17-OHP Concentrations Research Articles

6437 Analysis of 17-OH-Progesterone (17OHP) and Androstenedione (A4) Profiles To Rationalise Biochemical Monitoring Of CAH Patients

Abstract Disclosure: N.R. Lawrence: None. N.P. Krone: None. J.F. Dawson: None. Z. Lang: None. W. Arlt: Advisory Board Member; Self; Diurnal. A. Brac de la Perriere: Research Investigator; Self; Diurnal. A.L. Hirschberg: Research Investigator; Self; Diurnal. A. Juul: Research Investigator; Self; Diurnal. D.P. Merke: Research Investigator; Self; Diurnal. J.D. Newell-Price: Research Investigator; Self; Diurnal. A. Prete: Research Investigator; Self; Diurnal. A.E. Taylor: None. A. Rees: Research Investigator; Self; Diurnal. N. Reisch: Research Investigator; Self; Diurnal. M. Stikkelbroeck: Research Investigator; Self; Diurnal. P.A. Touraine: Research Investigator; Self; Diurnal. G. Collins: None. R.J. Ross: Consulting Fee; Self; Diurnal. Background: There is no consensus on how to monitor biochemical control of CAH as excess ACTH overnight is common on standard therapy. Modified-release hydrocortisone (MRHC) gives potential for improved disease control and the need to understand this impact on disease monitoring.Aim: To define the relationship between 17OHP and A4, in healthy controls and CAH patients. Methods: Analysis of 24-hour 2-hourly 17OHP and A4 endocrine profiles in patients randomised to either standard treatment or MRHC (n=122, mean age 36.3 (SD 11.6) years, 78 female, 44 male) and a population of healthy controls (n=20, age 30.0 (12.3), 13 male, 7 female). Markers were regressed, cross correlated and assessed by Bayesian Change Point analysis using R. Results: Regressing the natural logarithms of 17OHP on A4, Bayesian multiple change point analysis converged on a stable changepoint equivalent to 17OHP of 4.5 nmol/L 149 ng/dl (95%, CI:4.2 to 4.7 nmol/L, Rhat < 1.008). Below the 17OHP change point A4 is proportionally lower than above, with no evidence of difference between sexes or steroid preparation. The relationship between 17OHP and A4 was different for healthy subjects, who for similar levels of A4 have lower levels of 17OHP than CAH patients in nmol/L (ng/dl): A4 of 1 (29) and 10 (286) associated with 17OHP 1.5 (50) and 8.8 (291) in healthy subjects’ vs 3.3 (109) and 71.2 (2353) in CAH patients, respectively. There was no consistent rhythm in either marker in healthy patients, shown by an autocorrelation function (ACF) that falls quickly to zero. ACF approaches zero more quickly in CAH patients on MRHC than those on standard therapy who more commonly exhibit a diurnal rhythm with an 0700hrs peak. There was no lag between markers, with cross-correlation function plots symmetrical. Conclusions: The relationship between 17OHP and A4 is different in CAH than healthy controls. In CAH, high A4 suggests poor control but a low A4 may still be associated with a raised 17OHP. In CAH patients, 17OHP concentrations within the healthy subject range are associated with A4 below levels seen in healthy subjects. When elevated in CAH patients, A4 and 17OHP rise and fall together with no lag; thus, A4 does not give more information on control over time. On MRHC the biomarkers show a reduced diurnal rhythm; therefore, individual measurements taken in clinic are more likely to reflect the daily average than measurements taken when on standard therapy. Presentation: 6/1/2024

8665 Female Fertility In Patients With Classic And Non-Classic Congenital Adrenal Hyperplasia Under Modified-Release Hydrocortisone (MR-HC)

