Mitochondrial dysfunction results in enhanced adrenal androgen production in H295R cells.

The role of mitochondria in steroidogenesis is well established. However, the specific effects of mitochondrial dysfunction on androgen synthesis are not fully understood. In this study, we investigate the effects of various mitochondrial and metabolic inhibitors in H295R adrenal cells and perform a comprehensive analysis of steroid and metabolite profiling. We report that mitochondrial complex I inhibition by rotenone shifts cells toward anaerobic metabolism with a concomitant hyperandrogenic phenotype characterized by rapid stimulation of dehydroepiandrosterone (DHEA, 2 h) and slower accumulation of androstenedione and testosterone (24 h). Screening of metabolic inhibitors confirmed DHEA stimulation, which included mitochondrial complex III and mitochondrial pyruvate carrier inhibition. Metabolomic studies revealed truncated tricarboxylic acid cycle with an inverse correlation between citric acid and DHEA production as a common metabolic marker of hyperandrogenic inhibitors. The current study sheds light on a direct interplay between energy metabolism and androgen biosynthesis that could be further explored to identify novel molecular targets for efficient treatment of androgen excess disorders.

Clinical and genetic characteristics of a large international cohort of individuals with rare NR5A1/SF-1 variants of sex development.

BACKGROUND: Steroidogenic factor 1 (SF-1/NR5A1) is essential for human sex development. Heterozygous NR5A1/SF-1 variants manifest with a broad range of phenotypes of differences of sex development (DSD), which remain unexplained. METHODS: We conducted a retrospective analysis on the so far largest international cohort of individuals with NR5A1/SF-1 variants, identified through the I-DSD registry and a research network. FINDINGS: Among 197 individuals with NR5A1/SF-1 variants, we confirmed diverse phenotypes. Over 70% of 46, XY individuals had a severe DSD phenotype, while 90% of 46, XX individuals had female-typical sex development. Close to 100 different novel and known NR5A1/SF-1 variants were identified, without specific hot spots. Additionally, likely disease-associated variants in other genes were reported in 32 individuals out of 128 tested (25%), particularly in those with severe or opposite sex DSD phenotypes. Interestingly, 48% of these variants were found in known DSD or SF-1 interacting genes, but no frequent gene-clusters were identified. Sex registration at birth varied, with <10% undergoing reassignment. Gonadectomy was performed in 30% and genital surgery in 58%. Associated organ anomalies were observed in 27% of individuals with a DSD, mainly concerning the spleen. Intrafamilial phenotypes also varied considerably. INTERPRETATION: The observed phenotypic variability in individuals and families with NR5A1/SF-1 variants is large and remains unpredictable. It may often not be solely explained by the monogenic pathogenicity of the NR5A1/SF-1 variants but is likely influenced by additional genetic variants and as-yet-unknown factors. FUNDING: Swiss National Science Foundation (320030-197725) and Boveri Foundation Zürich, Switzerland.

The serum steroid signature of PCOS hints at the involvement of novel pathways for excess androgen biosynthesis.

