Case of Non-Classic Congenital Adrenal Hyperplasia with Compound CYP21A2 Mutations Combined with CYP11B1 Mutation

Introduction:Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder, caused by a deficiency in one of the enzymes involved in adrenal steroid synthesis. Homozygotes usually have a severe classical CAH phenotype. Heterozygotes, carrying only one abnormal copy of the gene, are thought to be generally asymptomatic, although could be associated with hyperandrogenism, decreased fertility, adrenal incidentalomas.21-hydroxylase deficiency (21OHD) accounts for 90% of all CAH cases, while 11 β-hydroxylase deficiency (11OHD) accounts for 4–8% of CAH cases.The nature and mechanism of a combined enzymatic defect are unknown. The coincidental presence of gene mutation for both 21OHD and 11OHD CAH in a single individual is very unlikely to occur.Clinical case:A 22-year old female with no significant past medical history presented to endocrinologist for evaluation of facial hirsutism.Patient had menarche at age 11, and menstrual cycle was regular since. No concerns for virilization of external genitalia. She was not sexually active, no pregnancies. No Family history of infertility or genetic conditions. Patient’s father was Jewish, and mother was Slavic.Physical examination revealed female phenotype, normal Blood pressure and BMI, acne on the back and upper arm, Ferriman-Gallwey hirsutism score 5. Labs: AM cortisol, CMP, CBC and TSH were normal. Total testosterone 68 ng/dL (2–45), free testosterone 7 pg/mL (0.1 - 6.4), FSH 5.7 mIU/mL (2.5–10.2), LH 10.6 mIU/mL (1.9–12.5), Progesterone 2.1 ng/mL (<1), Estradiol 51 pg/mL (19–144), 17-OH Progesterone 6728 ng/dL (45–285), Androstenedione 710 ng/dL (35–250), DHEA 1216 ng/dL (102 - 1185), 11-Deoxycortisol 204 ng/dL (<107), Pregnenolone 661 ng/dL(22–237), DHEAS 435 ng/dL (18–391).Elevated 11-Deoxycortisol level raised a suspicion for 11-OHD CAH, or adrenal vs ovarian hormone-producing mass.Abdominal CT and pelvic US were negative for adrenal or ovarian masses.3-day dexamethasone suppression test completely normalized all biochemical abnormalities the patient had.Genetic testing showed: CYP21A2 c.844G>T (non-classic 21OHD CAH mutation), CYP21A2 c.923dupT (classic 21OHD CAH mutation), CYP11B1 c.953C>G mutation.Thus diagnosis of non-classic 21OHD CAH, and carrier status of 11OHD CAH was made. She was started on oral Dexamethasone 0.25 mg every other day.11-Deoxycortisol elevation could not be explained by 21OHD alone. Her carrier state of the CYP11B1 mutation also cannot cause elevated 11-Deoxycortisol level.We hypothesize that 11-Deoxycortisol was elevated either from extra adrenal conversion of 17-hydroprogesterone to 11-Deoxycortisol, or from 11 β-hydroxylase inhibition by excess intra-adrenal androgens. Conclusion: Our case reports a rare finding of both CYP21A2 and CYP11B1 mutations in the same individual, which has implications for relatives, family planning and partner genetic screening.

Mutational analysis of SRD5A2: From gene to functional kinetics in individuals with steroid 5α-reductase 2 deficiency

Human steroid 5α-reductase 2 (SRD5A2) plays a determinative role in the masculinization of external genitalia. To date, approximately 114 different mutations of the SRD5A2 gene have been reported; however, little information is available about their impact on catalytic function or their three-dimensional (3D) structures. We determined the effect of point mutations on the testosterone-depend kinetic constants (Km,app and Vmax,app) and structural characteristics of SRD5A2 from Mexican patients with 46,XY-steroid 5α-reductase 2 deficiency. PCR-SSCP assays identified ten distinct gene variants and sequencing analysis identified missense mutations [p.V3I, p.S14R, p.A52T, p.F118L, p.R145W, p.R171S, p.L226P, p.F229S, p.S245Y, and p.A248V]. Mutations were re-created by site-directed mutagenesis and expressed in HEK293 cells. Functional studies demonstrated that 8 variants led to partial (Km,app = 0.16–2.6 μM; Vmax,app = 224−2640 pmol/mg P/min) or complete losses of activity compared to the wild-type enzyme (Km,app = 0.7 μM; Vmax,app = 4044 pmol/mg P/min). All the mutations were assessed using multiple software tools and the results predicted that all of the mutations were associated with disease or damage. Mapping mutations on the model of a 3D structure of SRD5A2 demonstrated alterations in contact sites with their proximal amino acids. Our data show that mutations affect the catalytic efficiency (Vmax/Km) or result in residual enzymatic activity, which could be due to erroneous interactions between amino acid residues, the substrate testosterone, or NADPH.

