Measurement Of 17-OHP Research Articles

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

ContextMeasurement 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.MethodsClinical 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 11β-hydroxylase deficiency (11βOHD), 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.ResultsA cascade logistic regression model, we named the “Steroidogenesis Score”, was able to discriminate the 3 most common CAH subtypes: 11βOHD, 17OHD, and 21OHD. In the perspective application cohort, the steroidogenesis score had a high diagnostic accuracy for all 3 subtypes, 11βOHD (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).ConclusionsThe 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.

Abnormal Newborn Screen During a Pandemic.

Abnormal Newborn Screen During a Pandemic.

First Morning Pregnanetriol and 17-Hydroxyprogesterone Correlated Significantly in 21-Hydroxylase Deficiency.

BackgroundBiochemically monitoring 21-hydroxylase deficiency (21-OHD) is challenging. Serum/blood 17-hydroxyprogesterone (17OHP) measurements are normally used for this purpose. Urinary pregnanetriol (PT), a urinary metabolite of 17OHP, may also be used. Based on auxological data, we previously reported that the optimal first morning PT value fell in the range of 2.2–3.3 mg/gCr (95% confidence interval of the mean) and 0.59-6.0 mg/gCr (10th – 90th percentile) for monitoring 21-OHD treatment. No report thus far has directly compared the first morning urinary PT value with the 17OHP value at various times during the day.ObjectiveTo explore the correlation between the first morning urinary PT value before glucocorticoid administration and the serum/blood 17OHP value at three time points, namely, before and two and four hours after glucocorticoid administration.DesignThis was a prospective study done at two children’s hospitals.MethodsIn total, 25 patients with 21-OHD aged 3-25 years were recruited. Their urinary PT levels and 17OHP levels were measured for three days within a total period of one week. The first morning PT value was collected on all three days. Dried blood spots and serum were used to measure 17OHP.ResultsThe range for the first morning PT value for all the samples (n=69) was 0.10-56.1 mg/gCr. A significant, positive correlation was found between the first morning PT and 17OHP values before medication (r=0.87, p<0.01), and weaker correlation was observed between the first morning PT and 17OHP values after medication.ConclusionsThe first morning PT correlated more significantly with 17OHP before the morning medication. Measuring the first morning PT value may be more practical and useful for monitoring 21-OHD biochemically.

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

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. 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. 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. 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). 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.

Is there an association between prostate-specific antigen and androgen levels in 46, XX patients with congenital adrenal hyperplasia?

Congenital adrenal hyperplasia (CAH), a genetic disease characterized by defective cortisol synthesis and excessive levels of sex hormones, can cause precocious puberty in both sexes in untreated individuals and virilization in female patients with a 46, XX karyotype. The female paraurethral (Skene's) gland has been reported as prostate analogous. Growth of prostate tissue is associated with androgen production; therefore, prostate-specific antigen (PSA) levels may represent a marker of virilization in 46, XX patients with CAH. To describe PSA levels in 46, XX patients and evaluate whether higher PSA levels are associated with androgenization and the severity of the disease. Sixty-six patients with CAH and a 46, XX karyotype were included, irrespective of age. Serum PSA, testosterone, 17-hydroxyprogesterone (17-OHP) and androstenedione levels were measured. Patients' age, age at diagnosis, forms of the disease, Prader classification, bone age assessment, sex of rearing, surgery, and the presence of clinical complications were obtained from their medical records. The mean age of patients was 11.45±10.74 years. Forty-three patients (65%) were diagnosed neonatally at a median of 0.08 years (mean 1.47±2.34 years), with registers of 17-OHP measurements (Guthrie test) being available in 51%. Testosterone, 17-OHP and androstenedione were significantly high. PSA was detectable in 25% of cases (levels >0.01ng/ml), with a mean of 0.03±0.09ng/ml, and only in patients over five years of age. A correlation was found between PSA and age (p<0.001), age at diagnosis (p=0.002), testosterone (p=0.001) and androstenedione (p=0.023). There was no correlation between PSA and the forms of CAH or Prader classification. A sub-analysis of the patients over five years of age in whom PSA was detectable also showed that there was a correlation between PSA (p<0.05) and age at analysis, age at diagnosis, testosterone and androstenedione levels. Limitations of this study include the small sample size due to the rareness of the disease, its retrospective nature, the absence of a control group, the fact that the sample was selected at two referral centers, which could have resulted in a selection bias, and the use of different reference values in the different laboratories conducting the PSA tests. PSA is detectable in 25% of 46, XX patients with CAH, only after five years of age. PSA level increases significantly with age, age at diagnosis, and testosterone and androstenedione levels, confirming a correlation between PSA levels and elevated androgen levels.