CONTEXT: Polycystic ovary syndrome (PCOS) is defined by androgen excess and ovarian dysfunction in the absence of a specific physiological diagnosis. The best clinical marker of androgen excess is hirsutism, while the best biochemical parameter is still a matter of debate. Current consensus guidelines recommend, among other hormones, serum free testosterone as an important serum parameter to measure androgen excess. Recently, however, novel active androgens and androgen metabolic pathways have been discovered. OBJECTIVE: To assess the contribution of novel androgens and related steroid biosynthetic pathways to the serum steroid pool in PCOS women in comparison to healthy controls. DESIGN: This is a case control study, wherein PCOS was diagnosed according to the AE-PCOS 2009 criteria. Serum steroid profiling was performed by liquid chromatography high-resolution mass spectrometry. SETTING: Yeditepe University and associated clinics in Istanbul, Turkey, together with Bern University Hospital Inselspital, Bern, Switzerland. PARTICIPANTS: 42 PCOS women and 42 matched, healthy control women. MAIN OUTCOME MEASURES: Assessment of 34 steroids compartmentalized in four androgen related pathways: the classic androgen pathway, the backdoor pathway, the C11-oxy backdoor pathway, and the C11-oxy (11ß-hydroxyandrostenedione) pathway. RESULTS: Metabolites of all four pathways were identified in healthy and PCOS women. Highest concentrations were found for progesterone in controls and androstenedione in PCOS. Lowest levels were found for 11-ketotestosterone in controls compared to PCOS, and for 20α-hydroxyprogesterone in PCOS compared to controls. PCOS also had higher serum testosterone levels compared to the controls. PCOS women had overall higher levels of steroid metabolites of all four androgen pathways compared to healthy controls. CONCLUSIONS: Novel alternative pathways contribute to the androgen production in healthy and PCOS women. Hyperandrogenism in PCOS is characterized by an overall increase of serum androgens in the classic, backdoor and C11-oxy pathways. While monogenetic disorders of steroid biosynthesis can be recognized by a specific pattern in the steroid profile, no diagnostic pattern or classifier was found in the serum for PCOS.

Effects of 2-Year Physical Activity and Dietary Intervention on Adrenarchal and Pubertal Development: The PANIC Study.

CONTEXT: Childhood overweight has been linked to earlier development of adrenarche and puberty, but it remains unknown if lifestyle interventions influence sexual maturation in general populations. OBJECTIVE: To investigate if a 2-year lifestyle intervention influences circulating androgen concentrations and sexual maturation in a general population of children. METHODS: We conducted a 2-year physical activity and dietary intervention study in which 421 prepubertal and mostly normal-weight 6- to 9-year-old children were allocated either to a lifestyle intervention group (119 girls, 132 boys) or a control group (84 girls, 86 boys). The main outcome measures were serum dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androstenedione (A4), and testosterone concentrations, and clinical adrenarchal and pubertal signs. RESULTS: The intervention and control groups had no differences in body size and composition, clinical signs of androgen action, and serum androgens at baseline. The intervention attenuated the increase of DHEA (P = .032), DHEAS (P = .001), A4 (P = .003), and testosterone (P = .007) and delayed pubarche (P = .038) in boys but it only attenuated the increase of DHEA (P = .013) and DHEAS (P = .003) in girls. These effects of lifestyle intervention on androgens and the development of pubarche were independent of changes in body size and composition, but the effects of intervention on androgens were partly explained by changes in fasting serum insulin. CONCLUSION: A combined physical activity and dietary intervention attenuates the increase of serum androgen concentrations and sexual maturation in a general population of prepubertal and mostly normal-weight children, independently of changes in body size and composition.

Natural History of Obesity Due to POMC, PCSK1, and LEPR Deficiency and the Impact of Setmelanotide.

Context: Rare homozygous or biallelic variants in POMC, PCSK1, and LEPR can disrupt signaling through the melanocortin-4 receptor (MC4R) pathway, resulting in hyperphagia and severe early-onset obesity. In pivotal Phase 3 clinical trials, treatment with the MC4R agonist setmelanotide reduced hunger and weight in patients with obesity due to proopiomelanocortin (POMC), proprotein convertase subtilisin/kexin type 1 (PCSK1), or leptin receptor (LEPR) deficiency. Objective: To characterize the historical weight trajectory in these patients. Methods: This analysis included data from 2 pivotal single-arm, open-label, Phase 3 trials (NCT02896192, NCT03287960). These were multicenter trials. Patients had obesity due to POMC/PCSK1 or LEPR deficiency. During the trial, patients were treated with setmelanotide. Historical data on measured weight and height were obtained during screening. Results: A total of 17 patients (POMC, n = 8; PCSK1, n = 1; LEPR, n = 8) with historical weight and height data were included in this analysis. Before setmelanotide treatment, patients with obesity due to POMC/PCSK1 or LEPR deficiency were above the 95th percentile for weight throughout childhood, demonstrated continuous weight gain, and did not show long-term weight loss upon interventions (eg, diet, surgery, exercise). Setmelanotide treatment attenuated weight and body mass index trajectories over the observation period of 1 year. Conclusion: In patients with POMC, PCSK1, or LEPR deficiency, traditional interventions for weight loss had limited impact on the trajectory of severe early-onset obesity. However, setmelanotide treatment attenuated weight and body mass index trajectories and led to weight loss associated with health benefits in most individuals.

