Liquid chromatography-mass spectrometry, the gold standard method for cortisol measurement, is expensive and not widely available in the developing countries. Chemiluminescent immunoassay, commonly used for cortisol measurement is prone to clinically meaningful inter-assay variability in some analysers. This occurs due to non-specific nature of anticortisol antibodies used in different platforms, having cross reactivity with structurally similar cortisol precursors like 17α-hydroxyprogesterone (<em>17OHP</em>), 11-deoxycortisol and 21-deoxycortisol. In patients with 21-hydroxylase deficiency, where <em>17OHP</em> and 21-deoxycortisol are significantly elevated, older generation machines like Siemens Advia Centaur XP provide spuriously high cortisol concentration compared with values measured by Roche Cobas e 411 or Siemens Immulite 1000. Diagnosis of potentially life-threatening salt-wasting 21-hydroxylase deficiency may be missed and treatment may be delayed due to such interference. Two children with classic 21-hydroxylase deficiency are being reported here, in whom high cortisol values were observed in Siemens Advia Centaur XP system.

Keywords: adrenal disorders; congenital disorders; neonatal and paediatric intensive care; paediatric intensive care.

Considerable researches on sex steroids and insulin action have suggested a mutual interaction between hyperandrogenemia and insulin resistance (IR). The objective of present study was to evaluate the androgens levels in young females with emphasis on the association of <em>17OHP</em> with IR. Serum concentrations of glucose, insulin, and androgens in 80 young females were measured by standard routine procedures. Total testosterone (TT), dihydrotestosterone (DHT), dehydroepiandrosterone sulfate (DHEAS), androstenedione (ASD), and 17-hydroxyprogesterone (<em>17OHP</em>) levels were higher in patients with IR compared to healthy controls (<i>p</i> < .05). <em>17OHP</em> was associated with IR and other androgens tested in young females. According to the results, androgen excess was associated with IR in young females and TT appeared to be independent predictor of IR in these patients. These data may suggest that simultaneous quantification of an androgen profile including at least TT, DHT, and <em>17OHP</em> can present useful clinical information for assessment of androgen excess.

Purpose: We compared the sexual function in women with classic forms of congenital adrenal hyperplasia (CAH) and polycystic ovary syndrome (PCOS) to find if the cause of androgen excess determines sexual functioning.

Methods: Hundred and four women (21 with CAH, 63 with PCOS and 20 healthy controls) aged 18-40 years were included into the study. All participants completed a questionnaire regarding their sociodemographic background and underwent anthropometric and basic biochemical measurements. Plasma levels of total testosterone, androstenedione, and 17-hydroxyprogesterone were measured with immunoassay. To assess the sexual functions, the Female Sexual Function Index (FSFI) questionnaire was applied.

<strong class="sub-title"> Results: </strong> Apart from the higher physical activity in PCOS patients (P = 0.017), we found no significant sociodemographic differences between the studied groups. In clinical assessment, women with CAH had a lower incidence of acne (P = 0.006). Their plasma levels of <em>17OHP</em> (P = 0.005) and insulin resistance index (P = 0.0248) were higher, while total testosterone (P = 0.0495) and glucose (P = 0.0061) was lower compared to the PCOS group. Significantly more women with CAH were homosexual (P = 0.003) and bisexual (P = 0.006). CAH group showed a lower total FSFI score (P = 0.0043) and lower scores in three domains: lubrication (P = 0.0131), sexual satisfaction (P = 0.0006), and dyspareunia (P < 0.0001). Higher physical activity was associated in all women with higher total FSFI score (P = 0.009) and scores in the domain of desire (P = 0.034) and sexual satisfaction (P = 0.01), while in CAH women apart from the total score (P = 0.03) and sexual satisfaction (P = 0.002) also in the domains of orgasm (P = 0.005), and pain (P = 0.03).

Conclusions: CAH women present more often homosexual and bisexual orientation, while their sexual functions are impaired compared to PCOS patients.

Keywords: Androgen excess disorders (AED); Androgens; Congenital adrenal hyperplasia (CAH); Polycystic ovary syndrome (PCOS); Sexual function.

