Service evaluation suggests variation in clinical care provision in adults with congenital adrenal hyperplasia in the UK and Ireland.

BACKGROUND: Congenital adrenal hyperplasia (CAH) encompasses a rare group of autosomal recessive disorders, characterised by enzymatic defects in steroidogenesis. Heterogeneity in management practices has been observed internationally. The International Congenital Adrenal Hyperplasia registry (I-CAH, https://sdmregistries.org/) was established to enable insights into CAH management and outcomes, yet its global adoption by endocrine centres remains unclear. DESIGN: We sought (1) to assess current practices amongst clinicians managing patients with CAH in the United Kingdom and Ireland, with a focus on choice of glucocorticoid, monitoring practices and screening for associated co-morbidities, and (2) to assess use of the I-CAH registry. MEASUREMENTS: We designed and distributed an anonymised online survey disseminated to members of the Society for Endocrinology and Irish Endocrine Society to capture management practices in the care of patients with CAH. RESULTS: Marked variability was found in CAH management, with differences between general endocrinology and subspecialist settings, particularly in glucocorticoid use, biochemical monitoring and comorbidity screening, with significant disparities in reproductive health monitoring, notably in testicular adrenal rest tumours (TARTs) screening (p = .002), sperm banking (p = .0004) and partner testing for CAH (p < .0001). Adoption of the I-CAH registry was universally low. CONCLUSIONS: Differences in current management of CAH continue to exist. It appears crucial to objectify if different approaches result in different long-term outcomes. New studies such as CaHASE2, incorporating standardised minimum datasets including replacement therapies and monitoring strategies as well as longitudinal data collection, are now needed to define best-practice and standardise care.

Emergency and perioperative management of adrenal insufficiency in children and young people: British Society for Paediatric Endocrinology and Diabetes consensus guidance.

Adrenal insufficiency (AI) is characterised by lack of cortisol production from the adrenal glands. This can be a primary adrenal disorder or secondary to adrenocorticotropic hormone deficiency or suppression from exogenous glucocorticoids. Symptoms of AI in children may initially be non-specific and include growth faltering, lethargy, poor feeding, weight loss, abdominal pain, vomiting and lingering illnesses. AI is treated with replacement doses of hydrocortisone. At times of physiological stress such as illness, trauma or surgery, there is an increased requirement for exogenous glucocorticoids, which if untreated can lead to an adrenal crisis and death. There are no unified guidelines for those <18 years old in the UK, leading to substantial variation in the management of AI. This paper sets out guidance for intercurrent illness, medical, dental and surgical procedures to allow timely and appropriate recognition and treatment of AI and adrenal crisis for children and young people.

Analysis of therapy monitoring in the International Congenital Adrenal Hyperplasia Registry.

OBJECTIVE: Congenital adrenal hyperplasia (CAH) requires exogenous steroid replacement. Treatment is commonly monitored by measuring 17-OH progesterone (17OHP) and androstenedione (D4). DESIGN: Retrospective cohort study using real-world data to evaluate 17OHP and D4 in relation to hydrocortisone (HC) dose in CAH patients treated in 14 countries. PATIENTS: Pseudonymized data from children with 21-hydroxylase deficiency (21OHD) recorded in the International CAH Registry. MEASUREMENTS: Assessments between January 2000 and October 2020 in patients prescribed HC were reviewed to summarise biomarkers 17OHP and D4 and HC dose. Longitudinal assessment of measures was carried out using linear mixed-effects models (LMEM). RESULTS: Cohort of 345 patients, 52.2% female, median age 4.3 years (interquartile range: 3.1-9.2) were taking a median 11.3 mg/m2 /day (8.6-14.4) of HC. Median 17OHP was 35.7 nmol/l (3.0-104.0). Median D4 under 12 years was 0 nmol/L (0-2.0) and above 12 years was 10.5 nmol/L (3.9-21.0). There were significant differences in biomarker values between centres (p < 0.05). Correlation between D4 and 17OHP was good in multiple regression with age (p < 0.001, R2 = 0.29). In longitudinal assessment, 17OHP levels did not change with age, whereas D4 levels increased with age (p < 0.001, R2 = 0.08). Neither biomarker varied directly with dose or weight (p > 0.05). Multivariate LMEM showed HC dose decreasing by 1.0 mg/m2 /day for every 1 point increase in weight standard deviation score. DISCUSSION: Registry data show large variability in 17OHP and D4 between centres. 17OHP correlates with D4 well when accounting for age. Prescribed HC dose per body surface area decreased with weight gain.