Short-Term Effects of Elexacaftor/Tezacaftor/Ivacaftor Combination on Glucose Tolerance in Young People With Cystic Fibrosis-An Observational Pilot Study.

Background: The effect of elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) on glucose tolerance and/or cystic-fibrosis-related diabetes (CFRD) is not well understood. We performed an observational study on the short-term effects of ELX/TEZ/IVA on glucose tolerance. Methods: Sixteen adolescents with CF performed oral glucose tolerance tests (OGTT) before and 4-6 weeks after initiating ELX/TEZ/IVA therapy. A continuous glucose monitoring (CGM) system was used 3 days before until 7 days after starting ELX/TEZ/IVA treatment. Results: OGTT categories improved after initiating ELX/TEZ/IVA therapy (p = 0.02). Glucose levels of OGTT improved at 60, 90, and 120 min (p < 0.05), whereas fasting glucose and CGM measures did not change. Conclusion: Shortly after initiating ELX/TEZ/IVA therapy, glucose tolerance measured by OGTT improved in people with CF. This pilot study indicates that ELX/TEZ/IVA treatment has beneficial effects on the endocrine pancreatic function and might prevent or at least postpone future CFRD.

Basics of androgen synthesis and action.

Androgens are essential sex steroid hormones for both sexes. Testosterone (T) is the predominant androgen in males, while in adult females, T concentrations are about 15-fold lower and androgen precursors are converted to estrogens. T is produced primarily in testicular Leydig cells in men, while in women precursors are biosynthesised in the adrenal cortex and ovaries and converted into T in the periphery. The biosynthesis of T occurs via a series of enzymatic reactions in steroidogenic organs. Notably, the more potent androgen, dihydrotestosterone, may be synthesized from T in the classic pathway, however, alternate metabolic pathways also exist. The classic action of androgens on target organs is mediated through the androgen receptor, which regulates nuclear receptor gene transcription. However, the androgen-androgen receptor complex may also interact directly with membrane proteins or signaling molecules to exert more rapid effects. This review summarizes the current knowledge of androgen biosynthesis, mechanisms of action and endocrine effects in human biology, and relates these effects to respective human congenital and acquired disorders.

Surgical Practice in Girls with Congenital Adrenal Hyperplasia: An International Registry Study.

In this article international trends in surgical practice in girls with congenital adrenal hyperplasia (CAH) are evaluated. All cases that had been classified in the I-CAH/I-DSD registry as 46,XX CAH and who were born prior to 2017 were identified. Centers were approached to obtain information on surgical decision making. Of the 330 included participants, 208 (63.0%) presented within the first month of life, and 326 (98.8%) cases were assigned female. Genital surgery had been performed in 250 (75.8%). A total of 64.3, 89.2, and 96.8% of cases residing in Europe, South America and Asia, respectively, had at least one surgery. In a logistic regression model for the probability of surgery before the second birthday (early surgery) over time an increase of probability for early vaginal surgery could be identified, but not for clitoral surgery or both surgeries combined. Genitoplasty in girls with CAH remains controversial. This large international study provides a snapshot of current practice and reveals geographical and temporal differences. Fewer surgeries were reported for Europe, and there seems to be a significant trend towards aiming for vaginal surgery within the first 2 years of life.

Health behaviour of women with Turner Syndrome.