A 30-year-old woman with a virilizing lipid tumor was initially suspected of having adult-onset congenital adrenal hyperplasia (CAH) when her plasma 17-hydroxyprogesterone (<em>17OHP</em>) concentration was found to be very high (298-3,170 ng/dL), to drop in response to an overnight dexamethasone (dex) suppression test (79 ng/dL) and to rise briskly 15 minutes after ACTH administration (751 ng/dL). However, the effect of dex was not sustained or complete: the pregnanetriol excretion dropped only from 5.5 to 4.4 mg daily. Furthermore, the plasma testosterone was inappropriately high (235-537 ng/dL) for adult-onset CAH and was more responsive to endogenous and exogenous gonadotropin stimulation than to ACTH. In addition, there was no evidence of 11 beta-hydroxylation of 21-deoxycorticoids, as would be expected in CAH. Removal of the tumor completely reversed the virilization and the abnormal responses to ACTH and human chorionic gonadotropin. The contralateral ovary bore lipid-laden stromal cells in the deep paracortex that bore a striking resemblance to the tumor cells, and a cyst in that ovary had fluid with a steroid pattern virtually identical to that of the tumor, with an androstenedione: <em>17OHP</em> ratio of 5:1. There was not evidence of polycystic ovary disease. The clinical picture of type II polycystic ovary syndrome (PCOS) gradually evolved over a one-year period postoperatively: plasma-free testosterone became mildly elevated and was not dex suppressible. The <em>17OHP</em> response to ACTH became slightly excessive. However, there was no evidence of tumor on computed tomography.(ABSTRACT TRUNCATED AT 250 WORDS)

<AbstractText>Non-classic 21-hydroxylase deficiency is usually diagnosed in post-pubertal women because of androgen excess. Indication of systematic steroid replacement therapy is controversial because the risk of acute adrenal insufficiency is unknown. In order to specify this risk we evaluated the cortisol and aldosterone secretions in response to appropriate pharmacological challenges.</AbstractText><AbstractText>In this prospective case-control non-inferiority study we investigated 20 women with non-classic 21-hydroxylase deficiency carrying biallelic CYP21A2 mutations and with serum 17-hydroxyprogesterone (<em>17OHP</em>) >10ng/mL after stimulation with Synacthen® (tetracosactrin) and 20 age- and BMI-matched healthy women with <em>17OHP</em> after Synacthen® <2ng/mL. Each participant underwent sequentially an insulin tolerance test to evaluate cortisol secretion and a sodium depletion test, obtained by oral administration of 40 mg furosemide under low sodium diet (< 20 mmol during 24 hours), to evaluate renin and aldosterone secretion. The trial was registered in ClinicalTrials.gov, number NCT01862380.</AbstractText><AbstractText>The peak serum cortisol concentration after insulin hypoglycemia was lower in patients than in controls (mean difference -47 ng/mL, 90%CI: -66, p=0.0026). A peak serum cortisol above a cutoff value of 170 ng/mL was obtained in all controls but only in 55% of patients (p=0.0039). 24-hours after sodium depletion, blood pressure, plasma sodium, potassium and serum aldosterone concentrations were comparable between the two groups, but patients had higher stimulated renin concentrations than controls (p=0.0044).</AbstractText><AbstractText>Patients with non-classic 21-hydroxylase deficiency frequently display partial cortisol insufficiency and compensated defect in aldosterone secretion. Their clinical management should systematically include assessment of adrenal functions.</AbstractText>

It is known that the prevalence of cardiovascular diseases, hypertension, noninsulin dependent diabetes mellitus and dyslipidemia in the late adulthood are in connection with intrauterine retardation, characterized by low birth weight. One possible explanation of this phenomenon is the abnormality of hypothalamus-hypophysis-adrenal cortex axis due to the accelerated growth. The authors investigated the steroid levels of young adults; whom birth weight were under 2500 g, and examined the relationship between hormone levels and some parameters of glucose metabolism and cardiovascular system. 75 subjects (43 female and 32 male patients, mean age: 19.6 and 19.8 years, respectively; range 18-22 ys) with low birth weight and without any sign of chronic disease, and 30 healthy, age-matched controls with normal birth weight were investigated. The basal serum cortisol, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEAS), androstenedione (AD), 17-hydroxyprogesterone (<em>17OHP</em>), estradiol (OE), sex-hormone binding globulin (SHBG), FSH, LH and insulin levels were determined. Moreover, oral glucose tolerance test with 75 g glucose (OGTT), impedance cardiography as well as ambulatory blood pressure monitoring were done by all subjects. In both sexes in subjects with low birth weight the mean serum cortisol level was significantly higher, than in the normal controls. In female patients the serum DHEA, DHEAS, AD, and <em>17OHP</em> levels were significantly higher than in the controls. Moreover, among these females a relationship was found between the elevations of adrenal and gonadal steroids and hyperinsulinemia, characterized by increased insulin response during OGTT. In male subjects a significant correlation was found between serum cortisol levels and systolic blood pressure and heart rate. In females there was a positive relationship between serum DHEA and heart rate. Summarized, the basic abnormality in patients with low birth weight seems to be a relative hypercortisolism, and in females because of hyperinsulinemia exists a mild hyperandrogenism as well. The hypercortisolism may cause cardiovascular abnormalities in males directly, while in females indirectly through the hyperinsulinemia and hyperandrogenism. These subtle abnormalities can be detected when no clinical signs present themselves, in young adulthood, giving the opportunity of taking preventive actions.

