Determinación de valores de corte de 17 hidroxiprogesterona ajustados por edad gestacional para la pesquisa neonatal de la hiperplasia suprarrenal congénita / Assessment of gestational age-adjusted 17-hydroxyprogesterone cut-off values for neonatal screening of congenital adrenal hyperplasia

La pesquisa neonatal de hiperplasia suprarrenal congénita se realiza mediante la determinación de 17 hidroxiprogesterona (17OHP) en gotas de sangre seca en papel de filtro. Los bebés prematuros presentan valores más elevados que los bebés de término, siendo de utilidad contar con límites de corte apropiados. Nuestro objetivo fue actualizar los valores de corte de 17OHP ajustados por edad gestacional para la metodología en uso a nivel nacional por las jurisdicciones asistidas por el "Programa Nacional de Fortalecimiento de la Detección Precoz de Enfermedades Congénitas". La 17OHP se determinó utilizando el kit comercial de enzimo-inmunoanálisis (ELISA competitivo), Elizen Neonatal 17OHP Screening (Zentech, Bélgica). Se obtuvieron límites de corte utilizando percentiles de la distribución de los valores de 17OHP para cada edad gestacional. La sensibilidad obtenida fue 100%, especificidad 98,76 %, tasa de falsos positivos 1,24 % y el valor predictivo positivo 1,12 %. Destacamos la importancia de disponer de límites de corte adecuados a la población. La armonización de los mismos permitirá resultados comparables entre los programas regionales de pesquisa neonatal (AU)

Newborn screening for congenital adrenal hyperplasia is performed by the measurement of 17-hydroxyprogesterone (17OHP) in dried blood spots on filter paper. Premature infants have higher values than full-term infants, and appropriate cutoff values are useful. Our aim was to update the cut-off values of 17OHP adjusted for gestational age for the methodology used at a national level in regions assisted by the "National Program for Strengthening the Early Detection of Congenital Diseases". 17OHP was determined using the commercial enzyme-linked immunosorbent assay (competitive ELISA) kit, Elizen Newborn 17OHP Screening (Zentech, Belgium). Cut-off values were obtained using percentiles of the distribution of 17OHP values for each gestational age. Sensitivity was 100%, specificity 98.76%, false positive rate 1.24%, and positive predictive value 1.12%. It is important to have cut-off values that are adjusted to the population. Harmonization will allow for the comparison of results among regional newborn screening programs (AU)

An unusual cause of adrenal insufficiency with elevation of 17-hydroxyprogesterone: case report.

BACKGROUND: We present an intriguing case of primary adrenal lymphoma, with associated primary adrenal insufficiency (PAI), in a patient presenting a transitory partial 21-hydroxylase deficiency during the active phase of the adrenal disease. CASE PRESENTATION: An 85-years old woman was referred because of worsening asthenia, lumbar pain, generalized myalgia and arthralgia. During investigations a computed tomography (CT) scan evidenced two large bilateral adrenal masses, highly suspicious for primary adrenal tumor. The hormonal assessment revealed very low levels of morning plasma cortisol and 24-h urinary cortisol, elevated ACTH levels with low plasma concentration of aldosterone, pointing to the diagnosis of PAI. After diagnosis of PAI our patient started glucocorticoid and mineralcorticoid replacement therapy with clinical benefit. In order to further characterize the adrenal lesions, adrenal biopsy, was performed. The histology revealed a high grade non-Hodgkin lymphoma with an immunophenotype consistent with intermediate aspects between diffuse large B-cell and Burkitt lymphoma, with a high proliferation index (KI-67 > 90%). The patient received chemotherapy with epirubicin, vincristine, cyclophosphamide, and rituximab, associated with methylprednisolone that resulted in a complete clinical and radiological remission within one year. After 2 years from the diagnosis and a total of 6 cycles of rituximab, the patient was in good clinical condition and was taking only the replacement therapy for PAI. The patient initially presented also a slight increase of 17-hydroxyprogesterone (17-OHP) for age that normalize after resolution of lymphoproliferative disease. CONCLUSIONS: In the presence of bilateral adrenal disease and/or in the presence of signs and symptoms of PAI clinicians must exclude the presence of PAL. The evidence of elevated ACTH-stimulated 17-OHP levels also in patients with other adrenal masses, together with the detection of elevated basal 17-OHP levels in our patient make it more plausible, in our view, an effect of the lesion on the "healthy" adrenal tissue residue than a direct secretory activity by the adrenal tumor.