AIM: This study assessed lifestyle-related risk factors for cardiovascular disease in young women with Turner syndrome. METHODS: In 2012, we sent a questionnaire to women with Turner syndrome aged ≥18 years and living in Switzerland with questions on socio-demographic and medical data as well as health behaviour. We compared the reported lifestyle with that of women from the Swiss Health Survey 2012, a representative survey of the general population. RESULTS: Fifty-seven per cent (45/79) of women with Turner syndrome answered the questionnaire (mean age: 24 years). Eighty per cent (36/45) had never smoked compared with 58% (1156/1972) of the general population (p < 0.01). Women with Turner syndrome engaged less often in binge drinking (34% vs. 71%) (p < 0.001), but consumed alcohol equally often as the general population (p = 0.327). They performed sports as often as the general population (p = 0.34), but only one quarter (11/45) of women with Turner syndrome adhered to official physical activity recommendations. CONCLUSION: Although most women with Turner syndrome had a healthy lifestyle, only a minority had sufficient physical activity. Paediatricians should promote structured physical activity in girls with Turner syndrome from early childhood onwards to reduce their cardiovascular risk in adulthood and to increase long-term health-related quality of life.

Approach to the Virilizing Girl at Puberty.

Virilization is the medical term for describing a female who develops characteristics associated with male hormones (androgens) at any age, or when a newborn girl shows signs of prenatal male hormone exposure at birth. In girls, androgen levels are low during pregnancy and childhood. A first physiologic rise of adrenal androgens is observed at the age of 6 to 8 years and reflects functional activation of the zona reticularis of the adrenal cortex at adrenarche, manifesting clinically with first pubic and axillary hairs. Early adrenarche is known as "premature adrenarche." It is mostly idiopathic and of uncertain pathologic relevance but requires the exclusion of other causes of androgen excess (eg, nonclassic congenital adrenal hyperplasia) that might exacerbate clinically into virilization. The second modest physiologic increase of circulating androgens occurs then during pubertal development, which reflects the activation of ovarian steroidogenesis contributing to the peripheral androgen pool. However, at puberty initiation (and beyond), ovarian steroidogenesis is normally devoted to estrogen production for the development of secondary female bodily characteristics (eg, breast development). Serum total testosterone in a young adult woman is therefore about 10- to 20-fold lower than in a young man, whereas midcycle estradiol is about 10- to 20-fold higher. But if androgen production starts too early, progresses rapidly, and in marked excess (usually more than 3 to 5 times above normal), females will manifest with signs of virilization such as masculine habitus, deepening of the voice, severe acne, excessive facial and (male typical) body hair, clitoromegaly, and increased muscle development. Several medical conditions may cause virilization in girls and women, including androgen-producing tumors of the ovaries or adrenal cortex, (non)classical congenital adrenal hyperplasia and, more rarely, other disorders (also referred to as differences) of sex development (DSD). The purpose of this article is to describe the clinical approach to the girl with virilization at puberty, focusing on diagnostic challenges. The review is written from the perspective of the case of an 11.5-year-old girl who was referred to our clinic for progressive, rapid onset clitoromegaly, and was then diagnosed with a complex genetic form of DSD that led to abnormal testosterone production from a dysgenetic gonad at onset of puberty. Her genetic workup revealed a unique translocation of an abnormal duplicated Y-chromosome to a deleted chromosome 9, including the Doublesex and Mab-3 Related Transcription factor 1 (DMRT1) gene. LEARNING OBJECTIVES: Identify the precise pathophysiologic mechanisms leading to virilization in girls at puberty considering that virilization at puberty may be the first manifestation of an endocrine active tumor or a disorder/difference of sex development (DSD) that remained undiagnosed before and may be life-threatening. Of the DSDs, nonclassical congenital adrenal hyperplasia occurs most often.Provide a step-by-step diagnostic workup plan including repeated and expanded biochemical and genetic tests to solve complex cases.Manage clinical care of a girl virilizing at puberty using an interdisciplinary team approach.Care for complex cases of DSD manifesting at puberty, such as the presented girl with a Turner syndrome-like phenotype and virilization resulting from a complex genetic variation.