Measurement of serum 17-hydroxyprogesterone using isotope dilution liquid chromatography-tandem mass spectrometry candidate reference method and evaluation of the performance for three routine methods.

Accurate measurements of serum 17-hydroxyprogesterone (17OHP) are essential for diagnosis and treatment monitoring for congenital adrenal hyperplasia patients. The performance of serum 17OHP routine methods remains highly variable that calls for a candidate reference measurement procedure (cRMP) to improve the standardization of serum 17OHP measurements. Serum samples spiked with internal standards were extracted with a combination of solid-phase extraction and liquid-liquid extraction. The 17OHP was quantified by the isotope dilution coupled with liquid chromatography/tandem mass spectrometry (ID-LC/MS/MS) with electrospray ionization in positive ion mode. Nine structural analogs of 17OHP were evaluated for interferences. The precision and analytical recovery were assessed. Twenty native and 40 spiked serum for performance evaluation were measured by the cRMP and two clinical LC/MS routine methods. No apparent interferences were found with the 17OHP measurement. The within-run, between-run, and total precision for our method were 0.4-0.8%, 0.6-2.0%, and 1.0-2.1% for four pooled serum (2.46-102.72nmol/L), respectively. The recoveries of added 17OHP were 100.0-100.2%. For the performance of two LC/MS routine methods, they showed relative deviation ranges of-22.1 to 1.1% and-6.7 to 12.8%, respectively. We developed and validated a reliable serum 17OHP method using ID-LC/MS/MS. The desirable accuracy and precision of this method enable it to serve as a promising cRMP to improve the standardization for serum 17OHP routine measurements.

Congenital Adrenal Hyperplasia in Children: A Pilot Study of Steroid Hormones Expressed as Sex- and Age-Related Standard Deviation Scores

Introduction: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease predominantly caused by 21-hydroxylase deficiency. Clinical management in children includes glucocorticoid and often mineralocorticoid treatment alongside monitoring outcomes such as an­thro­po­metry, pubertal status, blood pressure, and biochemistry. Objective: The objective of this pilot study was to present the use of 17-hydroxyprogesterone (17-OHP) and androgen metabolites expressed as standard deviation (SD) scores rather than actual concentrations as a tool in the management of children with CAH as well as in research settings. Methods: The study was a retrospective, longitudinal study that took place in a single, tertiary center and included 38 children and adolescents aged 3–18 years with CAH due to 21-hydroxylase deficiency. Biochemical measurements of 17-OHP, androstenedione, dehydroepiandrosterone-sulphate (DHEAS), and testosterone using liquid chromatography-tandem mass spectrometry were expressed as SD scores, and outcomes such as genotype, height, bone maturation, blood pressure, and treatment doses were extracted from patient files. Results: The majority (86%) of CAH patients had 17-OHP measurements above +2 SD during standard hydrocortisone therapy, receiving an average daily hydrocortisone dose of 12.6 mg/m². Androstenedione concentrations were mostly within ±2 SD, whereas DHEAS values were below –2 SD in 47% of patients. Conclusions: Applying sex- and age-related SD scores to 17-OHP and androgen metabolite concentrations allows for monitoring of hydrocortisone treatment independent of age, sex, assay, and center. We propose that 17-OHP and androgen metabolites expressed as SD scores be implemented as a unifying tool that simplifies research and, in the future, also optimal management of treatment.

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

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. 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. 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). 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. 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.

Screening for Nonclassic Congenital Adrenal Hyperplasia in the Era of Liquid Chromatography-Tandem Mass Spectrometry.