Pregnanetriol-3 alpha-glucuronide (PTG) is the majority urinary metabolite of 17-hydroxyprogesterone (<em>17OHP</em>) and it typically increases in the commonest form of congenital adrenal hyperplasia (CAH), due to 21 hydroxylase deficiency. We developed a simple chemiluminescent immunoassay for the direct measurement of PTG in diluted urine in order to avoid the preliminary hydrolysis and extraction steps that are usually employed in gas-liquid chromatographic methods. The immunogenic complex PTG-bovine-serum-albumin was used to induce the formation of specific antibodies in New Zealand rabbits. In addition, PTG was conjugated to aminoethylethylisoluminol and the resulting tracer was characterized by mass spectrometry and used to monitor the immunological reaction. The characteristics of the antibody were determined with regard to specificity and sensitivity. The precision of the assay method was also established. PTG excretion was studied before and after the ACTH stimulation test (1 mg synthetic ACTH i.m.) in 11 normal women and in one subject affected by CAH due to 21-hydroxylase deficiency. PTG levels well correlated with <em>17OHP</em> plasma concentrations both under basal and stimulated conditions, in normal women as well as in the patient affected by CAH.

Adrenal responsiveness to ACTH stimulation (1 mg, i.m.) was assessed by measuring cortisol (Cort) and 17-hydroxyprogesterone (<em>17OHP</em>) at 0, 30, 60, and 90 minutes and progesterone (P), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), delta 4-androstenedione (delta 4A), and testosterone (T) at 0, 60, and 90 minutes post-ACTH in 30 women with polycystic ovary syndrome (PCO). The results were compared with those from 12 normally menstruating women. Three distinct patterns of responses of the adrenal steroids were observed in PCO patients, while cortisol response was similar to that of normal controls: (a) normal responders (n = 9, 30% of PCO patients), in whom a delta max response similar to that of normals was observed, although basal delta 4A and T levels were found to be elevated; (b) 21-OH dysfunction group (n = 6, 16.6%), in whom delta max <em>17OHP</em> levels and delta max <em>17OHP</em> to delta max Cort ratio were significantly higher than those of normals and the other PCO groups, indicating a dysfunction at the 21-hydroxylase level; (c) adrenarchal type of response group (n = 16, 53%), in whom statistically significant (P less than .0001) hyperresponsiveness of DHEA and, in 11 of them, of delta 4A, with high delta max delta 4A to delta max DHEA to delta max Cort ratios were found, indicative of a selective overproduction of the steroids during steroidogenesis. Moreover, the significantly higher delta max delta 4A to delta max <em>17OHP</em> ratio found in group c is a further indication of increased 17,20-lyase efficiency, as is encountered during adrenarche.(ABSTRACT TRUNCATED AT 250 WORDS)