Abstract Disclosure: L. Tschaidse: None. H.F. Nowotny: None. M. Auer: None. C. Lottspeich: None. M. Bidlingmaier: None. J. Hawley: None. B.G. Keevil: None. N. Reisch: None. Background: Female patients with classic and non-classic (NC) CAH struggle with fertility, due to androgen excess, elevated progesterone and 17-hydroxyprogesterone (17OHP) levels, causing anovulation, impairment of endometrial development and menstrual irregularities. The typical approach to treating infertility often includes an increase in glucocorticoid (GC) dose. However, this poses a potential risk of side effects. Limited observational data on modified-release hydrocortisone (MR-HC), which better mimics the physiological diurnal rhythm of cortisol secretion, indicates improved fertility in female and male patients with classic CAH. Therefore, the aim of this study was to investigate fertility in women with classic and non-classic CAH before and after switching to MR-HC. Methods: 21 adult female patients with CAH (14 classic; 5 NC) with a median (range) age of 33.0 (30.0) years, premenopausal and without hormonal contraception, were enrolled in this prospective, observational, single-centre study. The data collection took place before (conventional GC preparation) and after dose-equivalent switching the medication to MR-HC. In addition to clinical parameters such as menstrual cycle and desire to conceive, the collection of morning serum and daily saliva profiles was conducted, measuring progesterone, 17OHP, androstenedione (A4), testosterone, 11-ketotestosterone (11KT) as well as 11-hydroxyandrostenedione (11OHA4). Results: Before study inclusion, 4/21 patients had an irregular cycle, 5/21 were amenorrhoeic and 10/21 patients had a desire to have children. There was no difference in median (range) hydrocortisone dose equivalent before and after switching to MR-HC (25.0 (50.0) vs. 25.0 (30.0), p=.441). Median (range) morning serum concentrations between 8-10 am were not different before and after switch to MR-HC for progesterone (5.9 (55.5) vs. 5.0 (58.8), p=.823), 17OHP (5.9 (362.1) vs. 5.7 (73.7), p=.280), A4 (2.9 (25.6) vs. 2.5 (6.9), p=.106), testosterone (0.8 (3.5) vs. 0.7 (1.5), p=.063), 11KT (0.4 (7.4) vs. 0.2 (2.6), p=.242) and 11OHA4 (0.8 (12.9) vs. 0.9 (10.4), p=.231). Concerning saliva day profiles, we found a significantly lower median (range) 17OHP level in early morning saliva (43.0 (526.9) vs. 19.4 (264.3), p=.049). 5/9 patients with menstrual irregularities reported menstrual regularisation within and 5/10 patients with a desire to have children became pregnant after switching to MR-HC. Conclusion: Our preliminary data show clinical improvement in fecundity and fertility under MR-HC in both women with classic and NC CAH without a dose increase of glucocorticoids. Under MR-HC replacement lower 17OHP concentrations in early morning saliva reflect better hormonal control over night contributing to ovulatory cycle regulation and improved fertility. Presentation: 6/1/2024

7966 Long-Term Safety and Efficacy of a Hydrocortisone Oral Granules in a Young Child with Congenital Adrenal Hypertrophy

Abstract Disclosure: M. Bertossi: None. C. Lowe: None. Q.L. Van Meter: Consulting Fee; Self; Eton Pharmaceuticals. Speaker; Self; Eton Pharmaceuticals. Introduction: Congenital adrenal hyperplasia (CAH) is the most common cause of pediatric adrenal insufficiency. Children diagnosed with CAH need exogenous glucocorticoid therapy throughout their lifetime. Because low dose hydrocortisone (HC) tablets are not available, patients and their caregivers must either split/crush hydrocortisone tablets or rely on HC that has been compounded from adult medications. In late 2020, a pediatric specific HC formulation, an oral granule, became available with doses as low as 0.5 mg. There are minimal data concerning the long-term use of this novel HC formulation. Subject and Method: This case study is a girl (DOB 11/2016) with CAH who has been administered the HC oral granule formulation for over 3 years. Results: The subject is a 7-year-old female who was diagnosed at birth with CAH. At an outside institution, she administered 6.25 mg HC, divided into three doses of 2.5 mg in the morning, 1.25 mg in the afternoon, and 2.5 mg in the evening (2.5/1.25/2.5; 9 mg/m2). She was also administered 0.15 mg fludrocortisone qd. Her dose was increased to a total dose of 7.5 mg, administered 2.5 mg three times/day (10 mg/m2) because of a high 17-OHP concentration, 1048 mg/dL. Other hormone levels were unremarkable. Her 17-OHP levels increased to 2662 mg/dL after 3 months of the increased dose. Because of the development of Cushing’s symptoms, she was switched to the 7.0 mg HC granules (2.5/2.0/2.5; 9.5 mg/m2 total dose). After the switch to the HC granules, her 17-OHP levels was 293 ng/dL. Because of rising 17-OHP levels, her dose needed to be increased twice over the past 2 yrs. Her current dose is 12 mg HC granules (4/4/4; 11 mg/m2 total dose), and her three most recent 17-OHP levels were 73, 101, and 89 ng/dL, respectively. She is also taking 0.2 mg fludrocortisone po, q am. Throughout HC granule therapy, she had no incidence of adrenal crisis. Her most recent calculated bone age was 8 yr 10 mo, with her chronological age was 6 yr, 8 mo with no evidence of virilization. Growth curves suggest that she is neither under- nor over-dosed on her HC therapy. Conclusion: This case may be the longest, continuous administration of the novel HC granules in US for a child with CAH. The results show the potential to better titrate HC dosing, especially with the narrow therapeutic window of HC in children with CAH. Her daily dose was adjusted by titrating in small increments to result in good outcomes, such as better hormonal control, the lack of virilization and continuous growth. Further evaluations of the long-term use of HC granules are needed to occur to establish the long-term risks and benefits of this novel HC granule. Presentation: 6/3/2024

