Measurement Of 17α-hydroxyprogesterone Research Articles

Diagnosis and prevalence of Congenital Adrenal Hyperplasia (CAH) in Austrian children screened or not screened for CAH

Prevalence of Congenital Adrenal Hyperplasia (CAH) is not exactly known in the Austria; a number of patients with CAH might not be diagnosed, especially males. CAH is in about 95 % of the cases due to a defect in the 21-hydroxylation (‘classical CAH’). Newborn screening for CAH, based on the measurement of 17α-hydroxyprogesterone (17-OHP) was shown to be efficient for diagnosis, and is part of the newborn screening programme in Austria since April 2001. In our study we compared 2 groups of children: Group A, children born in Styria (a province of Austria), 1992 – 2001, n = 119.001, m 61.256, and f 57.745; Group B, children born in Styria 2002 – 2011, n = 103.228, m 52.722, and f 50.506 In group A (patients not screened), CAH was diagnosed in 8 children (m 4; f 4); 4 of them with simple virilising (SV) 21-OH deficiency (m 3; f 1) and 4 with salt wasting (SV) 21-OH deficiency (m 1; f 3). In group B, 98,7 % of all newborns born in Styria could be screened by measuring 17-OHP in a dried blood spot on filter paper. Recall rate was 0,578 %. CAH was diagnosed in 10 children (m 3: f 7). 8 of them with SW (m 2; f 6), and 2 with 11β-hydroxylase deficiency (m 1; f 1). Whereas group A displayed the expected Mendelian sex ratio, group B showed a strong female predominance (m 3; f 7) Prevalence of CAH was 1:14.875 newborns in group A (not screened). In group B (newborns screened) prevalence was 1:10.132. If one exclude the 2 patients with 11s-hydroxylase deficiency from group B, prevalence of ‘classical CAH’ was 1:14.875 in group A, compared to 1:12.903 in group B. These data show that newborn screening for CAH seems to increase the rate of detection of CAH.

The Human Placenta Expresses CYP17 and Generates AndrogensDe Novo

The human placenta is believed to have insignificant CYP17 expression, rendering it dependent on the maternal and fetal compartments for the necessary androgenic precursors to yield the high levels of estrogens seen in pregnancy. The aim of the study was to analyze whether the human trophoblast is capable of expressing CYP17 and producing androgens de novo. Human trophoblasts from fresh placentas and JEG-3 cells were used for all experiments. CYP17 mRNA analysis was performed via RT-PCR, and protein detection by Western blot and immunohistochemical staining. Steroid products were quantified using RIAs. CYP17 mRNA was expressed in both cell types. CYP17 protein was detected by Western blotting and localized by immunostaining mainly to the cytoplasm of syncytiotrophoblasts. Measurement of 17α-hydroxyprogesterone, androstenedione, and their aromatized products in the media further demonstrated CYP17 expression and activity in the human trophoblast. Baseline levels of CYP17 steroid products were higher in primary cells and significantly increased in the presence of 22-hydroxycholesterol. We have demonstrated CYP17 mRNA and protein expression and activity in human trophoblasts. Considering the precursor concentration, blood flow, and mass of the placenta, we suggest that its contribution of androgens is an important source of estrogen production in pregnancy.

Monitoring Medical Treatment in Adolescents and Young Adults with Congenital Adrenal Hyperplasia: Utility of Salivary 17α-Hydroxyprogesterone Day Profiles

Efficacy of medical treatment in patients with 21-hydroxylase deficiency is usually monitored by measurement of 17α-hydroxyprogesterone (17OHP). Saliva instead of serum sampling offers some advantages, such as painless handling and measurement of the bioactive free hormone. This study evaluated the diagnostic validity of salivary 17OHP for monitoring medical treatment, with samples collected at 7 time points throughout a day. Day profiles were performed in 23 adolescents and young adults with 21-hydroxylase deficiency and 43 healthy volunteers. During each profile, saliva and serum samples for 17OHP were simultaneously collected. With regard to the initial day profiles, samples were pathological in 63% (saliva) and 41% (serum). After the first day profile 14 patients underwent adjustment of medical treatment, either because of highly elevated 17OHP levels or with the aim of dose reduction. When comparing the best with the first day profile fewer samples were pathological (saliva: 32% vs. 71%; p<0.05; serum: 21% vs. 47%; n. s.), while the mean hydrocortisone equivalent dose was significantly reduced (20.09 mg vs. 27.27 mg; p<0.01). In 53% of profiles with controlled salivary 17OHP levels at 0700 h, the necessity for a treatment modification became only apparent when analyzing the whole day profile. A single 0700 h value within the reference range does not allow for a reliable assessment of therapeutic efficacy. We therefore suggest 17OHP day profiles for monitoring medical treatment. In this context, saliva analysis appears to be more sensitive in identifying patients who are inadequately treated.