ContextScreening for and diagnosing non classic congenital adrenal hyperplasia (NCCAH) uses serum 17-hydroxyprogesterone (17OHP) thresholds established from immunoassay data; however, a new liquid-chromatography tandem mass spectrometry (LC-MS/MS) method results in lower 17OHP values. The evolution of immunoassays is also challenging our diagnostic cut-off for glucocorticoid insufficiency and few data re-evaluate the utility of testing for glucocorticoid insufficiency in NCCAH.Objective(1) Evaluate the 17OHP threshold that predicts NCCAH in children using LC-MS/MS, and (2) determine the prevalence of glucocorticoid insufficiency in NCCAH.MethodsA retrospective chart review of pediatric patients who underwent ACTH stimulation tests with cortisol and 17OHP measurements from 2011 to 2018 for assessment of NCCAH. Other adrenal pathologies were excluded. A cortisol < 415 nmol/L defined glucocorticoid insufficiency. Published correlation data determined a 17OHP of 3.3 nmol/L by LC-MS/MS was equivalent to 6 nmol/L by immunoassay. Data analysis was by measures of diagnostic accuracy.ResultsOf 188 patients included, 23 (12%) had NCCAH (21/23 had genetic confirmation); the remaining 2 had peak 17OHP > 30 nmol/L. Baseline 17OHP ≥ 6 nmol/L most accurately screened for NCCAH—sensitivity and specificity 96%. Almost all genetically confirmed NCCAH (20/21) had peak 17OHP > 30 nmol/L; all subjects with other diagnoses peaked < 30 nmol/L. Glucocorticoid insufficiency was present in 55% with NCCAH.ConclusionsDespite the increased specificity of LC-MS/MS, a baseline 17OHP ≥ 6 nmol/L most accurately screened for NCCAH; this supports current practice guidelines. This threshold identified all with glucocorticoid insufficiency, notably prevalent in our cohort and for whom glucocorticoid stress dosing should be considered.

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

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

SAT-277 Re-Evaluation of the 17-Hydroxyprogesterone (17-OHP) Screening Threshold for Diagnosing Nonclassic Congenital Adrenal Hyperplasia (NCCAH) in the Era of Liquid Chromatography Tandem-Mass Spectrometry (LC-MS/MS)

Background: NCCAH is characterized by reduced 21-hydroxylase activity leading to elevations in adrenal androgens. It may be associated with an inadequate cortisol response to ACTH stimulation. Clinical diagnosis is problematic as signs and symptoms overlap with more common conditions such as premature adrenarche and polycystic ovarian syndrome. A screening 17-OHP >6 nmol/L (200 ng/dL) based on immunoassay prompts NCCAH diagnostic testing with an ACTH stimulation test. Peak 17-OHP <30 nmol/L (1000 ng/dL) post-ACTH excludes NCCAH. In 2011, we replaced immunoassay with LC-MS/MS for 17-OHP quantification which has greater specificity. A method comparison revealed a negative bias between LC-MS/MS and immunoassay measurements prompting us to evaluate the accuracy of the 17-OHP thresholds (1). Aims: To evaluate the 17-OHP thresholds that predict genetically-confirmed NCCAH. A secondary objective was to determine the prevalence of adrenal insufficiency (AI) in this population. Methods: A retrospective chart review was performed of clinical and genetic data for all patients <18 years who underwent ACTH stimulation tests with measurement of cortisol and 17-OHP from 2011 to 2018. NCCAH was genetically confirmed; other adrenal pathologies were excluded. AI was defined as peak cortisol < 500 nmol/L (18 µg/dL), measured by immunoassay. Using correlation data between immunoassay and LC-MS/MS, a new 17OHP threshold of 3.3 nmol/L (110 ng/dL) was calculated for the LC-MS/MS method and was considered equivalent to 6 nmol/L by immunoassay; similarly, 20 nmol/L (666 ng/dL) was considered equivalent to 30 nmol/L (1). Results: 188 patients met inclusion criteria. 23 (12%) had NCCAH of which 21/23 had genetic confirmation; the remaining 2 had peak 17-OHP >30 nmol/L by LC-MS/MS. Baseline 17-OHP > 6 nmol/L most accurately diagnosed NCCAH (sensitivity and specificity 96%) with no improvement using 17-OHP >3.3 nmol/L. 20/21 genetically confirmed NCCAH had peak 17-OHP >30 nmol/L. Of three with 17-OHP peak 20-30 nmol/L, one was a CAH carrier, one had other adrenal pathology, and another with unknown diagnosis. 87% with NCCAH had biochemical AI. Baseline 17-OHP >6 nmol/L predicted all with AI. Conclusion: Despite increased specificity of LC-MS/MS for steroid measurements, a baseline 17-OHP >6 nmol/L remained the most accurate predictor of NCCAH. This threshold also predicts all with AI, notably prevalent in our cohort. Analysis is limited by a small cohort, low NCCAH incidence and lack of genetic data in those presumed without NCCAH. However, this re-evaluation of screening and diagnostic thresholds is important in the era of evolving assays and these results support current practice guidelines. Reference: 1. Kyriakopoulou L. et al. Clin Biochem. 2013;46:642-651

Pain and Stress Response during Intravenous Access in Children with Congenital Adrenal Hyperplasia: Effects of EMLA and Nitrous Oxide Treatment.