8723 Glucose Metabolism Disturbances in Non-Classic Adrenal Hyperplasia (NCAH)

Abstract Disclosure: B. Domagała: None. A. Gamrat: None. A. Skalniak: None. E. Przybylik-Mazurek(†): None. M. Trofimiuk-Muldner: None. A. Hubalewska-Dydejczyk: None. Introduction: There is data reporting an increased risk of cardiovascular and metabolic complications in patients with NCAH. This is frequently attributed to glucocorticoid (over)use or misdosing. It seems that long-term exposure to increased androgens concentrations may also itself lead to diminished insulin sensitivity and increased risk of prediabetes and diabetes. Aim: The study aimed to assess the link between NCAH diagnosis and glucose metabolism disturbances. Material and methods: The study included 327 subjects (315 females and twelve males, median age 25 years) referred for a cosyntropin stimulation test due to the suspected NCAH. Serum 17-OH-progesterone (17-OHP) was assessed with an ELISA assay. The diagnosis of NCAH was confirmed if the initial or stimulated 17-OHP concentration exceeded 10.0 ng/mL. None of the tested subjects was treated with glucocorticoids. A history of preexisting prediabetes or diabetes was recorded. Anthropometric measurements were obtained to calculate body mass index (BMI). Fasting glucose was measured in each subject. Mann-Whitney U and Yates’ χ2 tests were used in statistical analyses, with p<0.05 as the cut-off value of statistical significance. Results: 62 patients (60 females, two males; median age 26 years) were diagnosed with NCAH; the rest of the tested subjects were considered the control group. Preexisting type 2 diabetes was recorded in ten subjects in the entire study group (6 of them were diagnosed with NCAH). There was no significant difference in the fasting glucose levels (p=0.08) and BMI (p=0.32) between subjects with and without NCAH diagnosis (median fasting glucose: 4.96 mmol/L and 4.86 mmol/L, respectively; median BMI: 23.82 kg/m2 and 23.26 kg/m2, respectively). Diabetes was significantly more frequent in patients with NCAH diagnosis (p = 0.004; power: 0.74). The median 17-OHP was higher in patients with diabetes compared to those without it at both 30 (12.25 ng/mL vs 3.25 ng/mL; p=0.003) and 60 minutes (14.93 ng/mL vs 3.95 ng/mL; p=0.046) after stimulation. Conclusions: NCAH seems to be linked to increased diabetes risk, even in glucocorticoid-naïve patients. It seems advisable to screen NCAH patients for glucose metabolism disorders. Presentation: 6/1/2024

A-062 Determination Neonatal 17-Hydroxyprogesterone Cutoff Levels for Congenital Adrenal Hyperplasia in Korea: 3 years' experience

Abstract Background Newborn screening for congenital adrenal hyperplasia (CAH) is a crucial practice with significant global adoption. While it offers substantial benefits, it also presents challenges due to high false positive rates, which can lead to mental strain for families and unnecessary hospital visits. Our research delved into the impact of birth weight (BW), gestational age (GA), and GA combined with sampling time on 17-hydroxyprogesterone (17-OHP) concentration. Specifically, we aimed to establish appropriate 17-OHP cutoff values for preterm, low birth weight weight (LBW), and sick newborns. Methods A total of 2,429 newborns born between October 2020 and January 2024 were screened for CAH. Samples were collected from neonates on a filter paper with a heel prick method. The 17-OHP concentration was measured by AutoDELFIA Neonatal 17-hydroxyprogesterone kit (PerkinElmer, Turku, Finland). This assay is a solid phase, time-resolved fluoroimmunoassay based on the competitive reaction between europium labeled 17-OHP and sample 17-OHP for a limited amount of binding sites on 17-OHP specific polyclonal antibodies. The 99.5 percentile was applied to the cutoff of 17-OHP for CAH. Results 17-OHP concentrations in newborns with lower BW and GA were significantly higher than those in normal BW and term newborns. 17-OHP concentration showed a statistically significant negative correlation with BW (r = -0.512, p < 0.001) and GA (r = -0.578, p < 0.001). Not only the full-term subgroup, but also preterm subgroups had a same tendency of 17-OHP concentration decrease with increasing sampling time. Application of newly establishing 99.5 percentile cutoff criteria significantly reduced recall rates to 1.02%, 0.81%, and 1.45% according to each criterion of BW, GA, and GA with sampling time, respectively. Conclusions This research introduces novel 17-OHP cutoff values for preterm, low birth weight (LBW), and sick newborns in the context of congenital adrenal hyperplasia (CAH). We analyzed the distribution of these cutoff values, considering factors such as birth weight, gestational age, and the timing of sample collection. Notably, applying the newly established cutoff criteria led to a significant reduction in false positive rates. Our laboratory’s data can serve as a valuable reference for other institutions aiming to set appropriate cutoffs, thereby minimizing unnecessary follow-up tests and enhancing the efficiency of CAH screening. As we continue to accumulate additional data, further reserch in this field remains promising.

Severe adrenal insufficiency in six neonates with normal newborn screening for CAH.