Integrative and Analytical Review of the 5-Alpha-Reductase Type 2 Deficiency Worldwide.

IntroductionThe conversion of testosterone into dihydrotestosterone is catalyzed by the 5α-reductase type 2 enzyme which plays a crucial role in the external genitalia virilization. It is encoded by the SRD5A2 gene. Allelic variants in this gene cause a 46,XY DSD with no genotype–phenotype relationship. It was firstly reported in the early 70s from isolated clusters. Since then, several cases have been reported. Putting together, it will expand the knowledge on the molecular bases of androgen milieu.MethodsWe searched for SRD5A2 allelic variants (AV) in the literature (PubMed, Embase, MEDLINE) and websites (ensembl, HGMD, ClinVar). Only cases with AV in both alleles, either in homozygous or compound heterozygous were included. The included cases were analyzed according to ethnicity, exon, domain, aminoacid (aa) conservation, age at diagnosis, sex assignment, gender reassignment, external genitalia virilization and functional studies. External genitalia virilization was scored using Sinnecker scale. Conservation analysis was carried out using the CONSURF platform. For categorical variables, we used X2 test and Cramer’s V. Continuous variables were analyzed by t test or ANOVA. Concordance was estimated by Kappa.ResultsWe identified 434 cases of 5ARD2 deficiencies from 44 countries. Most came from Turkey (23%), China (17%), Italy (9%), and Brazil (7%). Sixty-nine percent were assigned as female. There were 70% of homozygous allelic variants and 30% compound heterozygous. Most were missense variants (76%). However, small indels (11%), splicing (5%) and large deletions (4%) were all reported. They were distributed along with all exons with exon 1 (33%) and exon 4 (25%) predominance. Allelic variants in the exon 4 (NADPH-binding domain) resulted in lower virilization (p<0.0001). The codons 55, 65, 196, 235 and 246 are hotspots making up 25% of all allelic variants. Most of them (76%) were located at conserved aa. However, allelic variants at non-conserved aa were more frequently indels (28% vs 6%; p<0.01). The overall rate of gender change from female to male ranged from 16% to 70%. The lowest rate of gender change from female to male occurred in Turkey and the highest in Brazil. External genitalia virilization was similar between those who changed and those who kept their assigned gender. The gender change rate was significantly different across the countries (V=0.44; p<0.001) even with similar virilization scores.Conclusion5ARD2 deficiency has a worldwide distribution. Allelic variants at the NADPH-ligand region cause lower virilization. Genitalia virilization influenced sex assignment but not gender change which was influenced by cultural aspects across the countries. Molecular diagnosis influenced on sex assignment, favoring male sex assignment in newborns with 5α-reductase type 2 deficiency.

Assisted Reproduction in Congenital Adrenal Hyperplasia.