Background Congenital adrenal hyperplasia (CAH) is an endocrine condition that requires regularly blood samples for optimal treatment. The management of CAH in children is complex when intravenous access is one of the most stressful procedures for children. The purpose of this pilot study was to investigate the effects of nitrous oxide inhalation (N2O) in combination with cutaneous application of local anesthetics (EMLA) for improving intravenous access in children with CAH. Method Ten children (7–14 years) were studied. The children received two intravenous procedures: one with EMLA and one with EMLA + N2O. The order of priority was randomized. The outcomes were the children's pain experience (0–10) and an evaluation of satisfaction (1–5) after the procedure. Heart rate, blood pressure, saturation, and analyses of 17-hydroxyprogesterone (17-OHP), norepinephrine, and glucose were analyzed. Results Higher pain scores, heart rate, and glucose levels were reported after EMLA, compared to EMLA + N2O, but 17-OHP levels remained unchanged. The children's satisfaction with the intravenous procedure was more positive for EMLA + N2O. Conclusions EMLA + N2O offers the possibility of improving the intravenous procedure for anxious children with CAH. Although the quality of care was better with N2O treatment, it was not possible to demonstrate that this is a prerequisite for valid 17-OHP measurements.

Monitoring steroid replacement therapy in children with congenital adrenal hyperplasia.

The objective of this study was to compare the analysis of 17-hydroxyprogesterone (17-OHP) by radio-immunoassay (RIA) in serum with analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) on dried blood spot samples (DBSS) for monitoring therapy in children with congenital adrenal hyperplasia (CAH), and to investigate differences in 17-OHP values during the day. Fourteen children (8 females), median age 4.2 (0.3-16.0) years, were studied. Serum samples and DBSS were drawn before hydrocortisone dosing. 17-OHP by LC-MS/MS in DBSS were highly correlated to 17-OHP by RIA in serum, r=0.956, p<0.01. A total of 26 three-time-point series were investigated. Using only the afternoon 17-OHP values to determine the hydrocortisone doses would have led to overdosing seven times and underdosing six times. Good agreement was demonstrated between 17-OHP determination by RIA in serum and LC-MS/MS on DBSS. Multiple 17-OHP measurements per day are required to ensure sufficient hydrocortisone dose adjustment.

The diagnosis of nonclassic congenital adrenal hyperplasia due to 21-hydroxylase deficiency, based on serum basal or post-ACTH stimulation 17-hydroxyprogesterone, can lead to false-positive diagnosis.

As nonclassic congenital adrenal hyperplasia (NCCAH) needs to be taken into account in women with hyperandrogenism, we aimed to assess whether the recommended level of poststimulated 17OHP ≥30 nmol/l confirms NCCAH. Forty, consecutive women with biochemical and/or clinical hyperandrogenism (aged 25·4, 18-38) suspected of having NCCAH were recruited to the study. In patients with 17OHP level between 5·1 and 29·9 nmol/l an ACTH stimulation test was performed. In patients with basal or poststimulated 17OHP ≥30 nmol/l, twenty-four-hour urinary steroid profile (USP) analysis was performed and CYP21A2 mutation was assessed. In selected patients with poststimulated 17OHP <30 nmol/l USP was also performed. The group was divided into two subgroups with basal or poststimulated 17OHP ≥30 nmol/l (group A) and with poststimulated 17OHP <30 nmol/l (group B). Among 40 patients, basal or poststimulated 17OHP ≥30 nmol/l was found in 21, but NCCAH was confirmed by USP followed by genetic testing only in 5 (24%). Four patients were diagnosed as heterozygotes, and in twelve, no CYP21A2 mutation was detected. The diagnosis of NCCAH based only on serum 17OHP measurements (basal or poststimulated) may lead to false-positive diagnosis when performed by immunoassay with a cut-off value of ≥30 nmol/l. The definitive diagnosis can be established based on USP and/or genetic testing.