The ultrasonographic measurement of cervical length with a cutoff of 15mm is currently the best method to identify a group of asymptomatic women in the general population at risk of spontaneous preterm birth, especially among asymptomatic patients with a singleton pregnancy with no history of preterm birth. Cerclage and 17 alpha-hydroxyprogesterone caproate (<em>17OHP</em>-C) are ineffective to reduce the risk of preterm birth among asymptomatic patients with a short cervix in midtrimester. However, vaginal progesterone (200-mg capsules of micronized progesterone or gel containing 90mg progesterone) has been demonstrated effective in 2 large randomized trials to reduce the risk of preterm birth and possibly the composite morbidity and perinatal mortality associated among asymptomatic women with a short cervix in the general population screened by ultrasound of the cervix in midtrimester. Three cost-effectiveness analyses are converging to show that universal screening for cervical length with vaginal progesterone treatment seems to be cost-effective compared with no screening. However, it is too early to definitively conclude that universal screening is justified for several reasons: many women must be screened to prevent a relatively small number of preterm births. Moreover, the epidemiology of preterm delivery is such that the use of progesterone in asymptomatic women with a short cervix screened by ultrasound in midtrimester in the general population will not significantly reduce the prevalence of preterm births; there are no data comparing the effectiveness of universal ultrasound screening followed by vaginal progesterone treatment in case of short cervix versus no universal screening associated to a progesterone treatment in case of incidentally observed short cervix; the universal ultrasound screening may not produce the same results in practice than those observed in published randomized trials, due to population differences, "indication creep", or "stretching of the cutoff" defining the short cervix. Moreover, the implementation of unevaluated or not recommended treatments, such as bed rest, tocolytics, <em>17OHP</em>-C or cerclage, can potentially cause unintended deleterious consequences and reduce the cost-effectiveness; the cost-effectiveness analyses evaluating universal screening for cervical length present uncertainties on critical variables, notably the short cervix prevalence and the progesterone efficacy. In conclusion, although the implementation of such a screening strategy can be considered by individual practitioners, this screening cannot be universally mandated.

Background: Exposure to per-/polyfluoroalkyl substances (PFASs) can disrupt endocrine hormones in humans. Prior studies have focused on the harmful effects of the two traditional per-/polyfluoroalkyl substances (PFASs), perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA). Other PFASs, used as the replacements of PFOS and PFOA, are widely and increasingly detected in humans. Whether these replacements influence glucocorticoids and progestogens in newborns remains unknown.

Objective: To investigate the associations between exposures of PFOS, PFOA and their replacements and glucocorticoids and progestogens in newborns.

<strong class="sub-title">Methods:</strong> We measured the concentrations of 13 PFASs, 3 glucocorticoids (11-deoxycortisol, cortisol and cortisone) and 2 progestogens [progesterone, 17-hydroxyprogesterone (<em>17OHP</em>)] in the cord sera of 374 neonates in a birth cohort from Wuhan, China, between 2013 and 2014. We evaluated the associations of each PFAS with glucocorticoids and progestogens using multiple linear regression models, and multiple comparisons were additionally corrected via false discovery rates (FDR).

<strong class="sub-title">Results:</strong> Out of the 13 PFASs, 9 were detected in over 95% of cord sera. The Chinese specific PFOS replacement - 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA, trade name F-53B) was positively associated with 13.13% change in cortisol in girls (95% CI = 4.47%, 22.52%, for each IQR increase in 6:2 Cl-PFESA). Seven PFASs had positive associations with the precursor of cortisol, namely 11-deoxycortisol (percent change ranged from 6.41% to 11.24%, for each IQR increase in PFASs). Perfluorobutane sulfonate (PFBS) in cord sera was positively associated with progesterone in the linear model, whereas PFOS and perfluorohexane sulfonate (PFHxS) levels were associated with progesterone in the quartile models. No PFASs were related to <em>17OHP</em> or cortisone.

Conclusions: In this study, PFOS, PFOA and/or their replacements were positively associated with progesterone, cortisol and 11-deoxycortisol in newborns. These results suggested that not only PFOS and PFOA, but also other PFASs have potential impacts on glucocorticoids and progestogens in newborns.

Keywords: Glucocorticoids; Newborn; Per-/polyfluoroalkyl substances; Progestogens.

High-performance liquid chromatography analysis of extracts of Arctic charr (salvelinus alpinus) eggs revealed the presence of several steroids, predominantly progestogens together with testosterone. Yolk sac embryos were incubated with tritiated progesterone ([3H]P4) or 17-hydroxyprogesterone ([3H]<em>17OHP</em>) to examine the ability of the embryos to metabolize progestogens in vitro; both progestogen precursors were converted to various free and conjugated steroids metabolites (sulfates and glucuronides). [3H]P4 was completely metabolized to form steroids that coeluted with standard 11-oxygenated androgens, corticosteroids, progestogens, and some other unidentified metabolites. This report also describes the biosynthesis of 17,20 beta-dihydroxy-4-pregnen-3-one and 17,20 beta-dihydroxy-4-pregnen-3-one 20-sulfate by embryos of Arctic charr that were incubated with <em>17OHP</em>.