The broad phenotypic spectrum of 17α-hydroxylase/17,20-lyase (CYP17A1) deficiency: a case series.

CONTEXT: 17α-Hydroxylase/17,20-lyase deficiency (17OHD) caused by mutations in the CYP17A1 gene is a rare form of congenital adrenal hyperplasia typically characterised by cortisol deficiency, mineralocorticoid excess and sex steroid deficiency. OBJECTIVE: To examine the phenotypic spectrum of 17OHD by clinical and biochemical assessment and corresponding in silico and in vitro functional analysis. DESIGN: Case series. PATIENTS AND RESULTS: We assessed eight patients with 17OHD, including four with extreme 17OHD phenotypes: two siblings presented with failure to thrive in early infancy and two with isolated sex steroid deficiency and normal cortisol reserve. Diagnosis was established by mass spectrometry-based urinary steroid profiling and confirmed by genetic CYP17A1 analysis, revealing homozygous and compound heterozygous sequence variants. We found novel (p.Gly111Val, p.Ala398Glu, p.Ile371Thr) and previously described sequence variants (p.Pro409Leu, p.Arg347His, p.Gly436Arg, p.Phe53/54del, p.Tyr60IlefsLys88X). In vitro functional studies employing an overexpression system in HEK293 cells showed that 17,20-lyase activity was invariably decreased while mutant 17α-hydroxylase activity retained up to 14% of WT activity in the two patients with intact cortisol reserve. A ratio of urinary corticosterone over cortisol metabolites reflective of 17α-hydroxylase activity correlated well with clinical phenotype severity. CONCLUSION: Our findings illustrate the broad phenotypic spectrum of 17OHD. Isolated sex steroid deficiency with normal stimulated cortisol has not been reported before. Attenuation of 17α-hydroxylase activity is readily detected by urinary steroid profiling and predicts phenotype severity. SIGNIFICANCE STATEMENT: Here we report, supported by careful phenotyping, genotyping and functional analysis, a prismatic case series of patients with congenital adrenal hyperplasia due to 17α-hydroxylase (CYP17A1) deficiency (17OHD). These range in severity from the abolition of function, presenting in early infancy, and unusually mild with isolated sex steroid deficiency but normal ACTH-stimulated cortisol in adult patients. These findings will guide improved diagnostic detection of CYP17A1 deficiency.

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.

Gonadectomy in conditions affecting sex development: a registry-based cohort study.

OBJECTIVES: To determine trends in clinical practice for individuals with DSD requiring gonadectomy. DESIGN: Retrospective cohort study. METHODS: Information regarding age at gonadectomy according to diagnosis; reported sex; time of presentation to specialist centre; and location of centre from cases reported to the International DSD Registry and who were over 16 years old in January 2019. RESULTS: Data regarding gonadectomy were available in 668 (88%) individuals from 44 centres. Of these, 248 (37%) (median age (range) 24 (17, 75) years) were male and 420 (63%) (median age (range) 26 (16, 86) years) were female. Gonadectomy was reported from 36 centres in 351/668 cases (53%). Females were more likely to undergo gonadectomy (n = 311, P < 0.0001). The indication for gonadectomy was reported in 268 (76%). The most common indication was mitigation of tumour risk in 172 (64%). Variations in the practice of gonadectomy were observed; of the 351 cases from 36 centres, 17 (5%) at 9 centres had undergone gonadectomy before their first presentation to the specialist centre. Median age at gonadectomy of cases from high-income countries and low-/middle-income countries (LMIC) was 13.0 years (0.1, 68) years and 16.5 years (1, 28), respectively (P < 0.0001) with the likelihood of long-term retention of gonads being higher in LMIC countries. CONCLUSIONS: The likelihood of gonadectomy depends on the underlying diagnosis, sex of rearing and the geographical setting. Clinical benchmarks, which can be studied across all forms of DSD will allow a better understanding of the variation in the practice of gonadectomy.

Bidirectional crosstalk between Hypoxia-Inducible Factor and glucocorticoid signalling in zebrafish larvae.

