Lessons Learned from 17 Years of Multidisciplinary Care for DSD Patients at A Single Indonesian Center.

BACKGROUND: Our Multidisciplinary Team (MDT) is a large specialized team based in Semarang, Indonesia that cares for a wide variety of pediatric and adult individuals with Differences of Sex Development (DSD) from across Indonesia. Here we describe our work over the last 17 years. METHODS: We analyzed phenotypic, hormonal and genetic findings from clinical records for all patients referred to our MDT during the period 2004 to 2020. RESULTS: Among 1184 DSD patients, 10% had sex chromosome DSD, 67% had 46,XY DSD and 23% had 46,XX DSD. The most common sex chromosome anomaly was Turner syndrome (45,X) (55 cases). For patients with 46,XY DSD under-masculinization was the most common diagnosis (311 cases) and for 46,XX DSD a defect of Müllerian development was most common (131 cases) followed by Congenital Adrenal Hyperplasia (CAH) (116 cases). Sanger sequencing, MLPA and targeted gene sequencing of 257 patients with 46,XY DSD found likely causative variants in 21% (55 cases), with 13 diagnostic genes implicated. The most affected gene coded for the Androgen Receptor. Molecular analysis identified a diagnosis for 69 of 116 patients with CAH, with 62 carrying variants in CYP21A2 including four novel variants, and seven patients carrying variants in CYP11B1. In many cases these genetic diagnoses influenced the clinical management of patients and families. CONCLUSIONS: Our work has highlighted the occurrence of different DSDs in Indonesia. By applying sequencing technologies as part of our clinical care, we have delivered a number of genetic diagnoses and identified novel pathogenic variants in some genes, which may be clinically specific to Indonesia. Genetics can inform many aspects of DSD clinical management, and whilst many of our patients remain undiagnosed, we hope that future testing may provide answers for even more.

CYP11B1 variants influence skeletal maturation via alternative splicing.

We performed genome-wide association study meta-analysis to identify genetic determinants of skeletal age (SA) deviating in multiple growth disorders. The joint meta-analysis (N = 4557) in two multiethnic cohorts of school-aged children identified one locus, CYP11B1 (expression confined to the adrenal gland), robustly associated with SA (rs6471570-A; ß = 0.14; P = 6.2 × 10-12). rs6410 (a synonymous variant in the first exon of CYP11B1 in high LD with rs6471570), was prioritized for functional follow-up being second most significant and the one closest to the first intron-exon boundary. In 208 adrenal RNA-seq samples from GTEx, C-allele of rs6410 was associated with intron 3 retention (P = 8.11 × 10-40), exon 4 inclusion (P = 4.29 × 10-34), and decreased exon 3 and 5 splicing (P = 7.85 × 10-43), replicated using RT-PCR in 15 adrenal samples. As CYP11B1 encodes 11-ß-hydroxylase, involved in adrenal glucocorticoid and mineralocorticoid biosynthesis, our findings highlight the role of adrenal steroidogenesis in SA in healthy children, suggesting alternative splicing as a likely underlying mechanism.

Levoketoconazole, the 2S,4R Enantiomer of Ketoconazole, a New Steroidogenesis Inhibitor for Cushing's Syndrome Treatment.

INTRODUCTION: Racemic ketoconazole (RK) is a steroidogenesis inhibitor used for treatment of Cushing's syndrome. Levoketoconazole (COR-003), the pure 2S,4R enantiomer, is potentially more potent and safe compared to RK. We compared in vitro effects of levoketoconazole and RK on adrenocortical and pituitary adenoma cells. MATERIALS AND METHODS: HAC15 cells and 15 primary human neoplastic adrenocortical cultures (+/- ACTH), and murine (AtT20) and human corticotroph adenoma cultures were incubated with levoketoconazole or RK (0.01-10 µM). Cortisol and ACTH were measured using a chemiluminescence immunoassay system, and steroid profiles by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: In HAC15, levoketoconazole inhibited cortisol at lower concentrations (IC50: 0.300 µM) compared to RK (0.611 µM; P < 0.0001). IC50 values of levoketoconazole for basal cortisol production in primary adrenocortical cultures varied over a 24-fold range (0.00578-0.140 µM), with 2 patients having a higher sensitivity for levoketoconazole vs RK (2.1- and 3.7-fold). LC-MS/MS analysis in selected cases revealed more potent inhibition of cortisol and other steroid profile components by levoketoconazole vs RK. In AtT20, levoketoconazole inhibited cell growth and ACTH secretion (10 µM: -54% and -38%, respectively), and levoketoconazole inhibited cell number in 1 of 2 primary human corticotroph pituitary adenoma cultures (-44%, P < 0.001). CONCLUSION: Levoketoconazole potently inhibits cortisol production in adrenocortical cells, with a variable degree of suppression between specimens. Levoketoconazole inhibits adrenal steroid production more potently compared to RK and might also inhibit ACTH secretion and growth of pituitary adenoma cells. Together with previously reported potential advantages, this indicates that levoketoconazole is a promising novel pharmacotherapy for Cushing's syndrome.