Development of an Indirect Enzyme Immunoassay using Monoclonal Antibodies for the Measurement of 17α-Hydroxyprogesterone in Human Serum

An indirect enzyme immunoassay for the measurement of total 17α-hydroxyprogesterone (17OHP) in serum using monoclonal antibodies generated in our laboratory was developed. Here, (a) instead of extraction with solvents, serum was heated to free protein-bound 17OHP and assay was performed at pH 9.6, (b) to ensure uniform assay conditions for both standards and samples, buffer for standards contained charcoal-stripped pre-heated pooled cord serum. Assays were done in 96-well EIA microplates pre-coated with 17α-hydroxyprogesterone-3-(o-carboxymethyl)oxime: bovine serum albumin. Secondary antibody was horseradish peroxidase-linked sheep anti-mouse IgG polyclonal antibody. The method was accurate and suitable for screening for congenital adrenal hyperplasia.

Estimation of the false-negative rate in newborn screening for congenital adrenal hyperplasia

Newborn screening based on measurement of 17alpha-hydroxyprogesterone (17-OHP) in a dried blood spot on filter paper is an effective tool for early diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Its most important rationale is prevention of a life-threatening salt-wasting (SW) crisis; in moderate forms of CAH, early diagnosis and treatment may prevent permanent negative effects of androgen overproduction. Our target was to analyse if all CAH patients who had been identified clinically before puberty would have been detected by the newborn screening. Newborn screening cards of 110 CAH patients born between 1988 and 2000 in five Middle-European countries and diagnosed prior to puberty (77 SW and 33 moderate) and cards from 920 random, healthy newborn controls were analysed. CAH screening had not yet been introduced during this time. The diagnosis was based on clinical and laboratory signs and, in most cases, on CYP21 gene mutation analysis. All 17-OHP measurements in dried blood spots were carried out using a time-resolved fluoroimmunoassay kit. In the newborn screening blood spots, the median of 17-OHP levels was 561 nmol/l (range 91-1404 nmol/l) in subjects with the SW form and 40 nmol/l (4-247 nmol/l) in the moderate form. All 77 SW patients would have been detected by newborn screening using the recommended cut-off limits (30 nmol/l). However, 10 of 33 patients with moderate CAH would have been missed. 17-OHP levels of all controls were below the cut-off. Newborn screening is efficient for diagnosing the SW form of CAH, but is inappropriate for identifying all patients with a moderate form of CAH. It appears that the false-negative rate is at least one-third in children with the moderate form of CAH.

Stability of 17α-Hydroxyprogesterone in Dried Blood Spots after Autoclaving and Prolonged Storage

In the last few decades, management of patients with inborn errors of metabolism or congenital endocrine disorders has been greatly improved by the introduction of neonatal mass screening programs in many parts of the world. For this purpose, blood from newborns, ideally 4–7 days of age, is collected on filter-paper cards by means of a heel prick. A standard set of up to 20 tests is usually carried out at a centralized laboratory within a few days. Until recently, either when the tests were being performed or during the final step, the entire filter-paper card was autoclaved and then stored (1). Thus, leftover blood spots on filter paper from the entire young population in many countries are available in native or autoclaved form, although autoclaving has now become outdated because of a change in analytical methods (1). The stored cards provide a potentially valuable source for population studies or retrospective examinations. Documentation of the long-term stability of the analytes to be examined is a prerequisite for such studies. In many countries, the latest addition to the neonatal screening program is the examination for congenital adrenal hyperplasia (CAH), by measurement of 17α-hydroxyprogesterone (17-OHP) in a dried blood spot (2)(3)(4). The technique, first described by Pang et al. in 1977 (5), is based on a direct immunoassay and is used as the standard procedure in programs screening for CAH (2). In several trials, 17-OHP in dried blood spots on filter-paper cards was resistant to freezing, high ambient temperatures, humidity, and short- or intermediate-term (up to 1 year) storage (6)(7)(8)(9)(10). In the present study, we examined the impact of autoclaving (i.e., extremely high temperature, high humidity and pressure) and prolonged (more than a decade) storage under typical room conditions on the …