In the last decades in vitro studies highlighted the potential for crosstalk between Hypoxia-Inducible Factor-(HIF) and glucocorticoid-(GC) signalling pathways. However, how this interplay precisely occurs in vivo is still debated. Here, we use zebrafish larvae (Danio rerio) to elucidate how and to what degree hypoxic signalling affects the endogenous glucocorticoid pathway and vice versa, in vivo. Firstly, our results demonstrate that in the presence of upregulated HIF signalling, both glucocorticoid receptor (Gr) responsiveness and endogenous cortisol levels are repressed in 5 days post fertilisation larvae. In addition, despite HIF activity being low at normoxia, our data show that it already impedes both glucocorticoid activity and levels. Secondly, we further analysed the in vivo contribution of glucocorticoids to HIF activity. Interestingly, our results show that both glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) play a key role in enhancing it. Finally, we found indications that glucocorticoids promote HIF signalling via multiple routes. Cumulatively, our findings allowed us to suggest a model for how this crosstalk occurs in vivo.

Exquisite sensitivity of adrenocortical carcinomas to induction of ferroptosis.

Adrenocortical carcinomas (ACCs) are rare and highly malignant cancers associated with poor survival of patients. Currently, mitotane, a nonspecific derivative of the pesticide DDT (1,1-(dichlorobiphenyl)-2,2-dichloroethane), is used as the standard treatment, but its mechanism of action in ACCs remains elusive. Here we demonstrate that the human ACC NCI-H295R cell line is remarkably sensitive to induction of ferroptosis, while mitotane does not induce this iron-dependent mode of regulated necrosis. Supplementation with insulin, transferrin, and selenium (ITS) is commonly used to keep NCI-H295R cells in cell culture. We show that this supplementation prevents spontaneous ferroptosis, especially when it contains polyunsaturated fatty acids (PUFAs), such as linoleic acid. Inhibitors of apoptosis (zVAD, emricasan) do not prevent the mitotane-induced cell death but morphologically prevent membrane blebbing. The expression of glutathione peroxidase 4 (GPX4) in H295R cells, however, is significantly higher when compared to HT1080 fibrosarcoma cells, suggesting a role for ferroptosis. Direct inhibition of GPX4 in H295R cells led to high necrotic populations compared to control, while cotreatment with ferrostatin-1 (Fer-1) completely reverted ferroptosis. Interestingly, the analysis of public databases revealed that several key players of the ferroptosis pathway are hypermethylated and/or mutated in human ACCs. Finally, we also detected that growth hormone-releasing hormone (GHRH) antagonists, such as MIA602, kill H295R cells in a nonapoptotic manner. In summary, we found elevated expression of GPX4 and higher sensitivity to ferroptosis in ACCs. We hypothesize that instead of treatment with mitotane, human adrenocortical carcinomas may be much more sensitive to induction of ferroptosis.

Causes, patterns and severity of androgen excess in 487 consecutively recruited pre- and post-pubertal children.

Objective Androgen excess in childhood is a common presentation and may signify sinister underlying pathology. Data describing its patterns and severity are scarce, limiting the information available for clinical decision processes. Here, we examined the differential diagnostic value of serum DHEAS, androstenedione (A4) and testosterone in childhood androgen excess. Design Retrospective review of all children undergoing serum androgen measurement at a single center over 5 years. Methods Serum A4 and testosterone were measured by tandem mass spectrometry and DHEAS by immunoassay. Patients with at least one increased androgen underwent phenotyping by clinical notes review. Results In 487 children with simultaneous DHEAS, A4 and testosterone measurements, we identified 199 with androgen excess (140 pre- and 59 post-pubertal). Premature adrenarche (PA) was the most common pre-pubertal diagnosis (61%), characterized by DHEAS excess in 85%, while A4 and testosterone were only increased in 26 and 9% respectively. PCOS was diagnosed in 40% of post-pubertal subjects, presenting equally frequent with isolated excess of DHEAS (29%) or testosterone (25%) or increases in both A4 and testosterone (25%). CAH patients (6%) predominantly had A4 excess (86%); testosterone and DHEAS were increased in 50 and 33% respectively. Concentrations increased above the two-fold upper limit of normal were mostly observed in PA for serum DHEAS (>20-fold in the single case of adrenocortical carcinoma) and in CAH for serum androstenedione. Conclusions Patterns and severity of childhood androgen excess provide pointers to the underlying diagnosis and can be used to guide further investigations.