Premature Adrenarche in Girls Characterized By Enhanced 17,20-Lyase and 17ß-Hydroxysteroid Dehydrogenase Activities.

CONTEXT: Girls with premature adrenarche (PA) may have a higher risk of developing polycystic ovary syndrome (PCOS) and metabolic syndrome. The biological purpose of adrenarche is unknown and the role of novel biosynthetic pathways remains unclear. OBJECTIVE: To compare the urinary steroid metabolome and enzyme activities of girls with PA to age-matched control girls and to published steroid values of girls with normal adrenarche and of women with PCOS and their newborn daughters. DESIGN: Prospective observational study from 2009 to 2014. SETTING: Academic pediatric endocrinology referral center. PARTICIPANTS: Twenty-three girls with PA and 22 healthy, age-matched girls. MAIN OUTCOME MEASURES: Steroid metabolites in 24-hour urine samples, including 4 progesterones, 5 corticosterones, aldosterone, 13 androgens, 2 estrogens, 14 glucocorticoids, and enzyme activities represented by metabolite ratios. RESULTS: Girls with PA had a higher body mass index (mean standard deviation scores 0.9 vs -0.3, P = 0.013). Androgen excretion was higher in PA girls than in control girls (median 3257 nmol/24 hours vs 1627 nmol/24 hours, P < 0.001), in particular metabolites from alternate androgen pathways. The amount of progesterone, corticosterone, aldosterone, estrogen, and cortisol metabolites were similar between groups. Activities of 17ß-hydroxysteroid-dehydrogenase and of 17,20-lyase were higher in girls with PA. Activities of 3ß-hydroxysteroid-dehydrogenase, 21-hydroxylase, and 5α-reductase activity were not different between groups, in contrast to published results on girls with normal adrenarche or PCOS females. CONCLUSIONS: Metabolites and enzymes involved in alternate androgen pathways appear to be markers of PA. Prospective studies should assess whether steroid production in PA also differs from adrenarche at normal timing and persists into adulthood.

Novel CYP19A1 Mutations Extend the Genotype-Phenotype Correlation and Reveal the Impact on Ovarian Function.

CONTEXT: The steroidogenic enzyme aromatase (CYP19A1) is required for estrogen biosynthesis from androgen precursors in the ovary and extragonadal tissues. The role of aromatase, and thus estrogens, is best illustrated by genetic variations of the CYP19A1 gene leading to aromatase deficiency or excess. OBJECTIVE: The objective of this work is to characterize novel CYP19A1 variants. DESIGN SETTING AND PATIENTS: Variants causing aromatase deficiency were suspected in four 46,XX children of African and Indian origin by careful clinical phenotyping. Sequencing of the CYP19A1 gene identified novel variants. Minigene experiments, aromatase activity assay, and computational, and histological analysis were used to characterize the variants. MAIN OUTCOME MEASURE AND RESULTS: CYP19A1 variants were found in all patients: a deletion in intron 9 leading to p.P423_H503del, a delins variant at p.P154, and point variants p.V161D, p.R264C, p.R375C. Except for R264C, all variants showed a loss of function. Protein structure and dynamics studies were in line with functional assays. The 2 female patients with delins variants manifested with ambiguous genitalia at birth. Histologic investigation revealed normal ovarian tissue on one side and a streak gonad on the other. Two female patients presented with abnormal pubertal development and polycystic ovaries. CONCLUSION: In girls, aromatase deficiency usually manifests at birth, but diagnosis may also be made because of abnormal pubertal development or ovarian torsion due to (poly)cystic ovaries. The ovary harboring CYP19A1 variants may present as streak gonad or appears normal at birth, but is then at very high risk to produce cysts with aging and is therefore prone to ovarian torsion.

Clinical but Not Histological Outcomes in Males With 45,X/46,XY Mosaicism Vary Depending on Reason for Diagnosis.