Osilodrostat Is a Potential Novel Steroidogenesis Inhibitor for the Treatment of Cushing Syndrome: An In Vitro Study.

CONTEXT: Metyrapone and ketoconazole, frequently used steroidogenesis inhibitors for treatment of Cushing syndrome, can be associated with side effects and limited efficacy. Osilodrostat is a CYP11B1 and CYP11B2 inhibitor, with unknown effects on other steroidogenic enzymes. OBJECTIVE: To compare the effects of osilodrostat, metyrapone, and ketoconazole on adrenal steroidogenesis, and pituitary adenoma cells in vitro. METHODS: HAC15 cells, 17 primary human adrenocortical cell cultures, and pituitary adenoma cells were incubated with osilodrostat, metyrapone, or ketoconazole (0.01 to 10 µM). Cortisol and ACTH were measured using chemiluminescence immunoassays, and steroid profiles by liquid chromatography-mass spectrometry. RESULTS: In HAC15 cells, osilodrostat inhibited cortisol production more potently (IC50: 0.035 µM) than metyrapone (0.068 µM; P < 0.0001), and ketoconazole (0.621 µM; P < 0.0001). IC50 values of osilodrostat and metyrapone for basal cortisol production varied with a 25- and 18-fold difference, respectively, with comparable potency. Aldosterone production was inhibited more potently by osilodrostat vs metyrapone and ketoconazole. Osilodrostat and metyrapone treatment resulted in strong inhibition of corticosterone and cortisol, 11-deoxycortisol accumulation, and modest effects on adrenal androgens. No pituitary-directed effects of osilodrostat were observed. CONCLUSIONS: Under our study conditions, osilodrostat is a potent cortisol production inhibitor in human adrenocortical cells, comparable with metyrapone. All steroidogenesis inhibitors showed large variability in sensitivity between primary adrenocortical cultures. Osilodrostat might inhibit CYP11B1 and CYP11B2, in some conditions to a lesser extent CYP17A1 activity, and a proximal step in the steroidogenesis. Osilodrostat is a promising treatment option for Cushing syndrome, and in vivo differences with metyrapone are potentially driven by pharmacokinetic differences.

Genetic Determinants of Circulating Estrogen Levels and Evidence of a Causal Effect of Estradiol on Bone Density in Men.

Context: Serum estradiol (E2) and estrone (E1) levels exhibit substantial heritability. Objective: To investigate the genetic regulation of serum E2 and E1 in men. Design, Setting, and Participants: Genome-wide association study in 11,097 men of European origin from nine epidemiological cohorts. Main Outcome Measures: Genetic determinants of serum E2 and E1 levels. Results: Variants in/near CYP19A1 demonstrated the strongest evidence for association with E2, resolving to three independent signals. Two additional independent signals were found on the X chromosome; FAMily with sequence similarity 9, member B (FAM9B), rs5934505 (P = 3.4 × 10-8) and Xq27.3, rs5951794 (P = 3.1 × 10-10). E1 signals were found in CYP19A1 (rs2899472, P = 5.5 × 10-23), in Tripartite motif containing 4 (TRIM4; rs17277546, P = 5.8 × 10-14), and CYP11B1/B2 (rs10093796, P = 1.2 × 10-8). E2 signals in CYP19A1 and FAM9B were associated with bone mineral density (BMD). Mendelian randomization analysis suggested a causal effect of serum E2 on BMD in men. A 1 pg/mL genetically increased E2 was associated with a 0.048 standard deviation increase in lumbar spine BMD (P = 2.8 × 10-12). In men and women combined, CYP19A1 alleles associated with higher E2 levels were associated with lower degrees of insulin resistance. Conclusions: Our findings confirm that CYP19A1 is an important genetic regulator of E2 and E1 levels and strengthen the causal importance of E2 for bone health in men. We also report two independent loci on the X-chromosome for E2, and one locus each in TRIM4 and CYP11B1/B2, for E1.