Newborn screening (NBS) reduces the risk of mortality in congenital adrenal hyperplasia (CAH), mainly due to the salt-wasting form of 21-hydroxylase deficiency. There is limited knowledge regarding the results of NBS in non-CAH primary adrenal insufficiency (non-CAH PAI). Clinical and NBS for CAH data of neonates who were diagnosed with non-CAH PAI between January and December 2022 were examined. Patients (n = 6, 4 females) were presented with severe hyperpigmentation (n = 6), hypoglycemia (n = 4), hyponatremia (n = 3), hyperkalemia (n = 1), respiratory distress syndrome (n = 1) between 3rd hour to 2 months of life. All had normal NBS results. The median first-tier 17-hydroxyprogesterone (17OHP) concentration in NBS for CAH was 0.14 ng/mL (range; 0.05-0.85). Molecular studies revealed biallelic mutations in the MC2R (n = 4; 3 homozygous, 1 compound heterozygous), MRAP (n = 1) and STAR (n = 1) genes. Glucocorticoid with or without mineralocorticoid replacement was initiated once the diagnosis ofnon-CAH PAI was established. Neonates with non-CAH PAI have always normal NBS due to persistently low 17OHP, even when these newborn infants are severely symptomatic for adrenal insufficiency. Clinicians should be alert for signs of adrenal insufficiency in neonates, even if the patient has a 'normal' screening for CAH, so as not to delay diagnosis and treatment. This fact should be kept in mind particularly in countries where these conditions are more common than elsewhere.

Model-Informed Target Morning 17α-Hydroxyprogesterone Concentrations in Dried Blood Spots for Pediatric Congenital Adrenal Hyperplasia Patients

Monitoring cortisol replacement therapy in congenital adrenal hyperplasia (CAH) patients is vital to avoid serious adverse events such as adrenal crises due to cortisol underexposure or metabolic consequences due to cortisol overexposure. The less invasive dried blood spot (DBS) sampling is an advantageous alternative to traditional plasma sampling, especially in pediatric patients. However, target concentrations for important disease biomarkers such as 17α-hydroxyprogesterone (17-OHP) are unknown using DBS. Therefore, a modeling and simulation framework, including a pharmacokinetic/pharmacodynamic model linking plasma cortisol concentrations to DBS 17-OHP concentrations, was used to derive a target morning DBS 17-OHP concentration range of 2-8 nmol/L in pediatric CAH patients. Since either capillary or venous DBS sampling is becoming more common in the clinics, the clinical applicability of this work was shown by demonstrating the comparability of capillary and venous cortisol and 17-OHP concentrations collected by DBS sampling, using a Bland-Altman and Passing-Bablok analysis. The derived target morning DBS 17-OHP concentration range is a first step towards providing improved therapy monitoring using DBS sampling and adjusting hydrocortisone (synthetic cortisol) dosing in children with CAH. In the future, this framework can be used to assess further research questions, e.g., target replacement ranges for the entire day.

Establishing 17-Hydroxyprogesterone Cutoff Values for Congenital Adrenal Hyperplasia in Preterm, Low Birth Weight, and Sick Newborns.

Newborn screening for congenital adrenal hyperplasia (CAH) has benefits with a high adoption rate worldwide. It also has problems of high false positives, which can cause stress to the patient's family with economic losses and unnecessary visits of newborns to hospitals. Therefore, we investigated the influence of birth weight (BW), gestational age (GA), and GA with sampling time on 17-hydroxyprogesterone (17-OHP) concentration and attempted to establish the 17-OHP cutoff values in preterm, low birth weight (LBW), and sick newborns. Newborns (n=1,071) born between October 2020 and January 2022 were screened for CAH. Samples from neonates were collected on filter paper with the heel prick method. 17-OHP concentration was measured by time-resolved immunofluorescence with an AutoDELFIA Neonatal 17-hydroxyprogesteron kit and grouped in relation to BW, GA, and GA with sampling time. The median age of newborns at neonatal sample collection was 6 days. 17-OHP concentration showed a statistically significant negative correlation with BW (r=-0.488, p<0.001) and GA (r=-0.560, p<0.001). Full-term and preterm subgroups had a similar decreasing tendency of 17-OHP concentration with increasing sampling time. Application of newly establishing cutoff criteria significantly reduced recall rates to 1.16%, 0.9%, and 1.75% according to each criterion of BW, GA, and GA with sampling time, respectively. This study presents new 17-OHP cutoff values for preterm, LBW, and sick newborns. These data in our laboratory can be used as a reference by other laboratories for establishing new cutoff criteria to help lower the high recall rate and reduce unnecessary follow-up tests.

Modified-release hydrocortisone is associated with lower plasma renin activity in patients with salt-wasting congenital adrenal hyperplasia.