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.

Cardiovascular health, growth and gonadal function in children and adolescents with congenital adrenal hyperplasia.

After the introduction of replacement therapy with glucocorticoids and mineralocorticoids in the 1950s, congenital adrenal hyperplasia (CAH) is no longer a life-limiting condition. However, due to the successful introduction of medical steroid hormone replacement, CAH has become a chronic condition, with associated comorbidities and long-term health implications. The aim of treatment is the replacement of mineralocorticoids and glucocorticoids and the normalisation of elevated androgen concentrations. Long-term consequences of the condition and current treatment regimens include unfavourable changes in the cardiovascular risk profile, impaired growth, testicular adrenal rest tumours (TART) in male and subfertility in both male and female patients with CAH. Optimising replacement therapy in patients with CAH remains challenging. On one hand, treatment with supraphysiological doses of glucocorticoids might be required to normalise androgen concentrations and decrease size or presence of TARTs. On the other hand, treatment with supraphysiological doses of glucocorticoids is associated with an increased prevalence of unfavourable cardiovascular and metabolic risk profiles as well as impaired longitudinal growth and gonadal function. Therefore, treatment of children and adults with CAH requires an individualised approach. Careful monitoring for early signs of complications is already warranted during paediatric healthcare provision to prevent and reduce the impact of comorbidities in later life.

Characterization of the molecular genetic pathology in patients with 11ß-hydroxylase deficiency.

OBJECTIVE: Steroid 11ß-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia. Nonclassic or mild 11OHD appears to be a rare condition. Our study assessed the residual CYP11B1 function of detected mutations, adding to the spectrum of mild 11OHD, and illustrates the variability of the clinical presentation of 11OHD. PATIENTS AND METHODS: Five patients presented with mild to moderate 11OHD. Two women presented with mild hirsutism and in one case with secondary amenorrhoea. Two men presented with precocious pseudopuberty, gynaecomastia and elevated blood pressure. One 46,XX female patient was diagnosed with virilization of the external genitalia 2 years after birth. Direct DNA sequencing was carried out to perform CYP11B1 mutation analysis. The CYP11B1 mutations were functionally characterized using an in vitro expression system. RESULTS: CYP11B1-inactivating mutations were detected in all patients. Two novel missense mutations (p.P42L and p.A297V) and the previously characterized p.R143W mutation had residual CYP11B1 activities between 10% and 27%. A novel p.L382R and the previously uncharacterized p.G444D mutation both caused complete loss of CYP11B1 enzymatic activity. CONCLUSION: Mutations causing partial impairment of 11ß-hydroxylase activity (residual activity of 10% or above) are associated with a less severe clinical presentation of 11OHD, which can be classified as a nonclassic form. Our data demonstrate that patients with nonclassic 11OHD can present with androgen excess, precocious pseudopuberty and increased blood pressure. Timely diagnosis of nonclassic 11OHD and consequently initiation of personalized treatment is essential to prevent co-morbidities caused by androgen excess and hypertension.

5α-Reductase Type 2 Regulates Glucocorticoid Action and Metabolic Phenotype in Human Hepatocytes.

Glucocorticoids and androgens have both been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); androgen deficiency in males, androgen excess in females, and glucocorticoid excess in both sexes are associated with NAFLD. Glucocorticoid and androgen action are regulated at a prereceptor level by the enzyme 5α-reductase type 2 (SRD5A2), which inactivates glucocorticoids to their dihydrometabolites and converts T to DHT. We have therefore explored the role of androgens and glucocorticoids and their metabolism by SRD5A2 upon lipid homeostasis in human hepatocytes. In both primary human hepatocytes and human hepatoma cell lines, glucocorticoids decreased de novo lipogenesis in a dose-dependent manner. Whereas androgen treatment (T and DHT) increased lipogenesis in cell lines and in primary cultures of human hepatocytes from female donors, it was without effect in primary hepatocyte cultures from men. SRD5A2 overexpression reduced the effects of cortisol to suppress lipogenesis and this effect was lost following transfection with an inactive mutant construct. Conversely, pharmacological inhibition using the 5α-reductase inhibitors finasteride and dutasteride augmented cortisol action. We have demonstrated that manipulation of SRD5A2 activity can regulate lipogenesis in human hepatocytes in vitro. This may have significant clinical implications for those patients prescribed 5α-reductase inhibitors, in particular augmenting the actions of glucocorticoids to modulate hepatic lipid flux.