A Multiclassifier System to Identify and Subtype Congenital Adrenal Hyperplasia Based on Circulating Steroid Hormones.

CONTEXT: Measurement of plasma steroids is necessary for diagnosis of congenital adrenal hyperplasia (CAH). We sought to establish an efficient strategy for detection and subtyping of CAH with a machine-learning algorithm. METHODS: Clinical phenotype and genetic testing were used to provide CAH diagnosis and subtype. We profiled 13 major steroid hormones by liquid chromatography-tandem mass spectrometry. A multiclassifier system was established to distinguish 11ß-hydroxylase deficiency (11ßOHD), 17α-hydroxylase/17,20-lyase deficiency (17OHD), and 21α-hydroxylase deficiency (21OHD) in a discovery cohort (n = 226). It was then validated in an independent cohort (n = 111) and finally applied in a perspective cohort of 256 patients. The diagnostic performance on the basis of area under receiver operating characteristic curves (AUCs) was evaluated. RESULTS: A cascade logistic regression model, we named the "Steroidogenesis Score", was able to discriminate the 3 most common CAH subtypes: 11ßOHD, 17OHD, and 21OHD. In the perspective application cohort, the steroidogenesis score had a high diagnostic accuracy for all 3 subtypes, 11ßOHD (AUC, 0.994; 95% CI, 0.983-1.000), 17OHD (AUC, 0.993; 95% CI, 0.985-1.000), and 21OHD (AUC, 0.979; 95% CI, 0.964-0.994). For nonclassic 21OHD patients, the tool presented with significantly higher sensitivity compared with measurement of basal 17α-hydroxyprogesterone (17OHP) (0.973 vs 0.840, P = 0.005) and was not inferior to measurement of basal vs stimulated 17OHP (0.973 vs 0.947, P = 0.681). CONCLUSIONS: The steroidogenesis score was biochemically interpretable and showed high accuracy in identifying CAH patients, especially for nonclassic 21OHD patients, thus offering a standardized approach to diagnose and subtype CAH.

Crinecerfont Lowers Elevated Hormone Markers in Adults With 21-Hydroxylase Deficiency Congenital Adrenal Hyperplasia.

CONTEXT: Classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21OHD) is characterized by impaired cortisol synthesis and excess androgen production. Corticotropin-releasing factor type 1 receptor (CRF1R) antagonism may decrease adrenal androgen production. OBJECTIVE: This work aimed to evaluate the safety, tolerability, and efficacy of crinecerfont (NBI-74788), a selective CRF1R antagonist, in 21OHD. METHODS: This open-label, phase 2 study, with sequential cohort design (NCT03525886), took place in 6 centers in the United States. Participants included men and women, aged 18 to 50 years, with 21OHD. Interventions included 4 crinecerfont regimens, each administered orally for 14 consecutive days: 50 or 100 mg once daily at bedtime (cohorts 1 and 2, respectively); 100 mg once daily in the evening (cohort 3); and 100 mg twice daily (cohort 4). Participants could enroll in more than 1 cohort. Main outcomes included changes from baseline to day 14 in adrenocorticotropin (ACTH), 17-hydroxyprogesterone (17OHP), androstenedione, and testosterone. RESULTS: Eighteen participants (11 women, 7 men) were enrolled: cohort 1 (n = 8), cohort 2 (n = 7), cohort 3 (n = 8), cohort 4 (n = 8). Mean age was 31 years; 94% were White. Median percent reductions were more than 60% for ACTH (-66%), 17OHP (-64%), and androstenedione (-64%) with crinecerfont 100 mg twice a day. In female participants, 73% (8/11) had a 50% or greater reduction in testosterone levels; male participants had median 26% to 65% decreases in androstenedione/testosterone ratios. CONCLUSION: Crinecerfont treatment for 14 days lowered ACTH and afforded clinically meaningful reductions of elevated 17OHP, androstenedione, testosterone (women), or androstenedione/testosterone ratio (men) in adults with 21OHD. Longer-term studies are required to evaluate the effects of crinecerfont on clinical end points of disordered steroidogenesis and glucocorticoid exposure in patients with 21OHD.