Poorly controlled salt-wasting (SW) congenital adrenal hyperplasia (CAH) patients often require high 9α-fluorocortisol doses as they show high levels of 17-hydroxyprogesterone (17OHP), which is a mineralocorticoid (MC)-receptor antagonist. We investigated the renin-angiotensin-aldosterone system in patients with SW-CAH receiving twice daily modified-release hydrocortisone (MR-HC, Efmody) compared with standard glucocorticoid (GC) therapy. Data were analyzed from the 6-month, phase 3 study of MR-HC (n = 42) versus standard GC therapy (n = 41). MC replacement therapy remained unchanged throughout the study. Blood pressure, serum potassium, serum sodium, plasma renin activity (PRA), and serum 17OHP and androstenedione concentrations were analyzed at baseline, 4, 12, and 24 weeks. The median serum 17OHP in the morning was significantly lower on MR-HC compared with standard GC at 24 weeks (2.5 nmol L-1 (IQR 8.3) versus 10.5 nmol L-1 (IQR 55.2), P = .001). PRA decreased significantly from baseline to 24 weeks in patients on MR-HC (0.83 ng L-1 s-1 (IQR 1.0) to 0.48 ng L-1 s-1 (IQR 0.61), P = .012) but not in patients on standard GC (0.53 ng L-1 s-1 (IQR 0.66) to 0.52 ng L-1 s-1 (IQR 0.78), P = .613). Serum sodium concentrations increased from baseline to 24 weeks in patients on MR-HC (138.8 ± 1.9 mmol L-1 to 139.3 ± 1.8 mmol L-1, P = .047), but remained unchanged on standard GC (139.8 ± 1.6 mmol L-1 to 139.3 ± 1.9 mmol L-1, P = .135). No significant changes were seen in systolic and diastolic blood pressure and serum potassium levels. 6 months of MR-HC therapy decreased PRA and increased sodium levels indicating a greater agonist action of the 9α-fluorocortisol dose, which may be due to the decreased levels of the MC-receptor antagonist 17OHP.

Multiple 17-OHP Cutoff Co-Variates Fail to Improve 21-Hydroxylase Deficiency Screening Accuracy.

To improve the positive predictive value (PPV) of newborn screening for 21-hydroxylase deficiency (21OHD), co-variates have been used to modify 17-hydroxyprogesterone (17OHP) cutoffs. The objective of this study is to evaluate whether 17OHP screening cutoffs adjusted for both collection time (CT) and birth weight (BW) improved the sensitivity and PPV of 21OHD screening. Unaffected newborn screening samples were stratified based on BW and CT to establish 17OHP concentration cutoffs at the 95th and 99th percentile. These cutoffs were applied to a cohort of confirmed cases of 21OHD to determine the sensitivity and PPV of the modified screening parameters. 17OHP cutoffs at the 99th percentile, adjusted for BW and CT, had a sensitivity of 96.3% and a specificity of 98.9%, but a relatively low PPV (0.130) for the identification of 21OHD and did not detect all cases. Use of the 95th percentile further increased sensitivity to 98.1% but resulted in a notably lower PPV (0.027). Alternative approaches that do not rely exclusively on 17OHP are needed to improve newborn screening accuracy for 21OHD.

Sex steroid profile during oocyte development and maturation in the intertidal worm Marphysa madrasi (Polychaeta: Eunicidae) from the east coast of India

Marphysa madrasi is a commercially valuable maturation diet in crustacean aquaculture. This study presents the first detailed investigation of oogenesis in the intertidal polychaete worm M. madrasi and reports the steroid profile during oocyte growth and development. Oogenesis is extraovarian type I, originating from coelomic epithelial cells, with four stages of development - primary growth, early vitellogenic, late vitellogenic, and maturation. The primary growth phase contains oogonial cells and previtellogenic oocyte clusters in the early, mid, and late stages of development form a dispersed ovary attached to blood vessels. The late previtellogenic oocytes detach from the ovary at the onset of vitellogenesis. The detached oocytes complete vitellogenesis and final maturation in the coelomic fluid as solitary free-floating cells without any connection with follicle cells. The worms display asynchronous reproduction with a heterogeneous population of developing oocytes. Steroid extracts from the polychaete homogenates in different stages of oogenesis were identified by HPLC and confirmed by LC-MS/MS. In M. madrasi, two vertebrate-type steroids, pregnenolone (P5) and 17α-hydroxyprogesterone (17-OHP) were detected and quantified. The P5 levels were low in immature worms but increased significantly by ∼ 8.3-fold in the previtellogenic stage and peaked during oocyte maturation. 17-OHP levels were low in immature worms but gradually increase as the oogenesis progress to the primary growth and early vitellogenic phase, with a significant increase (p < 0.001) during the late vitellogenic phase. Although an increase in the concentration of P5 and 17-OHP during vitellogenesis and maturation of oocytes points to a possible role in reproduction, the absence of other vertebrate-type steroids in the investigated polychaete signifies a plausible uptake of P5 and 17-OHP from the environment.

Measuring steroids in hair opens up possibilities to identify congenital adrenal hyperplasia in developing countries.