A diagnosis not to be missed: nonclassic steroid 11ß-hydroxylase deficiency presenting with premature adrenarche and hirsutism.

CONTEXT: Steroid 11ß-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia. Milder nonclassic forms are rare and at risk to be missed. OBJECTIVE: The objective of the study was to demonstrate the challenges in diagnosing nonclassic 11OHD. PATIENTS AND METHODS: Patient 1, a 10-year-old boy, presented with high-normal blood pressure and previously unexplained exaggerated adrenarche from age 4 years. Previous tests at the age of 8 years showed normal 17-hydroxyprogesterone concentrations with increased androgens. Patient 2, a 14-year-old female, presented with facial hirsutism, primary amenorrhea, and high-normal blood pressure. Novel CYP11B1 mutations were functionally analyzed in transiently transfected COS7 cells measuring the conversion of 11-deoxycortisol to cortisol by liquid chromatography-tandem mass spectrometry. RESULTS: Biochemical findings including urinary steroid metabolite analysis by gas chromatography-mass spectrometry were suggestive of 11OHD in all patients. CYP11B1 mutation analysis revealed compound heterozygosity in patient 1 (g.235T>A, p.F79I/g.2608C>T, p.R138C) and a homozygous mutation in patient 2 and two siblings (g.2623C>T, p.R143W). Functional in vitro analysis demonstrated partially impaired CYP11B1 activity compared with wild-type (p.F79I: 8.8% ± 0.8% (SEM); p.R138C: 9.8% ± 0.8%; p.R143W: 10.6% ± 1.2%). CONCLUSION: In addition to nonclassic 21-hydroxylase deficiency and steroid-secreting tumors, nonclassic 11OHD should be considered as an important differential diagnosis in patients with unexplained hyperandrogenism without 46,XX disorder of sex development. Nonclassic 11OHD is likely to be missed when relying on measuring standard steroid hormone panels. This diagnosis needs to be established early in life to avoid long-term health problems such as short stature, hyperandrogenism-related metabolic complications, potentially severe arterial hypertension, and cardiovascular consequences.

Testicular adrenal rest tumors develop independently of long-term disease control: a longitudinal analysis of 50 adult men with congenital adrenal hyperplasia due to classic 21-hydroxylase deficiency.

CONTEXT: Testicular adrenal rest tumors (TARTs) and hypogonadotropic hypogonadism are the two most common causes for male infertility in classic 21-hydroxylase deficiency. Current hypotheses suggest the quality of disease control to be one of the main pathogenic factors for TART development. OBJECTIVE: The aim was to study long-term predictors for TART development in a retrospective longitudinal study. DESIGN: Fifty men with classic 21-hydroxylase deficiency (31 salt wasting, 19 simple virilizing) were investigated. Testicular ultrasound at a median age at investigation of 27 years detected TARTs in 28 of 50 subjects (19 salt wasting, 9 simple virilizing). TART presence was correlated with long-term parameters of disease control during childhood and adolescence obtained from patients' charts: 24-hour urine pregnanetriol, serum 17-hydroxyprogesterone, onset and stage of pubic hair development, testicular growth, and bone age in relation to chronological age. RESULTS: There was no difference in pregnanetriol excretion over lifetime between patients with and without TARTs. Similarly, neither development of pubic hair and testicular volume (Tanner) nor bone age in relation to chronological age differed between the two groups. Furthermore, the two groups had the same body mass index and the same impairment of final height in relation to midparental target height. CONCLUSION: Our longitudinal analysis demonstrates no association between TART presence and parameters of disease control. These data, therefore, argue for other mechanisms more relevant for TART induction including those occurring during fetal development.

Prenatal diagnosis of congenital adrenal hyperplasia caused by P450 oxidoreductase deficiency.