The Application of Principal Component Analysis on Clinical and Biochemical Parameters Exemplified in Children With Congenital Adrenal Hyperplasia.

Purpose: Principal component analysis (PCA) is a mathematical model which simplifies data into new, combined variables. Optimal treatment of pediatric congenital adrenal hyperplasia (CAH) remains a challenge and requires evaluation of all biochemical and clinical markers. The aim of this study was to introduce PCA methodology as a tool to optimize management in a cohort of pediatric and adolescent patients with CAH by including adrenal steroid measurements and clinical parameters. Methods: This retrospective, longitudinal cohort of 33 children and adolescents with CAH due to 21-hydroxylase deficiency included 406 follow-up observations. PCAs were applied to serum hormone concentrations and compared to treatment efficacy evaluated by clinical parameters. Results: We provide and describe the first PCA models with hormone parameters denoted in sex- and age-adjusted standard deviation (SD) scores to comprehensibly describe the combined 'endocrine profiles' of patients with classical and non-classical CAH, respectively. Endocrine profile scores were predictive markers of treatment efficacy for classical (AUC=92%; accuracy 95%; p=1.8e-06) and non-classical CAH (AUC=80%; accuracy 91%; p=0.004). A combined PCA demonstrated clustering of patients with classical and non-classical CAH by serum 17-hydroxyprogesterone (17-OHP) and dehydroepiandrosterone-sulphate (DHEAS) concentrations. Conclusion: As an example of the possibilities of PCA, endocrine profiles were successfully able to distinguish between patients with CAH according to treatment efficacy and to elucidate biochemical differences between classical and non-classical CAH.

Reverse circadian glucocorticoid treatment in prepubertal children with congenital adrenal hyperplasia.

OBJECTIVES: Children with salt-wasting congenital adrenal hyperplasia (CAH) have an impaired function of steroid synthesis pathways. They require therapy with glucocorticoid (GC) and mineralocorticoid hormones to avoid salt-wasting crisis and other complications. Most commonly, children receive hydrocortisone thrice daily with the highest dose in the morning, mimicking the regular physiology. However, reverse circadian treatment (RCT) had been suggested previously. In this study, we aimed to determine the efficacy of RCT in prepubertal children with CAH by comparing the salivary 17-hydroxyprogesterone (s17-OHP) levels individually. METHODS: In this retrospective study, we analyzed the records of children with classical CAH and RCT who were monitored by s17-OHP levels. The study included 23 patients. We identified nine prepubertal children with RCT schemes (three boys and six girls) and compared the s17-OHP levels in the morning, afternoon, and evening. The objective of this study was to demonstrate the non-effectiveness of RCT in terms of lowering the morning s17-OHP concentration. In addition, we compared s17-OHP day profiles in six patients on RCT and non-RCT therapy (intraindividually). RESULTS: Eight of nine children with RCT showed higher s17-OHP levels in the morning compared to the evening. In addition, none of the children showed a significant deviation of development. Three children were overweight. No adrenal crisis or pubertal development occurred. Comparison of RCT and non-RCT regimens showed no difference in 17-OHP profiles. CONCLUSIONS: Our data do not support the use of RCT schemes for GC replacement in children with CAH due to lack of benefits and unknown long-term risks.

Endocrine disruption caused by the aquatic exposure to aluminum and manganese in Astyanax altiparanae (Teleostei: Characidae) females during the final ovarian maturation.

Aluminum (Al) and manganese (Mn) can be toxic to aquatic biota and cause endocrine disruption in fish, affecting reproduction. This study evaluates the physiological responses of the ray-finned teleost fish Astyanax altiparanae vitellogenic females after acute exposure (96 h) to Al and Mn (alone and combined) in acid pH followed by the same period of exposure to metal-free water in neutral pH. The aim of this second period of exposure was to assess the recovery capacity from the toxic effects these metals. Five experimental groups were established: a control in neutral pH (Ctrl), and acidic pH (Ac), aluminum (Al), manganese (Mn), and Al + Mn groups, maintaining the acidic pH in the groups to which metals were added. The following biological parameters were evaluated: metal tissue concentration, relative fecundity (RF: absolute fecundity/body mass). Plasma levels of cortisol (proxy for stress) and 17α hydroxyprogesterone (17α-OHP), and gene expression of pituitary lhß mRNA (proxies for final maturation) were measured to evaluate endocrine disruption. In the synchronic exposure, the presence of Mn potentiated the accumulation of Al in gills. The females from acidic pH and Al groups showed a reduced RF. Exposure to Al and Mn triggered an endocrine disruption response, evidenced by a decrease in the plasma concentration of 17α-OHP and cortisol. Despite this anti-steroidogenic effect, no changes occurred in the pituitary gene expression of lhß. The endocrine changes and the metal accumulation were temporary, while the impacts on RF under the experimental conditions suggest permanent impairment in the reproduction of this species.