Patients with congenital adrenal hyperplasia (CAH) in developing countries have limited access to appropriate laboratory facilities for diagnosis and follow-up. The aim of this study is to evaluate steroid measurement in hair as a diagnostic tool to identify and monitor CAH in these patients. A method was developed to measure steroids in hair, the stability of steroids in hair was assessed, and the concentration range in healthy volunteers was determined. Hair samples of patients, before and after starting therapy, were transported at ambient temperature to The Netherlands for analysis. Twenty-two Indonesian CAH patients and 84 healthy volunteers participated. Cortisol, 17-hydroxyprogesterone (17OHP), androstenedione, and testosterone in hair were measured by liquid chromatography with tandem mass spectrometry. Steroids in hair could be measured and remained stable (<4.9% deviation) for at least 3 weeks at 4°C and 30°C. In each of the untreated patients, hair concentrations of 17OHP (9.43-1135 pmol/g), androstenedione (36.1-432 pmol/g), and testosterone (2.85-69.2 pmol/g) were all above the upper limit of the corresponding range in healthy volunteers; 5.5 pmol/g, 13 pmol/g, and 1.8 pmol/g, respectively. After starting glucocorticoid treatment, the steroid concentrations in the hair of CAH patients decreased significantly for androstenedione (73%) and testosterone (59%) after 6 months. CAH could be confirmed in Indonesian patients based on the concentration of 17OHP, androstenedione, and testosterone in hair, and a treatment effect was observed. These findings open up opportunities to diagnose and/or monitor CAH in developing countries with a simple noninvasive technique.

Twenty Years of Neonatal Screening for Congenital Adrenal Hyperplasia in North-Eastern Italy: Role of Liquid Chromatography-Tandem Mass Spectrometry as a Second-Tier Test

Background: Newborn screening for congenital adrenal hyperplasia (CAH) based on 17-hydroxyprogesterone (17-OHP) concentration in dried blood spots has been taking place in North-Eastern Italy since 2001. Since 2017, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been introduced, for the first time in Italy, as a second-tier test. Aims: Our study aims to evaluate, on the one hand, the effectiveness of the newborn screening for CAH after 20 years of testing and, on the other, the impact that the introduction of the second-tier test had on the diagnostic accuracy of the screening program. Methods: Since 2001 dried blood spots taken from newborns have been screened with a time-resolved fluoroimmunoassay for 17-OHP determination. Over the years, the cut-off levels of 17-OHP were adjusted according to gestational age. Since 2017, a second-tier test in LC-MS/MS was introduced for samples displaying fluoroimmunoassay 17-OHP exceeding the cut-off. Results: In total, 862,521 newborns have been screened over a period of 20 years. The total incidence of 21-hydroxylase deficiency (21-OHD) was 1:25,368, moreover, a case of 11-β-hydroxylase deficiency was identified. All these diagnoses were genetically confirmed. The sensitivity and specificity of the screening program were 97% and 99.4%, respectively. The use of LC-MS/MS as a second-tier test significantly reduced the recall rate and increased the positive predictive value. Conclusions: Screening for CAH is useful in the neonatal diagnosis of a classic form of 21-OHD, allowing a precocious treatment of affected children. The introduction of an LC-MS/MS second-tier reduced the recall rate, avoiding unnecessary blood withdrawal and medical evaluations and preventing stress to families. Furthermore, it helped identify rarer forms of CAH.

Abnormal Newborn Screen During a Pandemic.

Abnormal Newborn Screen During a Pandemic.

Diurnal salivary androstenedione and 17-hydroxyprogesterone levels in healthy volunteers for monitoring treatment efficacy of patients with congenital adrenal hyperplasia.

ObjectiveTreatment of congenital adrenal hyperplasia (CAH) patients with glucocorticoids is often challenging since there is a delicate balance between over‐ and undertreatment. Treatment can be monitored noninvasively by measuring salivary androstenedione (A4) and 17‐hydroxyprogesterone (17‐OHP). Optimal treatment monitoring requires the establishment of reference values in saliva.DesignA descriptive study.PatientsFor this study saliva of 255 healthy paediatric and adult volunteers with an age range of 4–75 years old was used.MeasurementsWe developed a sensitive liquid chromatography‐tandem mass spectrometry method, assessed salivary A4 and 17‐OHP stability, and measured A4 and 17‐OHP concentrations in saliva collected in the morning, afternoon, and evening.ResultsWe quantified A4 and 17‐OHP concentrations in the morning, afternoon, and evening and demonstrated that there is a significant rhythm with the highest levels in the morning and decreasing levels over the day. A4 and 17‐OHP concentrations display an age‐dependent pattern. These steroids remain stable in saliva at ambient temperature for up to 5 days.ConclusionsGood stability of the steroids in saliva enables saliva collection by the patient at home. Since salivary A4 and 17‐OHP display a diurnal rhythm and age‐dependent pattern, we established reference values for both children and adults at three time points during the day. These reference values support treatment monitoring of children and adults with CAH.