CONTEXT: Larger studies on outcomes in males with 45,X/46,XY mosaicism are rare. OBJECTIVE: To compare health outcomes in males with 45,X/46,XY diagnosed as a result of either genital abnormalities at birth or nongenital reasons later in life. DESIGN: A retrospective, multicenter study. SETTING: Sixteen tertiary centers. PATIENTS OR OTHER PARTICIPANTS: Sixty-three males older than 13 years with 45,X/46,XY mosaicism. MAIN OUTCOME MEASURES: Health outcomes, such as genital phenotype, gonadal function, growth, comorbidities, fertility, and gonadal histology, including risk of neoplasia. RESULTS: Thirty-five patients were in the genital group and 28 in the nongenital. Eighty percent of all patients experienced spontaneous pubertal onset, significantly more in the nongenital group (P = 0.023). Patients were significantly shorter in the genital group with median adult heights of 156.7 cm and 164.5 cm, respectively (P = 0.016). Twenty-seven percent of patients received recombinant human GH. Forty-four patients had gonadal histology evaluated. Germ cells were detected in 42%. Neoplasia in situ was found in five patients. Twenty-five percent had focal spermatogenesis, and another 25.0% had arrested spermatogenesis. Fourteen out of 17 (82%) with semen analyses were azoospermic; three had motile sperm. CONCLUSION: Patients diagnosed as a result of genital abnormalities have poorer health outcomes than those diagnosed as a result of nongenital reasons. Most patients, however, have relatively good endocrine gonadal function, but most are also short statured. Patients have a risk of gonadal neoplasia, and most are azoospermic, but almost one-half of patients has germ cells present histologically and up to one-quarter has focal spermatogenesis, providing hope for fertility treatment options.

In silico and functional studies reveal novel loss-of-function variants of SRD5A2, but no variants explaining excess 5α-reductase activity.

Androgens are steroid hormones essential for human male and female development. Steroid reductases 5α (SRD5As) are key enzymes in androgen biosynthesis. Mutations in the human SRD5A2 are known to cause loss-of-function and severe 46,XY undervirilization. Gain-of-function variants have been suggested in androgen excess syndromes, but have not been found so far. Therefore we searched for gain-of-function mutations in the human SRD5A2 gene which might explain hyperandrogenic disorders such as the polycystic ovary syndrome, premature adrenarche and prostate cancer. We screened databases for candidate variants and characterised them in silico with the help of a novel SRD5A2 model. We selected 9 coding SNPs (A49T, R50A, P106L, P106A, N122A, L167S, R168C, P173S, R227Q) that have not been described in manifesting individuals, and assessed their enzyme kinetic properties in HEK293 cells. SRD5A2 activity was assessed by conversion of testosterone (T), progesterone (Prog) and androstenedione (Δ4A) to their 5α-reduced metabolites. Variants R50A and P173S showed partial activity with substrates T (34% and 28%) and Δ4A (37% and 22%). With substrate Prog variants P106L, P106A, L167S and R168C in addition showed partial activity (15% to 64%). Functional testing of all other variants showed loss-of-function. As predicted in our in silico analysis, all coding SNPs affected enzyme activity, however none of them showed gain-of-function. Thus excess 5α-reductase activity might be rather regulated at the (post)-transcriptional and/or post-translational level. However through this work seven new coding SNPs were characterised which might be of clinical relevance. It is possible that individuals carrying these SNPs show a minor phenotype that is not yet identified.

How high-resolution techniques enable reliable steroid identification and quantification.

Due to possible matrix interferences and artefact generation during sample preparation, careful method validation is required for quantitative bioanalytical methods, especially for analytes that are only present in low concentrations. Using the identification and quantification of progesterone metabolites in the urine of newborns as an example, we show how modern high-resolution instruments can be used to verify analyte assignment and avoid pitfalls commonly encountered by the use of low-resolution instruments.