LC-MS/MS improves screening towards 21-hydroxylase deficiency

Basal serum 17OHP measurement remains the first screening step for nonclassic congenital adrenal hyperplasia (NCCAH) and the accuracy of the test is of high value. The aim of this study was to compare the accuracy of immunoassays to LC-MS/MS in the assessment of serum 17OHP and androgens concentration in women with hyperandrogenism and controls.17OHP, total testosterone, androstendione and DHEA-S were measured in 39 women with clinically and/or biochemically evident hyperandrogenism and in 29 age-matched controls without clinical hyperandrogenism. 17OHP and androgens were measured by immunoassays and by LC-MS/MS. In patients group median 17OHP level measured by immunoassays was significantly higher compared to LC-MS/MS (5.49 nmol/l-ELISA NovaTec® and 3.57 nmol/l-ELISA DRG® versus 1.56 nmol/l-LC-MS/MS p < 0.0001) as well as in the control group (2.58 nmol/l-ELISA DRG® versus 1.14 nmol/l-LC-MS/MS p < 0.0001). Additional, unnecessary diagnostic procedures explaining elevated 17OHP level were undertaken in 85% of patients when NovaTec® test was used, in 50% when ELISA DRG® and in none when LC-MS/MS method was applied. Total testosterone, androstendione and DHEA-S concentrations in the patients and the controls assessed by the immunoassays were also significantly higher compared to LC-MS/MS. LC-MS/MS is more reliable diagnostic tool in the measurement of serum 17OHP and androgens concentrations compared to immunoassays in women with hyperandrogenism.

Ten-year evaluation of a Neonatal Screening Program for congenital adrenal hyperplasia.

Evaluate the Neonatal Screening Program (NSP) for congenital adrenal hyperplasia (CAH) of the Department of Health of the State of Santa Catarina (Secretaria de Estado da Saúde de Santa Catarina, SES/SC), and provide information to improve the program. Descriptive, retrospective study of 748,395 children screened between January 2001 and December 2010. We analyzed the coverage of the NSP-SES/SC prevalence of CAH, child's age when the first sample for 17-hydroxyprogesterone (17OHP) measurement was collected, levels of 17OHP, mean age at treatment onset and main clinical manifestations. The NSP-SES/SC covered 89% of the live newborns in the State. It diagnosed 50 cases of CAH, yielding an incidence of 1:14,967. Mean age at collection of the first sample was 7.3 days and mean level of 17OHP was 152.9 ng/mL. The most frequent manifestations were virilized genitalia with nonpalpable gonads, clitoromegaly and genital hyperpigmentation. In three girls, the genre established at birth was incorrect. The salt-wasting form was present in 74% of the cases. There was no occurrence of shock or death. Mean age at treatment onset in the salt-wasting form was 17.4 days compared with 54.9 days in those without the salt-wasting form of the disease. All children were treated with hydrocortisone, and those with salt-wasting CAH were also treated with fludrocortisone. The incidence of CAH was 1 case to 14,967 live newborns. Collection of the first sample occurred outside the recommended time, resulting in delays in treatment onset.

17-Hydroxyprogesterone in children, adolescents and adults

17-Hydroxyprogesterone (17-OHP) is an intermediate steroid in the adrenal biosynthetic pathway from cholesterol to cortisol and is the substrate for steroid 21-hydroxylase. An inherited deficiency of 21-hydroxylase leads to greatly increased serum concentrations of 17-OHP, while the absence of cortisol synthesis causes an increase in adrenocorticotrophic hormone. The classical congenital adrenal hyperplasia (CAH) presents usually with virilisation of a girl at birth. Affected boys and girls can have renal salt loss within a few days if aldosterone production is also compromised. Diagnosis can be delayed in boys. A non-classical form of congenital adrenal hyperplasia (NC-CAH) presents later in life usually with androgen excess. Moderately raised or normal 17-OHP concentrations can be seen basally but, if normal and clinical suspicion is high, an ACTH stimulation test will show 17-OHP concentrations (typically >30 nmol/L) above the normal response. NC-CAH is more likely to be detected clinically in females and may be asymptomatic particularly in males until families are investigated. The prevalence of NC-CAH in women with androgen excess can be up to 9% according to ethnic background and genotype. Mutations in the 21-hydroxylase genes in NC-CAH can be found that have less deleterious effects on enzyme activity. Other less-common defects in enzymes of cortisol synthesis can be associated with moderately elevated 17-OHP. Precocious puberty, acne, hirsutism and subfertility are the commonest features of hyperandrogenism. 17-OHP is a diagnostic marker for CAH but opinions differ on the role of 17OHP or androstenedione in monitoring treatment with renin in the salt losing form. This review considers the utility of 17-OHP measurements in children, adolescents and adults.