CONTEXT: Mutations in the electron donor enzyme P450 oxidoreductase (POR) result in congenital adrenal hyperplasia with apparent combined 17α-hydroxylase/17,20 lyase and 21-hydroxylase deficiencies, also termed P450 oxidoreductase deficiency (PORD). Major clinical features present in PORD are disordered sex development in affected individuals of both sexes, glucocorticoid deficiency, and multiple skeletal malformations. OBJECTIVE: The objective of the study was to establish a noninvasive approach to prenatal diagnosis of PORD including assessment of malformation severity to facilitate optimized prenatal diagnosis and timely treatment. DESIGN: We analyzed 20 pregnancies with children homozygous or compound heterozygous for disease-causing POR mutations and 1 pregnancy with a child carrying a heterozygous POR mutation by recording clinical and biochemical presentations and fetal ultrasound findings. In 4 of the pregnancies (3 homozygous and 1 heterozygous for disease-causing POR mutations), prenatal analysis of steroid metabolite excretion in maternal urine was carried out by gas chromatography/mass spectrometry during gestational weeks 11-23. RESULTS: Pregnancy complications in our cohort included maternal virilization (6 of 20) with onset in the second trimester. Seven pregnant women presented with low unconjugated estriol at prenatal screening (triple or quadruple antenatal screening test). Overt dysmorphic features were noted in 19 of the 20 babies at birth but observed in only 5 by prenatal ultrasound. These 5 had the most severe malformation phenotypes and poor outcome, whereas the other babies showed normal development. Steroid profiling of maternal urine revealed significantly increased steroids of fetal origin, namely the pregnenolone metabolite epiallopregnanediol and the androgen metabolite androsterone, with concomitant low values for estriol. Diagnostic steroid ratios conclusively indicated PORD as early as gestational week 12. In the heterozygous pregnancy, steroid ratios were only slightly elevated and estriol excretion was normal. CONCLUSION: Prenatal diagnosis in PORD is readily established via urinary steroid metabolite analysis of maternal urine. Visible malformations at prenatal ultrasound predict a severe malformation phenotype.

Urine steroid metabolomics as a biomarker tool for detecting malignancy in adrenal tumors.

CONTEXT: Adrenal tumors have a prevalence of around 2% in the general population. Adrenocortical carcinoma (ACC) is rare but accounts for 2-11% of incidentally discovered adrenal masses. Differentiating ACC from adrenocortical adenoma (ACA) represents a diagnostic challenge in patients with adrenal incidentalomas, with tumor size, imaging, and even histology all providing unsatisfactory predictive values. OBJECTIVE: Here we developed a novel steroid metabolomic approach, mass spectrometry-based steroid profiling followed by machine learning analysis, and examined its diagnostic value for the detection of adrenal malignancy. DESIGN: Quantification of 32 distinct adrenal derived steroids was carried out by gas chromatography/mass spectrometry in 24-h urine samples from 102 ACA patients (age range 19-84 yr) and 45 ACC patients (20-80 yr). Underlying diagnosis was ascertained by histology and metastasis in ACC and by clinical follow-up [median duration 52 (range 26-201) months] without evidence of metastasis in ACA. Steroid excretion data were subjected to generalized matrix learning vector quantization (GMLVQ) to identify the most discriminative steroids. RESULTS: Steroid profiling revealed a pattern of predominantly immature, early-stage steroidogenesis in ACC. GMLVQ analysis identified a subset of nine steroids that performed best in differentiating ACA from ACC. Receiver-operating characteristics analysis of GMLVQ results demonstrated sensitivity = specificity = 90% (area under the curve = 0.97) employing all 32 steroids and sensitivity = specificity = 88% (area under the curve = 0.96) when using only the nine most differentiating markers. CONCLUSIONS: Urine steroid metabolomics is a novel, highly sensitive, and specific biomarker tool for discriminating benign from malignant adrenal tumors, with obvious promise for the diagnostic work-up of patients with adrenal incidentalomas.

Pubertal presentation in seven patients with congenital adrenal hyperplasia due to P450 oxidoreductase deficiency.