Risk of Meningioma in European Patients Treated With Growth Hormone in Childhood: Results From the SAGhE Cohort.

Context: There has been concern that GH treatment of children might increase meningioma risk. Results of published studies have been inconsistent and limited. Objective: To examine meningioma risks in relation to GH treatment. Design: Cohort study with follow-up via cancer registries and other registers. Setting: Population-based. Patients: A cohort of 10,403 patients treated in childhood with recombinant GH in five European countries since this treatment was first used in 1984. Expected rates from national cancer registration statistics. Main Outcome Measures: Risk of meningioma incidence. Results: During follow-up, 38 meningiomas occurred. Meningioma risk was greatly raised in the cohort overall [standardized incidence ratio (SIR) = 75.4; 95% CI: 54.9 to 103.6], as a consequence of high risk in subjects who had received radiotherapy for underlying malignancy (SIR = 658.4; 95% CI: 460.4 to 941.7). Risk was not significantly raised in patients who did not receive radiotherapy. Risk in radiotherapy-treated patients was not significantly related to mean daily dose of GH, duration of GH treatment, or cumulative dose of GH. Conclusions: Our data add to evidence of very high risk of meningioma in patients treated in childhood with GH after cranial radiotherapy, but suggest that GH may not affect radiotherapy-related risk, and that there is no material raised risk of meningioma in GH-treated patients who did not receive radiotherapy.

Urinary steroid profiling in women hints at a diagnostic signature of the polycystic ovary syndrome: A pilot study considering neglected steroid metabolites.

BACKGROUND: Although the polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women with vast metabolic consequences, its etiology remains unknown and its diagnosis is still made by exclusion. This study aimed at characterizing a large number of urinary steroid hormone metabolites and enzyme activities in women with and without PCOS in order to test their value for diagnosing PCOS. METHODS: Comparative steroid profiling of 24h urine collections using an established in-house gas-chromatography mass spectrometry method. Data were collected mostly prospectively. Patients were recruited in university hospitals in Switzerland. Participants were 41 women diagnosed with PCOS according to the current criteria of the Androgen Excess and PCOS Society Task Force and 66 healthy controls. Steroid profiles of women with PCOS were compared to healthy controls for absolute metabolite excretion and for substrate to product conversion ratios. The AUC for over 1.5 million combinations of metabolites was calculated in order to maximize the diagnostic accuracy in patients with PCOS. Sensitivity, specificity, PPV, and NPV were indicated for the best combinations containing 2, 3 or 4 steroid metabolites. RESULTS: The best single discriminating steroid was androstanediol. The best combination to diagnose PCOS contained four of the forty measured metabolites, namely androstanediol, estriol, cortisol and 20ßDHcortisone with AUC 0.961 (95% CI 0.926 to 0.995), sensitivity 90.2% (95% CI 76.9 to 97.3), specificity 90.8% (95% CI 81.0 to 96.5), PPV 86.0% (95% CI 72.1 to 94.7), and NPV 93.7% (95% CI 84.5 to 98.2). CONCLUSION: PCOS shows a specific 24h urinary steroid profile, if neglected metabolites are included in the analysis and non-conventional data analysis applied. PCOS does not share a profile with hyperandrogenic forms of congenital adrenal hyperplasias due to single steroid enzyme deficiencies. Thus PCOS diagnosis by exclusion may no longer be warranted. Whether these findings also apply to spot urine and serum, remains to be tested as a next step towards routine clinical applicability.

Management of Gonads in Adults with Androgen Insensitivity: An International Survey.