Congenital Adrenal Hyperplasia (CAH) is a group of autosomal recessive disorders characterized by defects of adrenal steroidogenesis due to mutations in one of the following enzymes: 21-hydroxylase (21OH), 11β-hydroxylase (11βOH), 17α-hydroxylase (17OH; also known as 17, 20-lyase), 3β hydroxysteroid dehydrogenase type 2 (3βHSD2), steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage (P450scc), and P450 oxidoreductase (POR). More than 95% of congenital adrenal hyperplasia cases are due to mutations in CYP21A2, the gene encoding the adrenal steroid 21-hydroxylase enzyme (P450c21). This work focuses on this type of CAH given that it is the most frequent one. This disease is characterized by impaired cortisol and aldosterone production as well as androgen excess. A variant of the CAH is the non-classic type of CAH (NCCAH), usually asymptomatic before the 5th year of age, diagnosed during puberty especially in patients visiting a fertility clinic. NCCAH is characterized mainly by anovulatory cycles and/or high androgen concentrations. Both types of CAH are associated with infertility. Given that the incidence of NCCAH is greater than that of CAH, patients suffering from NCCAH are more often diagnosed for the first time in a fertility clinic. Thus, screening for NCCAH should always be considered. The causes of infertility in CAH patients are multi-factorial including virilization of external genitalia, altered psychosocial development, and hormonal disorders. The main challenges encountered in assisted reproduction are the androgen excess-associated anovulatory cycles as well as the increased circulating progesterone concentrations during the follicular phase which impact endometrial receptivity, tubal motility, and cervical thickness. Administration of sufficient substitution dose of glucocorticoids usually resolves these problems and leads not only to successful assisted reproduction treatment but also to spontaneous pregnancy. Patients with CAH should be followed by a multidisciplinary team including gynecologist, endocrinologist, and pediatrician.

Somatic mosaicism of androgen receptor gene in an androgen insensitivity syndrome patient conceived through assisted reproduction technique.

BackgroundMutations of human androgen receptor (AR) gene are responsible for androgen insensitivity syndrome (AIS). Variable phenotypes and androgen receptor binding activity have permitted the classification of AIS into complete (CAIS), partial (PAIS), and minimal or mild (MAIS) forms. Somatic mosaicism in AIS is a rare condition which happened when de novo mutations occur after the zygotic stage.MethodsClinical evaluation, hormone measurements, and molecular analysis were performed to diagnose the patient in the study.ResultsA 46, XY girl who conceived through in vitro fertilization (IVF), presented with partial virilization of external genitalia, was found to have the p.C620R in heterozygosity. The variant p.C620R of AR has been previously reported in a patient with completely feminized external genitalia, which was inherited from the heterozygote carrier mother. Mutation analysis of the mother of our patient revealed that the variant was de novo and presented as a somatic mosaicism which indicated an insufficient amount of wild‐type AR in our patient.ConclusionThis is the first case that AIS was caused by de novo mutation of AR in a 46, XY Disorder of Sexual Development (DSD) patient by the assisted reproduction technique (ART). The phenotype of partial virilization could be explained by AR mutation in somatic mosaicism.

MALE PSEUDOHERMAPHRODITISM: CASE REPORT

A pseudohermaphrodite represents a human being whose gonads are consistent with the chromosomal sex but who has external genitalia resembling the opposite sex. Male pseudohermaphrodites are characterized by normal testes with insufficient masculinization of the wolffian duct system and external genitalia. Patients with female pseudohermaphroditism have female internal genitalia and karyotype (XX) and various degree of external genitalia virilization. We present the case of a fetus with male pseudohermaphroditism, detected at 21 weeks of pregnancy during the second trimester morphology exam, with no other anomalies present. The peculiarity of this case is represented by the fact that the initial supposition was of clitoral hypertrophy (female pseudohermaphroditism), but after amniocentesis incomplete masculinization causes are being explored (male pseudohermaphroditism).