Tildacerfont in Adults With Classic Congenital Adrenal Hyperplasia: Results from Two Phase 2 Studies.

CONTEXT: Congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21OHD) is typically treated with lifelong supraphysiologic doses of glucocorticoids (GCs). Tildacerfont, a corticotropin-releasing factor type-1 receptor antagonist, may reduce excess androgen production, allowing for GC dose reduction. OBJECTIVE: Assess tildacerfont safety and efficacy. DESIGN AND SETTING: Two Phase 2 open-label studies. PATIENTS: Adults with 21OHD. INTERVENTION: Oral tildacerfont 200 to 1000 mg once daily (QD) (n = 10) or 100 to 200 mg twice daily (n = 9 and 7) for 2 weeks (Study 1), and 400 mg QD (n = 11) for 12 weeks (Study 2). MAIN OUTCOME MEASURE: Efficacy was evaluated by changes from baseline at 8 am in adrenocorticotropic hormone (ACTH), 17-hydroxyprogesterone (17-OHP), and androstenedione (A4) according to baseline A4 ≤ 2× upper limit of normal (ULN) or A4 > 2× ULN. Safety was evaluated using adverse events (AEs) and laboratory assessments. RESULTS: In Study 1, evaluable participants with baseline A4 > 2× ULN (n = 11; 19-67 years, 55% female) had reductions from baseline in ACTH (-59.4% to -28.4%), 17-OHP (-38.3% to 0.3%), and A4 (-24.2% to -18.1%), with no clear dose response. In Study 2, participants with baseline A4 > 2× ULN (n = 5; 26-63 years, 40% female) had ~80% maximum mean reductions in biomarker levels. ACTH and A4 were normalized for 60% and 40%, respectively. In both studies, participants with baseline A4 ≤ 2× ULN maintained biomarker levels. AEs (in 53.6% of patients overall) included headache (7.1%) and upper respiratory tract infection (7.1%). CONCLUSIONS: For patients with 21OHD, up to 12 weeks of oral tildacerfont reduced or maintained key hormone biomarkers toward normal.

Second-tier Testing for 21-Hydroxylase Deficiency in the Netherlands: A Newborn Screening Pilot Study.

CONTEXT: Newborn screening (NBS) for classic congenital adrenal hyperplasia (CAH) consists of 17-hydroxyprogesterone (17-OHP) measurement with gestational age-adjusted cutoffs. A second heel puncture (HP) is performed in newborns with inconclusive results to reduce false positives. OBJECTIVE: We assessed the accuracy and turnaround time of the current CAH NBS algorithm in comparison with alternative algorithms by performing a second-tier 21-deoxycortisol (21-DF) pilot study. METHODS: Dried blood spots (DBS) of newborns with inconclusive and positive 17-OHP (immunoassay) first HP results were sent from regional NBS laboratories to the Amsterdam UMC Endocrine Laboratory. In 2017-2019, 21-DF concentrations were analyzed by LC-MS/MS in parallel with routine NBS. Diagnoses were confirmed by mutation analysis. RESULTS: A total of 328 DBS were analyzed; 37 newborns had confirmed classic CAH, 33 were false-positive and 258 were categorized as negative in the second HP following the current algorithm. With second-tier testing, all 37 confirmed CAH had elevated 21-DF, while all 33 false positives and 253/258 second-HP negatives had undetectable 21-DF. The elevated 21-DF of the other 5 newborns may be NBS false negatives or second-tier false positives. Adding the second-tier results to inconclusive first HPs reduced the number of false positives to 11 and prevented all 286 second HPs. Adding the second tier to both positive and inconclusive first HPs eliminated all false positives but delayed referral for 31 CAH patients (1-4 days). CONCLUSION: Application of the second-tier 21-DF measurement to inconclusive first HPs improved our CAH NBS by reducing false positives, abolishing the second HP, and thereby shortening referral time.