INSL3 and AMH in patients with previously congenital or acquired undescended testes.

BACKGROUND: In previous reports no differences in Leydig and Sertoli cell function were found between congenital undescended testis (CUDT) and acquired UDT (AUDT) on the basis of serum levels of LH, testosterone, FSH or inhibin B. This study tried to detect differences in Leydig and Sertoli cell function between CUDT and AUDT using insulin-like peptide 3 (INSL3) and anti-Müllerian hormone (AMH). METHOD: 118 men with a history of UDT (CUDT N=55 (6/55 bilateral), AUDT N=63 (15/63 bilateral)) were investigated. Differences between CUDT and AUDT, influence of age at surgery in CUDT, and effect of spontaneous descent or orchiopexy in AUDT were evaluated. RESULTS: For INSL3, no significant differences were found. AMH levels in bilateral CUDT were significantly lower compared with bilateral AUDT (6.4 (1.7-11.4) vs 13.2 (6.1-30.1) µg/l, p=0.02). AMH levels in unilateral CUDT were significantly higher than in bilateral CUDT (12.1 (2.4-43.7) vs. 6.4 (1.7-11.4) µg/l, p=0.02). CONCLUSION: No differences in Leydig cell function on the basis of INSL3 levels between the different UDT groups were found. Sertoli cell function evaluated by AMH, was more negatively affected in bilateral CUDT in comparison with bilateral AUDT and unilateral CUDT. LEVEL OF EVIDENCE RATING: Level III Treatment Study.

Adrenocorticotropic hormone elicits gonadotropin secretion in premenopausal women.

STUDY QUESTION: Does adrenocorticotropic hormone (ACTH) induce gonadotropin release in premenopausal women? SUMMARY ANSWER: Administration of ACTH stimulates gonadotropin release, most likely by stimulation of the production of cortisol, in premenopausal women. WHAT IS KNOWN ALREADY: In animal models, acute activation of the hypothalamic-pituitary-adrenal (HPA) axis has been shown to induce gonadotropin release in the presence of sufficiently high estrogen levels. However, it is unknown whether the HPA axis has a similar influence on gonadotropin release in humans. STUDY DESIGN, SIZE, DURATION: This study had a mixed factorial design. A total of 60 healthy female participants participated in the experimental study. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study sample comprised three distinct hormonal-based populations according to their levels of progesterone (PROG) and estradiol (E2): (i) low-PROG-low-E2, (ii) low-PROG-high-E2 and (iii) high-PROG-high-E2 women. A low dose (1 µg) of ACTH was administered to all study participants. Serum steroid and gonadotropin concentrations were measured prior to, and at 30 and 90 minutes after, intravenous ACTH administration. MAIN RESULTS AND THE ROLE OF CHANCE: Mean serum cortisol levels increased significantly following ACTH administration in all groups (P < 0.001). Similarly, the serum levels of 17-OH-PROG, androstenedione, dehydroepiandrosterone and testosterone increased significantly in all groups (P < 0.01). The low-PROG-high-E2 and high-PROG-high-E2 groups exhibited a significant increase in LH and FSH levels (P < 0.001), whereas the low-PROG-low-E2 group demonstrated blunted LH and FSH responses to ACTH administration (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: Testing was performed during the luteal phase of the natural menstrual cycle. Testing during the follicular phase might have elicited premature, or more pronounced, LH surges in response to ACTH administration. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest a novel mechanism by which the adrenal cortex functions as a mediator of gonadotropin release. These findings contribute to a greater understanding of the influence of acute stress on reproductive endocrinology. STUDY FUNDING/COMPETING INTERESTS: Funding was received from the Erasmus University Medical Center. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: EudraCT Number 2012-005640-14.