Serum Concentration of 17α-Hydroxyprogesterone in Children from Birth to Adolescence

Background: Reference intervals (RI) of serum 17α- hydroxyprogesterone (17OHP) are useful to confirm congenital adrenal hyperplasia (CAH) in neonates with abnormal screening results and nonclassical forms of CAH in symptomatic children. We aimed to establish serum 17OHP RI in normal children and adolescents using a current 17OHP radioimmunoassay (RIA). Methods: Serum 17OHP was measured via a current RIA (Diasource) in children, i.e. 111 infants aged <1 year [before (NE-17OHP) and after extraction (E-17OHP)] and 216 children aged 1-17 years. Forty NE serum samples from subjects aged >1 year, covering the whole analytical range, were simultaneously measured to compare 17OHP RIA from Diagnostic System Laboratories (DSL) (withdrawn) and Diasource by Passing Bablok linear regression and ratio plot. The equation obtained was used to correct our own previous RI (DSL RIA) for infancy for the Diasource RIA. Samples from infants aged <1 year were used to verify the calculated RI with evaluator protocol C28-A3. The influence of age, gender, and Tanner's classification (T) was assessed in children aged >1 year by ANOVA. Results: E-17OHP as measured via the Diasource RIA was significantly lower than NE-17OHP in infants aged <1 year (p < 0.0001). The 17OHP measurement from the Diasource RIA was negatively biased compared to the value obtained using the DSL RIA (Diasource (ng/ml) = 0.85 DSL (ng/ml) -0.32 ng/ml, r = 0.952). Most infants (93%) had age- and gender-adjusted NE-17OHP and E-17OHP levels within the recalculated RI. Serum 17OHP significantly increased throughout prepuberty (p < 0.001). Sexual dimorphism was only observed at T IV-V. Conclusion: When evaluating 17OHP during childhood, we recommend taking into account the extraction procedure in neonates, the method used, age, and the Tanner's stage.

The comparative assessment of the effectiveness of immunoanalysis and tandem mass spectrometry applied for re-testing the children with suspected congenital adrenal cortical hyperplasia

Congenital adrenal cortical hyperplasia (CAH) is one of the commonest endocrine pathologies in the children. Screening newborn infants for CAH is currently based on the measurement of 17-hydroxyprogesterone (17-OHP) levels in blood spots using the immunoenzymatic assay. However, this techniques is known to suffer relatively low specificity accounting for the high percentage of false-positive results. We compared two 17-OHP measurement tecniques, immunoenzymatic assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS) employed for the additional examination of the children in whom screening for CAH revealed the enhanced blood 17-OHP levels. The study included 50 patients at the age from 7 days to 1.5 months born at different gestational ages. 17-OHP levels were measured in all of them using the immunoenzymatic assay and LC-MS/MS. The results of the study indicate that the latter technique should be regarded as the method of choice for the confirmation of diagnosis of CAH. The absence of clinical manifestations of 21-hydroxylase deficiency in the patients in whom the immunoenzymatic assay reveals the enhanced levels of 17-OHP whereas the LC-MS/MS method demonstrates the normal or only slightly elevated concentrations of 17-OHP allows to exclude diagnosis of CAH. The gestational age of the infant is the key factor in the choice of the upper threshold level of 17-OHP for the purpose of screening; the child's body weight is of low diagnostic value. The 17-OHP levels measured by LC-MS/MS in the boys may be slightly higher than the reference values compared with the girls.

Assessment of theca cell function: a prerequisite to androgen or luteinizing hormone supplementation in poor responders

Poor responders are a heterogeneous population, with some patients displaying a diminished ovarian reserve and others a poor ovarian reserve with preserved granulosa cell function. Androgen and LH/hCG supplementation has been advocated for poor responders, mainly those >40 years old. Although androgens synergistically act with FSH to support folliculogenesis, and ovarian androgen secretion declines with age, there is still no evidence that androgen therapy is actually effective to improve ovarian FSH sensitivity. The main reason seems to be that theca cell function has not been appropriately assessed in patients at risk of poor response. The definition of theca insufficiency is hampered by methodologic shortcomings in routine bioassays. Provocative tests for theca cells might help to identify those patients who could benefit from androgen supplementation. The lack of data regarding theca cells in these patients might contribute to explaining the absence of evidence for a positive effect of androgen therapy.