Comparison of different preparation techniques of dried blood spot quality controls in newborn screening for congenital adrenal hyperplasia.

In newborn screening, samples suspected for congenital adrenal hyperplasia (CAH), a potentially lethal inborn error of steroid biosynthesis, need to be confirmed using liquid chromatography-tandem mass spectrometry. Daily quality controls (QCs) for the 2nd-tier CAH assay are not commercially available and are therefore generally prepared within the laboratory. For the first time, we aimed to compare five different QC preparation approaches used in routine diagnostics for CAH on the concentrations of cortisol, 21-deoxycortisol, 11-deoxycortisol, 4-androstenedione and 17-hydroxyprogesterone in dried blood spots. The techniques from Prep1 to Prep5 were tested at two analyte concentrations by spiking aliquots of a steroid-depleted blood, derived from washed erythrocyte suspension and steroid-depleted serum. The preparation processes differed in the sequence of the preparation steps and whether freeze-thaw cycles were used to facilitate blood homogeneity. The five types of dried blood spot QCs were assayed and quantitated in duplicate on five different days using a single calibration row per day. Inter-assay variations less than 15% and concentrations within ±15% of the nominal values were considered acceptable. Results obtained by means of the four dried blood spot QC preparation techniques (Prep1, Prep2, Prep4 and Prep5) were statistically similar and remained within the ±15% ranges in terms of both reproducibility and nominal values. However, concentration results for Prep3 (spiking prior to three freeze-thaw cycles) were significantly lower than the nominal values in this setting, with differences exceeding the ±15% range in many cases despite acceptable inter-assay variations. These findings have implications for the in-house preparation of QC samples in laboratory developed tests for CAH, including 2nd-tier assays in newborn screening.

Birth Weight- or Gestational Age-adjusted Second-tier LCMSMS Cutoffs Improve Newborn Screening for CAH in New Zealand.

CONTEXT: The positive predictive value of newborn screening for congenital adrenal hyperplasia (CAH) in New Zealand is approximately 10%. The use of a second tier liquid chromatography-tandem mass spectrometry bloodspot steroid profile test with birth weight- or gestational age-adjusted screening cutoffs may result in further screening improvements. METHODS: Three years of newborn screening data with additional second-tier steroid metabolites was evaluated (n = 167 672 births). Data from babies with a negative screening test and confirmed CAH cases were compared. First- and second-tier steroid measurements were correlated with both birth weight and gestational age. Analysis of variance was used to determine birth weight and gestational age groups. Screening cutoffs were determined and applied retrospectively to model screening performance. RESULTS: First-tier immunoassay data correlated better with gestational age than with birth weight, but there was no difference with second-tier steroid measurements. Four distinct birth weight and gestational age groups were established for 17-hydroxyprogesterone and a steroid ratio measurement. Application of 97.5th percentile second-tier birth weight- or gestational age-adjusted cutoffs would result in 10 positive tests over the period of the study with 8 true-positive screens and 2 false-positive tests. The positive predictive value of screening would be increased from 10.8% to 80%. CONCLUSIONS: The use of either birth weight- or gestational age-adjusted cutoffs for second-tier screening tests can significantly reduce the false positive rate of newborn screening for CAH in New Zealand without loss in screening sensitivity.

11-oxygenated androgens and their relation to hypothalamus-pituitary-gonadal-axis disturbances in adults with congenital adrenal hyperplasia.

CONTEXT: Hypothalamus-pituitary-gonadal (HPG)-axis disturbances are a common phenomenon in patients with classic congenital adrenal hyperplasia (CAH). 11-oxygenated androgens have been suggested to play a role in this context. DESIGN: Cross-sectional single center study including 89 patients (N = 42 men, N = 55 women) with classic CAH. MAIN OUTCOME MEASURES: Differences in steroid markers in men with hypogonadism and women with secondary amenorrhea with a special focus on 11-ketotestosterone (11KT) and 11ß-hydroxyandrostenedione (11OHA4). RESULTS: Hypogonadotropic hypogonadism was present in 23 % of men and 61 % of those women currently not on contraceptives suffered from irregular menstrual cycles or amenorrhea. Testicular adrenal rest tumor (TART) was documented in 28 % of men. 11KT (3.5x) and 11OHA4 (5.7x) among other adrenal steroids were significantly elevated in men with hypogonadism and in women with amenorrhea in comparison to those with a regular cycle (11KT: 5.2x; 11OHA4: 3.7x). 11-oxygenated androgens were not higher in men with TART than in those without. There was a negative association of 11KT and 11OHA4 with FSH but not with LH in men. As expected, all steroids were strongly correlated with each other and cases of disproportionally elevated 11-oxygenated androgens that could explain for HPG-disturbances or TART in otherwise controlled patients were rare and also found in eugonadal individuals. CONCLUSIONS: In CAH, 11-oxygenated androgens are elevated in women with menstrual disturbances and in men with hypogonadotropic hypogonadism. Due to the close correlation of 11-oxygenated androgens with other adrenal steroids it remains to be shown if their measurement is superior to conventional markers of androgen control.