Hormonal evaluation in relation to phenotype and genotype in 286 patients with a disorder of sex development from Indonesia.

OBJECTIVE: The objective of this study was to determine the aetiological spectrum of disorders of sex development (DSD) in a large cohort of underprivileged and undiagnosed patients from Indonesia. METHODS: A total of 286 patients with atypical external and/or internal genitalia were evaluated using clinical, hormonal, molecular genetic and histological parameters. RESULTS: The age (years) at presentation was 0-0·5 in 41 (14·3%), >0·5-12 in 181 (63·3%) and >12 in 64 cases (22·4%). 46,XY DSD was most common (68·2%, n = 195), 46,XX DSD was found in 23·4% (n = 67) and sex chromosomal DSD in 8·4% (n = 24). In 61·2% of 46,XX DSD patients, 17·9% of 46,XY DSD patients and all sex chromosome DSD patients (29·4% in total), a final diagnosis was reached based on genetic or histological gonadal tissue evaluation. 17-hydroxyprogesterone and androstenedione levels were the most distinctive parameters in 46,XX DSD patients. In 46,XY DSD, diagnostic groups were identified based on the external masculinization score: androgen action disorder (AAD), unknown male undermasculinization (UMU), and gonadal dysgenesis (GD). LH, FSH and testosterone levels were most informative especially in the older age group. HCG tests were of no additional value as no patients with androgen synthesis disorders were found. Hormonal profiles of patients with sex chromosome DSD and a Y-chromosome sequence containing karyotype showed high levels of LH and FSH, and low levels of AMH, inhibin B and testosterone compared with the normal male range. Gene mutations were found in all patients with CAH, but in only 24·5% and 1·8% of patients with AAD and UMU. In 32% of 46,XY GD patients, copy number variants of different genes were found. CONCLUSION: A stepwise diagnostic approach led to a molecularly or histologically proven final diagnosis in 29·4% of the patients. The most informative parameters were serum levels of 17-hydroxyprogesterone and androstenedione in 46,XX DSD patients, and serum LH, FSH and testosterone levels in 46,XY DSD patients.

Use of statins is associated with lower serum total and non-sex hormone-binding globulin-bound testosterone levels in male participants of the Rotterdam Study.

OBJECTIVE: Statins, or HMG-CoA reductase inhibitors, decrease cholesterol production. Because cholesterol is a precursor of the testosterone biosynthesis pathway, there is some concern that statins might lower serum testosterone levels. The objective of the present study was to investigate the association between the use of statins and serum testosterone levels in men. DESIGN: Cross-sectional study within the prospective population-based Rotterdam Study. SUBJECTS AND METHODS: We included 4166 men with available data on total testosterone, non-sex hormone-binding globulin (SHBG)-bound testosterone, and medication use. Multivariable linear regression analysis was used to compare the differences in serum testosterone levels (nmol/l) between current, past, and never statin users. We considered dose and duration of use. Analyses were adjusted for age, BMI, cardiovascular disease, diabetes mellitus, hypertension, and estradiol levels. RESULTS: We identified 577 current (mean age 64.1 years), 148 past (mean age 64.6 years), and 3441 never (mean age 64.6 years) statin users. Adjusted for all covariables, current statin use of 1-≤ 6 months or >6 months was significantly associated with lower total testosterone levels as compared to non-users (ß -1.24, 95% CI -2.17, -0.31, and ß -1.14, 95% CI -2.07, -0.20 respectively). Current use of 1-≤ 6 months was also associated with significantly lower non-SHBG-bound testosterone levels (ß -0.42, 95% CI -0.82, -0.02). There was a trend toward lower testosterone levels at higher statin doses both for total (P(trend) 2.9 × 10(-5)) and non-SHBG-bound (P(trend) 2.0 × 10(-4)) testosterone. No association between past statin use and testosterone levels was found. CONCLUSION: We showed that current use of statins was associated with significantly lower serum total and non-SHBG-bound testosterone levels. The clinical relevance of this association should be further investigated.