21-Deoxycortisol is a Key Screening Marker for 21-Hydroxylase Deficiency

To assess whether 21-deoxycortisol (21deoxy) can be used to predict 21-hydroxylase deficiency (21OHD) in newborns and to evaluate the influence of gestational age and the timing of collection on 21deoxy concentrations. 17-hydroxyprogesterone (17OHP) and 21deoxy levels were measured in 906 newborn screening specimens (851 unaffected newborns, 55 confirmed cases of 21OHD) to compare their ability to identify babies with 21OHD. In addition, these 2 steroids were assessed in the unaffected cohort to determine the influence of gestational age (ranging from 23 to 42weeks) and the timing of specimen collection on the measured concentrations. The gestational age of the newborn impacted both 17OHP and 21deoxy concentrations, but the degree of influence was more substantial for 17OHP. Timing of collection did not affect 21deoxy concentration. Moreover, 21deoxy was a better predictor of 21OHD status compared with 17OHP, with little overlap in concentrations between the unaffected population and confirmed cases of 21OHD. A streamlined decision tree using solely 21deoxy (cutoff value, 0.85ng/mL) yielded a 91.7% positive predictive value for 21OHD screening. Our findings demonstrate that 21deoxy is a key disease marker of 21OHD and can be used to improve the accuracy of newborn screening for this disorder.

Salivary Profiles of 11-oxygenated Androgens Follow a Diurnal Rhythm in Patients With Congenital Adrenal Hyperplasia.

ContextSeveral studies have highlighted the importance of the 11-oxygenated 19-carbon (11oxC19) adrenal-derived steroids as potential biomarkers for monitoring patients with 21-hydroxylase deficiency (21OHD).ObjectiveTo analyze circadian rhythmicity of 11oxC19 steroids in saliva profiles and evaluate their relevance as potential monitoring parameters in 21OHD.Design, Setting, and ParticipantsCross-sectional single-center study including 59 patients with classic 21OHD (men = 30; women = 29) and 49 body mass index- and age-matched controls (men = 19; women = 30).Outcome MeasuresSalivary concentrations of the following steroids were analyzed by liquid chromatography-tandem mass spectrometry: 17-hydroxyprogesterone (17OHP), androstenedione (A4), testosterone (T), 11β-hydroxyandrostenedione (11OHA4), and 11-ketotestosterone (11KT).ResultsSimilar to the previously described rhythmicity of 17OHP, 11OHA4 and 11KT concentrations followed a distinct diurnal rhythm in both patients and controls with highest concentrations in the early morning and declining throughout the day (11-OHA4: mean reduction of hormone concentrations between timepoint 1 and 5 (Δ mean) in male patients = 66%; male controls Δ mean = 83%; female patients Δ mean = 47%; female controls Δ mean = 86%; 11KT: male patients Δ mean = 57%; male controls Δ mean = 63%; female patients Δ mean = 50%; female controls Δ mean = 76%). Significant correlations between the area under the curve for 17OHP and 11KT (rpmale = 0.773<0.0001; rpfemale = 0.737<0.0001), and 11OHA4 (rpmale = 0.6330.0002; rpfemale = 0.5640.0014) were observed in patients but not present or reduced in controls.ConclusionsAdrenal 11oxC19 androgens are secreted following a diurnal pattern. This should be considered when evaluating their utility for monitoring treatment control.

Salivary profiles of 11-oxygenated androgens follow a diurnal rhythm in patients with congenital adrenal hyperplasia

CONTEXT Several studies have highlighted the importance of the 11-oxygenated 19-carbon (11oxC19) adrenal-derived steroids as potential biomarkers for monitoring patients with 21-hydroxylase deficiency (21OHD). OBJECTIVE To analyze circadian rhythmicity of 11oxC19 steroids in saliva profiles and evaluate their relevance as potential monitoring parameters in 21OHD. DESIGN, SETTING, AND PARTICIPANTS Cross-sectional single-center study including 59 patients with classic 21OHD (men = 30; women = 29) and 49 body mass index- and age-matched controls (men = 19; women = 30). OUTCOME MEASURES Salivary concentrations of the following steroids were analyzed by liquid chromatography-tandem mass spectrometry: 17-hydroxyprogesterone (17OHP), androstenedione (A4), testosterone (T), 11β-hydroxyandrostenedione (11OHA4), and 11-ketotestosterone (11KT). RESULTS Similar to the previously described rhythmicity of 17OHP, 11OHA4 and 11KT concentrations followed a distinct diurnal rhythm in both patients and controls with highest concentrations in the early morning and declining throughout the day (11-OHA4: mean reduction of hormone concentrations between timepoint 1 and 5 (Δ mean) in male patients = 66%; male controls Δ mean = 83%; female patients Δ mean = 47%; female controls Δ mean = 86%; 11KT: male patients Δ mean = 57%; male controls Δ mean = 63%; female patients Δ mean = 50%; female controls Δ mean = 76%). Significant correlations between the area under the curve for 17OHP and 11KT (rpmale = 0.773<0.0001; rpfemale = 0.737<0.0001), and 11OHA4 (rpmale = 0.6330.0002; rpfemale = 0.5640.0014) were observed in patients but not present or reduced in controls. CONCLUSIONS Adrenal 11oxC19 androgens are secreted following a diurnal pattern. This should be considered when evaluating their utility for monitoring treatment control.