Preliminary evaluation of eight less frequent endocrine assays designed for MAGLUMI 800 chemiluminescence immunoanalyzer.

Transition to new analytical systems and methods requires end-user verification to ensure acceptability for routine use. Our aim was to verify precision of MAGLUMI 800 immunoassay analyzer for 17-hydroxyprogesterone (17-OHP), 25-hydroxy vitamin D (25(OH)D), aldosterone, androstenedione, growth hormone (GH), insulin-like growth factor 1 (IGF-1), insulin-like growth factor-binding protein 3 (IGFBP-3) and renin, as well as to assess their comparability with the routinely used assays. Precision was evaluated at two levels following the CLSI EP15-A2 protocol. Method comparison included parallel analysis of 40 routine samples for each assay on MAGLUMI 800 and the routinely used automated or manual immunoassays. Within-run coefficients of variation (CV) ranged from 0.8% (androstenedione) to 14.5% (aldosterone), between-run CVs from 1.0% (IGFBP-3) to 12.8% (renin), while within-laboratory (total) precision CVs were from 2.1% (IGFBP-3) to 14.9% (renin). All assays with the exception of IGF-1 and 25(OH)D at the low concentration control level, satisfied biological variation criteria for imprecision. Passing-Bablok regression showed proportional difference for 17-OHP and aldosterone, constant for androstenedione, while both constant and proportional difference was revealed for 25(OH)D, GH and IGF-1. Statistically significant relative biases higher than the desirable biological variation acceptance criteria were observed for 17-OHP, 25(OH)D, aldosterone, androstenedione and IGF-1. The evaluated assays need further assessment as well as verification of reference intervals in order to be suitable for introduction into routine practice in our laboratory. Our study clearly demonstrates that we are still far from achieving immunoassay standardization and comparability of results.

[Clinical characteristics of 21 infertile women with non-classic 21-hydroxylase deficiency].

Objective: To investigate the clinical features of infertile women with non-classic 21-hydroxylase deficiency (21-OHD). Methods: The study enrolled 21 infertile women with non-classic 21-OHD in Sun Yat-sen Memorial Hospital of Sun Yat-sen University from January 2009 to December 2018. The clinical presentation, endocrine hormone, glucolipid metabolism and treatment outcome were retrospectively analyzed. The diagnosis of non-classic 21-OHD was comprehensively based on clinical and hormonal characteristics. Results: Among 21 cases, the age was (29.9±2.9) years, the mean age at menarche was (13.6±2.0) years, body mass index was (22.1±2.9) kg/m2, and 38% (8/21) had oligomenorrhea. Hirsutism was diagnosed in 3 cases (14%, 3/21). Clitoromegaly was seen in 14% (3/21) and polycystic ovarian morphology was found in 33% (7/21) of the patients. The mean serum level of basal progesterone was (11.3±21.0) nmol/L, with 48% (10/21) having high basal progesterone level; after therapy by glucocorticoid, the level of progesterone was (1.9±2.0) nmol/L. Serum 17-hydroxyprogesterone concentration was (66.4±123.6) nmol/L; after therapy by glucocorticoid, it was (2.4±1.8) nmol/L. In the study increased testosterone, androstenedione and dehydroepiandrosterone sulfate were present in 62% (13/21), 52% (11/21) and 43% (9/21), respectively; and 52% (11/21) of patients manifested androgen excess and basal progesterone elevation; androgen levels decreased after therapy by glucocorticoid. The pregnancy rate was 76% (16/21). Out of 19 pregnancies, 6/19 ended in spontaneous miscarriages. Conclusions: Infertile women with non-classic 21-OHD are characterized by hyperandrogenism and basal progesterone elevation, whereas gonad axis disorder is not apparent. After no response to conventional therapy, adult infertile women with non-classic 21-OHD could achieve a desirable pregnancy outcome with proper treatment of glucocorticoid.