CONTEXT: P450 oxidoreductase (POR) is a crucial electron donor to all microsomal P450 cytochrome (CYP) enzymes including 17α-hydroxylase (CYP17A1), 21-hydroxylase (CYP21A2) and P450 aromatase. Mutant POR causes congenital adrenal hyperplasia with combined glucocorticoid and sex steroid deficiency. P450 oxidoreductase deficiency (ORD) commonly presents neonatally, with disordered sex development in both sexes, skeletal malformations, and glucocorticoid deficiency. OBJECTIVE: The aim of the study was to describe the clinical and biochemical characteristics of ORD during puberty. DESIGN: Clinical, biochemical, and genetic assessment of seven ORD patients (five females, two males) presenting during puberty was conducted. RESULTS: Predominant findings in females were incomplete pubertal development (four of five) and large ovarian cysts (five of five) prone to spontaneous rupture, in some only resolving after combined treatment with estrogen/progestin, GnRH superagonists, and glucocorticoids. Pubertal development in the two boys was more mildly affected, with some spontaneous progression. Urinary steroid profiling revealed combined CYP17A1 and CYP21A2 deficiencies indicative of ORD in all patients; all but one failed to mount an appropriate cortisol response to ACTH stimulation indicative of adrenal insufficiency. Diagnosis of ORD was confirmed by direct sequencing, demonstrating disease-causing POR mutations. CONCLUSION: Delayed and disordered puberty can be the first sign leading to a diagnosis of ORD. Appropriate testosterone production during puberty in affected boys but manifest primary hypogonadism in girls with ORD may indicate that testicular steroidogenesis is less dependent on POR than adrenal and ovarian steroidogenesis. Ovarian cysts in pubertal girls may be driven not only by high gonadotropins but possibly also by impaired CYP51A1-mediated production of meiosis-activating sterols due to mutant POR.

Gas chromatography/mass spectrometry (GC/MS) remains a pre-eminent discovery tool in clinical steroid investigations even in the era of fast liquid chromatography tandem mass spectrometry (LC/MS/MS).

Liquid chromatography tandem mass spectrometry (LC/MS/MS) is replacing classical methods for steroid hormone analysis. It requires small sample volumes and has given rise to improved specificity and short analysis times. Its growth has been fueled by criticism of the validity of steroid analysis by older techniques, testosterone measurements being a prime example. While this approach is the gold-standard for measurement of individual steroids, and panels of such compounds, LC/MS/MS is of limited use in defining novel metabolomes. GC/MS, in contrast, is unsuited to rapid high-sensitivity analysis of specific compounds, but remains the most powerful discovery tool for defining steroid disorder metabolomes. Since the 1930s almost all inborn errors in steroidogenesis have been first defined through their urinary steroid excretion. In the last 30 years, this has been exclusively carried out by GC/MS and has defined conditions such as AME syndrome, glucocorticoid remediable aldosteronism (GRA) and Smith-Lemli-Opitz syndrome. Our recent foci have been on P450 oxidoreductase deficiency (ORD) and apparent cortisone reductase deficiency (ACRD). In contrast to LC/MS/MS methodology, a particular benefit of GC/MS is its non-selective nature; a scanned run will contain every steroid excreted, providing an integrated picture of an individual's metabolome. The "Achilles heel" of clinical GC/MS profiling may be data presentation. There is lack of familiarity with the multiple hormone metabolites excreted and diagnostic data are difficult for endocrinologists to comprehend. While several conditions are defined by the absolute concentration of steroid metabolites, many are readily diagnosed by ratios between steroid metabolites (precursor metabolite/product metabolite). Our work has led us to develop a simplified graphical representation of quantitative urinary steroid hormone profiles and diagnostic ratios.

Inactivating PAPSS2 mutations in a patient with premature pubarche.

Dehydroepiandrosterone (DHEA) sulfotransferase, known as SULT2A1, converts the androgen precursor DHEA to its inactive sulfate ester, DHEAS [corrected], thereby preventing the conversion of DHEA to an active androgen. SULT2A1 requires 3'-phosphoadenosine-5'-phosphosulfate (PAPS) for catalytic activity. We have identified compound heterozygous mutations in the gene encoding human PAPS synthase 2 (PAPSS2) in a girl with premature pubarche, hyperandrogenic anovulation, very low DHEAS levels, and increased androgen levels. In vitro coincubation of human SULT2A1 and wild-type or mutant PAPSS2 proteins confirmed the inactivating nature of the mutations. These observations indicate that PAPSS2 deficiency is a monogenic adrenocortical cause of androgen excess.