Assessment of Steroid Hormones in Both Saliva and Blood During a Phase 2 Clinical Trial for the Use of Tildacerfont in Adults With Classic Congenital Adrenal Hyperplasia

Background: Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is an autosomal recessive disorder characterized by an inability to synthesize endogenous cortisol resulting in overproduction of 17-hydroxyprogesterone (17-OHP) and androgens such as androstenedione (A4). Serum-based methods to measure steroid hormones typically require clinic visits and are not amenable to frequent, serial measurements when patient daily glucocorticoid doses are adjusted. Reliable, non-invasive new methodologies that can easily be adopted in a clinical trial setting are needed to complement existing measures of disease control. Here we report on the correlation between salivary and serum steroid hormone data from a Phase 2 proof-of-concept study for tildacerfont, a second generation corticotropin-releasing factor-1 (CRF1) receptor antagonist, which previously showed an ability to reduce excess adrenocorticotropic hormone (ACTH), 17-OHP, and A4 concentrations. Methods: Subjects with CAH under evaluation for tildacerfont underwent concurrent salivary and serum concentration measurements of androstenedione (A4), 17-hydroxyprogesterone (17-OHP) and testosterone (T) at approximately 8 am every 2 weeks for up to 6 weeks. Both serum and saliva samples were measured using liquid chromatography-tandem mass spectrometry. Results: 25 subjects (16 females) with a median age of 31 years (range 19-67) participated; median body mass index (BMI) was 27.8 kg/m2 (22-62 kg/m2). The number of matched samples across time points were: n=106 (17-OHP), n=106 (A4) and n=98 (T). 17-OHP and A4 were well correlated between serum and salivary assessments, r=0.80 (p<0.001) and r=0.80 (p<0.001), respectively, using kendall rank tests. T measurements had a lower correlation, r=0.67 (p<0.001). Over the duration of treatment, correlations between saliva and serum for 17-OHP and A4 were generally stable with only small differences across sex and clinic visit. Correlations between saliva and serum for T showed a moderate degree variability in women and a high degree of variability in men across visits. While correlations were high for A4 and 17-OHP, the magnitude of change, as a percentage of baseline across time points, was less pronounced with saliva as compared to serum. Conclusions: The ability to employ salivary steroid hormone measurements in CAH patients was demonstrated in a clinical trial setting. These data show that the measurement of the hormones 17-OHP and A4 in saliva may offer a promising, non-invasive approach to more frequently assessing response to therapy in patients with CAH.

Prenatal paraben exposure and anogenital distance and reproductive hormones during mini-puberty: A study from the Odense Child Cohort

BackgroundParabens are added to personal care products as antimicrobial preservatives. They have been suggested to have endocrine disrupting abilities. Prenatal exposure to parabens has been associated with reproductive endpoints including reduced male anogenital distance (AGD, distance from anus to genitals), which is sensitive to prenatal anti-androgenic exposure. ObjectivesTo study the associations between maternal paraben concentrations in second trimester urine and AGD and reproductive hormone concentrations at 3 months of age in offspring. MethodsPregnant women from Odense, Denmark were included in early pregnancy from 2010 to 12, and their children are being followed up. Fasting spot urine samples from 536 pregnant women were analyzed for methylparaben (MeP), ethyl-paraben (EtP), iso-propylparaben (i-PrP), n-propylparaben (n-PrP), n-butylparaben (n-BuP) and benzylparaben (BzP) by liquid chromatography tandem mass spectrometry and thereafter osmolarity adjusted. Three months after expected date of birth, AGD was measured in 452 children, and serum concentrations of follicle stimulating hormone (FSH), luteinizing (LH), testosterone, dehydroepiandrosterone-sulphate (DHEAS), androstenedione and 17-hydroxyprogesterone (17-OHP) were measured in 198 children. Maternal paraben exposure was categorized into tertiles or below and above level of detection, and sex-stratified multiple linear regression analyses were performed with AGD or reproductive hormones as outcomes. ResultsMost pregnant women had low concentrations of parabens in urine, but 10% exceeded the threshold for adverse estrogenic effects. Higher maternal paraben exposure was associated with shorter AGD in male offspring and longer AGD in girls, although only significant for MeP in boys. In addition, FSH, LH, DHEAS, 17-OHP concentrations were lower in girls with high prenatal paraben exposure, whereas no consistent pattern was found in boys. DiscussionThe endocrine disrupting abilities of parabens may affect humans at vulnerable time periods during development, which may have long term impact on reproductive function. This is the first study to find these associations in girls and our findings need confirmation.