Establishment of Clinical and Lab Algorithms for the Identification of Carriers of Mutations in CYP21A2 - A Study of 365 Children and Adolescents.

BACKGROUND: Mutations of CYP21A2 encoding 21-hydroxylase are the most frequent cause of congenital adrenal hyperplasia (CAH) and are associated either with elevated basal or ACTH-stimulated levels of 17-hydroxyprogesterone (17OHP) in blood. OBJECTIVE: The study objective was to identify the most suitable of 12 different test algorithms and appropriate cut-off levels for that test to recognize patients with non-classical congenital adrenal hyperplasia (NCCAH) and carriers of clinically relevant mutations in CYP21A2. METHOD AND PATIENTS: Between July 2006 and July 2015 ACTH-tests were conducted in 365 children and adolescents (Age 1-20 y) suspected to have NCCAH. As a reference, results from subsequent gene sequencing of CYP21A2 was used. Inclusion criteria that were used were premature pubarche with accelerated bone age, hyperandrogenism, hirsutism, or menstrual irregularities. Receiver operating characteristics (ROC) were plotted. Evaluated test algorithms were composed around 17OHP measurements by radioimmunoassays. The most suitable test was identified by the greatest area under the curve (AUC). RESULTS: Among the 12 tested algorithms, the sum of 30 min and 60 min stimulated 17OHP values (sum17OHPstim) showed the highest AUC of 0.774 for identifying heterozygous and bi-allelic mutations. A cut-off of 10.1 µg/l was advisable. Bi-allelic mutations only were best identified calculating the difference between 30 min and basal 17OHP values (Δ17OHP30). A cut-off of 9.4 µg/l was most effective. CONCLUSION: Alternatively to the above mentioned cut-offs the difference of 60 min after stimulation to basal 17OHP (Δ17OHP60) can be used for the benefit of a combined test to identify both heterozygotes and bi-allelic patients. There are minimal decreases in sensitivity and specificity compared to an approach that applies two tests. However, it denotes a simpler approach in the clinical routine.

Bilateral microscopic Leydig cell ovarian tumors in the postmenopausal woman.

A 64-year-old postmenopausal female patient presented with approximately 5 years of intermittent spotting, progressive hirsutism and significantly increased libido and clitoral hypersensitivity with spontaneous orgasms multiple times a day beginning a few months prior. Initial hormone work-up revealed elevated total serum testosterone, androstenedione and 17-hydroxyprogesterone. Luteinising hormone, follicle stimulating hormone, estradiol, dehydroepiandrosterone-sulfate, thyroid stimulating hormone and prolactin were all within normal limits. Initial suspicions suggested an androgen-secreting tumour, likely in the ovary. The lesion was undetectable on transvaginal ultrasound and abdominal-pelvic CT scan. Laparoscopic bilateral salpingo-oophorectomy was performed to remove the likely source of excess androgens. Visible gross lesions were not observed intraoperatively; however, bilateral Leydig (hilus cell) tumours were confirmed by histopathology. Serum testosterone, androstenedione and 17-hydroxyprogesterone levels were normalised postoperatively within 2 weeks and 1 month, respectively.

Analytical bias of automated immunoassays for six serum steroid hormones assessed by LC-MS/MS.