The incorporation of 14C-glucosamine into glycoproteins (GP) and glycosaminoglycans (GAG), of 3H-leucine into protein, and the hexuronic acid (HA) content of polymerized GAG was determined in incubated placental tissue. In placentae of earlier gestational age (GA), incorporation of 14C-glucosamine was 2.6 times greater than other at 38-39 weeks GA. 19 of 26 placentae at 38-39 weeks GA responded by one or more parameters when incubated with 0.5-3 microM 17 alpha-OH-progesterone (<em>17OHP</em>). Those which did not respond were all of earlier GA; the placental content of GAG decreased in these from 31.1 +/- 1.8 nmol HA/mg protein to 16.4 +/- 1.5 nmol in placentae of GA 38-39 weeks. In the latter, <em>17OHP</em> increased GAG by 42.5% to 24.4 +/- 2.5 nmol HA/mg protein in a 2-hour incubation. Progesterone, oestriol, cortisol and testosterone were without effect. It is concluded that synthesis of placental GAG decreases toward the end of gestation, but can be increased by <em>17OHP</em> specifically. This indicates that <em>17OHP</em> is a biologically active steroid and might have a role in maintaining placental function.

Steroid 21-hydroxylase activity of the microsome-enriched fraction of follicular linings from equine ovaries has been demonstrated by gas chromatography-mass spectrometry. The 21-hydroxylated metabolites were quantified by isotope dilution with deuterated analogues. The two most abundant potential substrates for follicular steroid 21-hydroxylase, progesterone (P) and 17-hydroxyprogesterone (<em>17OHP</em>), were converted respectively to 11-deoxycorticosterone (DOC) and 11-deoxycortisol with corresponding apparent specific activities of 308 and 24 pmol/mg protein/h and apparent Km values of 1.1 and 6.4 microM. Competitive inhibition of the P-to-DOC conversion was exerted by <em>17OHP</em> and pregnenolone. Hence, the ovarian follicle of the mare is an extraadrenal site of preferential DOC biosynthesis by an enzyme having steroid 21-hydroxylase activity.

OBJECTIVE

To determine the 17OH progesterone (<em>17OHP</em>) levels in the neonatal screening for Congenital Adrenal Hyperplasia due to 21 hydroxylase deficiency (CAH-21OHD).

METHODS

The evaluation was performed using 76,360 paper filter samples, obtained from newborn screening in Rio de Janeiro from June 1992 to December 2000. The <em>17OHP</em> were assayed by fluoroimmunoassay method using the blood collected onto filter paper cards. The cut-off level was 10 ng/mL. The infants with <em>17OHP</em> levels above this cut-off were recalled to undergo a new dosage.

RESULTS

Thirty-eight patients presented <em>17OHP</em> levels above the cut-off limit. In 11 (4 males, 6 females, 1 undefined gender) the diagnoses of CAH-21OHD were confirmed. Their <em>17OHP</em> levels ranged from 25 to 254.5 ng/mL (mean: 133.84 ng/mL) on the first analysis and from 46.86 to 360 ng/mL (mean: 218.84 ng/mL) on the second analysis. The patients with the salt-wasting form showed higher <em>17OHP</em> levels when compared to simple virilizers, both on the first analysis (mean: 169.21 ng/mL vs 27.46 ng/mL) and on the second one (mean: 227.16 ng/mL vs 110.95 ng/mL). Among the 27 infants without confirmed disease (false-positives), <em>17OHP</em> levels ranged from 10.27 to 27.5 ng/mL (mean: 14.8 ng/mL) on the first sample and from 2.39 to 32.39 ng/mL (mean: 10.07 ng/mL) on the second. In this group, 8 children maintained borderline <em>17OHP</em> levels during a variable period, but in 7 of them it was normalized before the first year of life. In the remaining case, who was asymptomatic after 8 years of follow-up, a cortrosin stimulation test was compatible with the non-classic form of the disease.

CONCLUSIONS

These data confirm that <em>17OHP</em> analysis was a reliable test to CAH-21OHD neonatal screening and was able to differentiate between normal infants and those with the classical form of CAH-21OHD.