BACKGROUND: Complete and partial androgen insensitivity syndrome (CAIS, PAIS) are associated with an increased risk of gonadal germ cell cancer (GGCC). Recent guidelines recommend gonadectomy in women with CAIS in late adolescence. Nevertheless, many adult women prefer to retain their gonads. AIMS: This study aims to explore attitudes towards gonadectomy in AIS in centres around the world, estimate the proportion of adults with retained gonads and/or who developed GGCC, and explore reasons for declining gonadectomy. METHODS: A survey was performed among health care professionals who use the International DSD Registry (I-DSD). RESULTS: Data were provided from 22 centres in 16 countries on 166 women (CAIS) and 26 men (PAIS). In CAIS, gonadectomy was recommended in early adulthood in 67% of centres; 19/166 (11.4%) women refused gonadectomy. Among 142 women who had gonadectomy, evidence of germ cell neoplasm in situ (GCNIS), the precursor of GGCC, was reported in 2 (1.4%) out of 8 from whom pathology results were formally provided. Nine out of 26 men with PAIS (34.6%) had retained gonads; 11% of centres recommended routine gonadectomy in PAIS. CONCLUSION: Although development of GGCC seems rare, gonadectomy after puberty is broadly recommended in CAIS; in PAIS this is more variable. Overall, our data reflect the need for evidence-based guidelines regarding prophylactic gonadectomy in AIS.

Mechanism of the Dual Activities of Human CYP17A1 and Binding to Anti-Prostate Cancer Drug Abiraterone Revealed by a Novel V366M Mutation Causing 17,20 Lyase Deficiency.

The CYP17A1 gene regulates sex steroid biosynthesis in humans through 17α-hydroxylase/17,20 lyase activities and is a target of anti-prostate cancer drug abiraterone. In a 46, XY patient with female external genitalia, together with a loss of function mutation S441P, we identified a novel missense mutation V366M at the catalytic center of CYP17A1 which preferentially impaired 17,20 lyase activity. Kinetic experiments with bacterially expressed proteins revealed that V366M mutant enzyme can bind and metabolize pregnenolone to 17OH-pregnenolone, but 17OH-pregnenolone binding and conversion to dehydroepiandrosterone (DHEA) was impaired, explaining the patient’s steroid profile. Abiraterone could not bind and inhibit the 17α-hydroxylase activity of the CYP17A1-V366M mutant. Molecular dynamics (MD) simulations showed that V366M creates a “one-way valve” and suggests a mechanism for dual activities of human CYP17A1 where, after the conversion of pregnenolone to 17OH-pregnenolone, the product exits the active site and re-enters for conversion to dehydroepiandrosterone. The V366M mutant also explained the effectiveness of the anti-prostate cancer drug abiraterone as a potent inhibitor of CYP17A1 by binding tightly at the active site in the WT enzyme. The V366M is the first human mutation to be described at the active site of CYP17A1 that causes isolated 17,20 lyase deficiency. Knowledge about the specificity of CYP17A1 activities is of importance for the development of treatments for polycystic ovary syndrome and inhibitors for prostate cancer therapy.

Combined transcriptome and metabolome analyses of metformin effects reveal novel links between metabolic networks in steroidogenic systems.

Metformin is an antidiabetic drug, which inhibits mitochondrial respiratory-chain-complex I and thereby seems to affect the cellular metabolism in many ways. It is also used for the treatment of the polycystic ovary syndrome (PCOS), the most common endocrine disorder in women. In addition, metformin possesses antineoplastic properties. Although metformin promotes insulin-sensitivity and ameliorates reproductive abnormalities in PCOS, its exact mechanisms of action remain elusive. Therefore, we studied the transcriptome and the metabolome of metformin in human adrenal H295R cells. Microarray analysis revealed changes in 693 genes after metformin treatment. Using high resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS-NMR), we determined 38 intracellular metabolites. With bioinformatic tools we created an integrated pathway analysis to understand different intracellular processes targeted by metformin. Combined metabolomics and transcriptomics data analysis showed that metformin affects a broad range of cellular processes centered on the mitochondrium. Data confirmed several known effects of metformin on glucose and androgen metabolism, which had been identified in clinical and basic studies previously. But more importantly, novel links between the energy metabolism, sex steroid biosynthesis, the cell cycle and the immune system were identified. These omics studies shed light on a complex interplay between metabolic pathways in steroidogenic systems.