INTRODUCTION: There is a growing amount of evidence showing the significant analytical bias of steroid hormone immunoassays, but large number of available immunoassays makes conduction of a single comprehensive study of this issue hardly feasible. Aim of this study was to assess the analytical bias of six heterogeneous immunoassays for serum aldosterone, cortisol, dehydroepiandrosterone sulphate (DHEAS), testosterone, 17-hydroxyprogesterone (OHP) and progesterone using the liquid chromatography coupled to the tandem mass spectrometry (LC-MS/MS). MATERIALS AND METHODS: This method comparison study included 49 serum samples. Testosterone, DHEAS, progesterone and cortisol immunoassays were performed on the Abbott Architect i2000SR or Alinity i analysers (Abbott Diagnostics, Chicago, USA). DiaSorin's Liaison (DiaSorin, Saluggia, Italy) and DIAsource's ETI-Max 3000 analysers (DIAsource ImmunoAssays, Louvain-La-Neuve, Belgium) were chosen for aldosterone and OHP immunoassay testing, respectively. All immunoassays were evaluated against the LC-MS/MS assay relying on the commercial kit (Chromsystems, Gräfelfing, Germany) and LCMS-8050 analyser (Shimadzu, Kyoto, Japan). Analytical biases were calculated and method comparison was conducted using weighted Deming regression analysis. RESULTS: Depending on the analyte and specific immunoassay, mean relative biases ranged from -31 to + 137%. Except for the cortisol, immunoassays were positively biased. For none of the selected steroids slope and intercept 95% confidence intervals simultaneously contained 0 and 1, respectively. CONCLUSIONS: Evaluated immunoassays failed to satisfy requirements for methods' comparability and produced significant analytical biases in respect to the LC-MS/MS assay, especially at low concentrations.

Changes in Adrenal Androgens and Steroidogenic Enzyme Activities From Ages 2, 4, to 6 Years: A Prospective Cohort Study.

CONTEXT: The levels of adrenal androgens are increased through the action of steroidogenic enzymes with morphological changes in the adrenal zona reticularis. OBJECTIVE: We investigated longitudinal changes in androgen levels and steroidogenic enzyme activities during early childhood. DESIGN AND PARTICIPANTS: From a prospective children's cohort, the Environment and Development of Children cohort, 114 boys and 86 girls with available blood samples from ages 2, 4, and 6 years were included. OUTCOME MEASUREMENTS: Serum concentrations of adrenal androgens using liquid chromatography-tandem mass spectrometry and steroidogenic enzyme activity calculated by the precursor/product ratio. RESULTS: During ages 2 to 4 years, 17,20-lyase and dehydroepiandrosterone (DHEA) sulfotransferase activities increased (P < 0.01 for both in boys). During ages 4 to 6 years, 17,20-lyase activity persistently increased, but 3ß-hydroxysteroid dehydrogenase (HSD) and 17ß-HSD activities decreased (P < 0.01 for all). Serum DHEA sulfate (DHEA-S) levels persistently increased from 2, 4, to 6 years, and DHEA, 17-hydroxyprogesterone, and androstenedione levels increased during ages 4 to 6 years (P < 0.01 for all). Serum DHEA-S levels during early childhood were associated with body mass index z-scores (P = 0.001 in only boys). CONCLUSION: This study supports in vivo human evidence of increased 17,20-lyase and DHEA sulfotransferase activities and decreased 3ß-HSD activity during early childhood.

Simultaneous quantitation of four androgens and 17-hydroxyprogesterone in polycystic ovarian syndrome patients by LC-MS/MS.

BACKGROUND: Due to the low concentration of androgens in women and the limitation of immunoassays, it remains a challenge to accurately determine the levels of serum androgens in polycystic ovary syndrome (PCOS) patients for clinical laboratories. In this report, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous quantitation of testosterone (T), androstenedione (A4), dehydroepiandrosterone sulfate (DHEAS), dihydrotestosterone (DHT), and 17-hydroxyprogesterone (17-OHP) that are associated with PCOS. METHODS: The serum samples were processed by protein precipitation and solid phase extraction before analysis with the in-house developed LC-MS/MS. The chromatographic separation was achieved with a C18 column, using a linear gradient elution with two mobile phases: 0.02% formic acid in water (phase A) and 0.1% formic acid in methanol (phase B). The separated analytes were detected by positive or negative electrospray ionization mode under multiple reaction monitoring (MRM). RESULTS: The assay for all the five analytes was linear, stable, with imprecision less than 9% and recoveries within ±10%. The lower limits of quantification were 0.05, 0.05, 5, 0.025, and 0.025 ng/mL for T, A4, DHEAS, DHT, and 17-OHP, respectively. In the receiver operating characteristic curve (ROC) analyses with the PCOS (n = 63) and healthy (n = 161) subjects, the AUC of the four-androgen combined was greater than that of any single androgen tested in PCOS diagnosis. CONCLUSIONS: The LC-MS/MS method for the four androgens and 17-OHP showed good performance for clinical implementation. More importantly, simultaneous quantitation of the four androgens provided better diagnostic power for PCOS.