The impact of CYP21A2 (P30L/I172N) genotype on female fertility in one family

BackgroundThe simple virilizing (SV) form of congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder usually caused by steroid 21-hydroxylase deficiency due to I172N missense mutation at the CYP21A2 gene. Clinical presentation encompasses virilization of external genitalia in newborn females and pseudoprecocious puberty in both sexes, due to reactive androgen overproduction. The aim of this study was to present two sisters with an SV form of CAH and distinctive genotype, detected and treated since childhood with a poor compliance and poor metabolic control hindering the fertility.Case presentationWe retrospectively reviewed the clinical, biochemical, and molecular data of two sisters with CAH a 46,XX karyotype when they reached an age of 35 and 38 years, respectively, and were attempting conception for several years. They had been diagnosed with SV form of CAH at the age of 7 and 9 years, respectively, by the standard clinical and biochemical procedures, presenting with severe virilization due to androgen excess. Follow-up was performed through standard methods of measurement of 17-OHP, testosterone, and ACTH. Clitoroplasty with vaginoplasty was performed at the age of 18 in the older sister. Using PCR/ACRS, we performed molecular analysis of the nine most common point CYP21A2 mutations in the patients and family members. The P30L/II72N genotype was observed in both sisters. They had inadequate metabolic control due to noncompliance until decision to conceive. IVF was performed three times in the older sister without success. Sufficient follicles were harvested and fertilized; however, the embryos were lost 3–5 days after implantations. The younger sister is preparing for IVF. She underwent follicle harvesting and the embryos were frozen awaiting appropriate hormonal balance for embryo transfer. The I172N mutation in the heterozygote state was observed in their other two sisters, whose fertility was unaffected.ConclusionsDespite significant improvements over the last years in achieving fertility in female patients with SV CAH, it is highly dependent upon the severity of virilization and the metabolic control. The role of P30L mutation in infertility and unsuccessfully assisted reproduction remains to be elucidated.

Loss of Function of the Nuclear Receptor NR2F2, Encoding COUP-TF2, Causes Testis Development and Cardiac Defects in 46,XX Children

Emerging evidence from murine studies suggests that mammalian sex determination is the outcome of an imbalance between mutually antagonistic male and female regulatory networks that canalize development down one pathway while actively repressing the other. However, in contrast to testis formation, the gene regulatory pathways governing mammalian ovary development have remained elusive. We performed exome or Sanger sequencing on 79 46,XX SRY-negative individuals with either unexplained virilization or with testicular/ovotesticular disorders/differences of sex development (TDSD/OTDSD). We identified heterozygous frameshift mutations in NR2F2, encoding COUP-TF2, in three children. One carried a c.103_109delGGCGCCC (p.Gly35Argfs∗75) mutation, while two others carried a c.97_103delCCGCCCG (p.Pro33Alafs∗77) mutation. In two of three children the mutation was de novo. All three children presented with congenital heart disease (CHD), one child with congenital diaphragmatic hernia (CDH), and two children with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). The three children had androgen production, virilization of external genitalia, and biochemical or histological evidence of testicular tissue. We demonstrate a highly significant association between the NR2F2 loss-of-function mutations and this syndromic form of DSD (p = 2.44 × 10−8). We show that COUP-TF2 is highly abundant in a FOXL2-negative stromal cell population of the fetal human ovary. In contrast to the mouse, these data establish COUP-TF2 as a human “pro-ovary” and “anti-testis” sex-determining factor in female gonads. Furthermore, the data presented here provide additional evidence of the emerging importance of nuclear receptors in establishing human ovarian identity and indicate that nuclear receptors may have divergent functions in mouse and human biology.

5α-Dihydrotestosterone negatively regulates cell proliferation of the periurethral ventral mesenchyme during urethral tube formation in the murine male genital tubercle.

Androgen is an essential factor involved in masculinization of external genitalia. Failure of the exposure to 5α-dihydrotestosterone (DHT) causes a hypoplastic penile size and urethral abnormality. The main pathology of hypospadias is defective urethral closure on the ventral side of the penis. Hormone-dependent genes are suggested as the causative factors. However, the detailed mechanisms of DHT functions on urethral tube formation remain unknown. Androgen is both a positive and negative regulator of cell proliferation. The roles of locally converted DHT in cell proliferation at the periurethral mesenchyme have not been elucidated. We revealed the expression pattern of 5α-reductase type 2 mRNA (Srd5a2) and local DHT distribution by direct measurement in this study. We also analyzed periurethral mesenchymal cell proliferation status using systematic three-dimensional (3D) reconstruction analyses. A prominent Srd5a2 expression and localized DHT distribution on the ventral side of the genital tubercle were detected. Cell proliferation was reduced in this mesenchymal region during urethral formation. The current results suggest the presence of the possible negative regulation of cell proliferation by DHT. Moreover, cell proliferation related to urethral tube formation was revealed to be DHT dose dependent. These data are expected to contribute to the understanding of the mode of regulation of cell proliferation related to urethral tube formation by DHT. These findings may also offer insight into the understanding of human hypospadias and related hormone-dependent factors.