OBJECTIVE

Women who have had a spontaneous periviable delivery are at high risk for recurrent preterm delivery. The objective of our study was to determine interpregnancy interval (IPI) after periviable birth as well as percentage of women taking 17 alpha hydroxyprogesteronecaproate (<em>17OHP</em>-C) after periviable birth. We then examined the association between adherence with a postpartum visit after a periviable birth and IPI as well as receipt of <em>17OHP</em>-C.

METHODS

We included all women with a periviable delivery (20-26-week gestation) due to spontaneous preterm birth at Magee Women's Hospital between 2009 and 2014, who had their subsequent delivery at our institution during or before May of 2016. Information on maternal, fetal, and neonatal outcomes was obtained from the Magee Obstetrical Medical and Infant (MOMI) database as well as chart abstraction. We calculated IPI, proportion of women who received <em>17OHP</em>-C in their next pregnancy, and attendance rates with a postpartum visit. The relationship between attendance with a postpartum visit and IPI, and receipt of <em>17OHP</em>-C was examined with a logistic regression.

RESULTS

During the study period, 361 women had a spontaneous periviable birth. A total of 60 women had a subsequent delivery at Magee Women's Hospital. Only 33/60 (52.5%) presented for a postpartum visit after their periviable delivery. The median IPI for the cohort was 12.5 months (interquartile range: 6.4, 17.5 months) and 21.0% (n = 13) had an IPI less than 6 months. Adherence with the postpartum visit was not associated with an IPI less than 6 months. A total of 18.33% (11 women) did not receive <em>17OHP</em>-C in their subsequent pregnancy. Women who attended a postpartum visit were much more likely to receive <em>17OHP</em>-C (p = .001).

CONCLUSIONS

Many women with a history of a periviable birth do not optimize strategies to reduce their risk of recurrent preterm birth. While attendance with a postpartum visit was associated with greater receipt of <em>17OHP</em>-C in the subsequent pregnancy, given the overall poor rate of attendance with the postpartum visit in this cohort, novel strategies to counsel women about interpregnancy health are needed.

In this study, we established a method for the quantitative measurement of native adrenal steroids with GC-MS equipped with capillary column (cross-linked methyl silicone 25 m X 0.2 mm I.D., 0.11 m thin film). 1 ml of serum sample containing 5 alpha-cholestane as internal standard (IS) was elicited by organic solvent using extrelunt column. These samples were derived by n-butylboronic acid, o-methylhydroxylamine and trimethyl-silylating agents, then were finally applied to GC-MS. The intensities of molecular ions were used for the measurement of the serum concentration of steroids. The molecular ion peaks of steroids were obtained at m/z460 (17 alpha-hydroxyprogesterone; <em>17OHP</em>), m/z548 (corticosterone; B), m/z470 (11-deoxycortisol; S), m/z417 (pregnenolone; PL), m/z372 (progesterone; PT), m/z558 (cortisol; F), m/z389 (dehydroepiandrosterone; DHEA), m/z371 (estrone; E1), m/z416 (estradiol; E2), m/z504 (estriol; E3), m/z389 (testosterone; T), m/z344 (androstenedione; A) and m/z372 (IS). The curve of calibration for each steroid showed good linearity. The sensitivities of the GC/MS method were less than 5pg/one shot of each sample. The coefficients of variations of accuracies and precisions in this GC/MS method were less than 15% of each steroid. The samples from normal subjects after metyrapone and ACTH loading tests, and the patients of congenital adrenal hyperplasia showed a good correlationship between the data of GC/MS and the data of RIA after sephadex LH-20 column-chromatography. These results implied the usefulness of our system in clinical application. Moreover, this assay takes only 3 hrs. Thus it saves much time in comparison with the time-consuming radioimmunoassay system.

Objective: To evaluate the impact of a liquid chromatography tandem mass spectrometry (LCMSMS) second tier test on newborn screening for congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH) in New Zealand.

<strong class="sub-title"> Design: </strong> In a prospective study a LCMSMS method to measure 17-hydroxyprogesterone (<em>17OHP</em>) was adapted to measure four additional steroids. Steroid concentrations were collected on all second tier CAH screening tests while protocols remained unchanged. Steroid ratio parameters with recommended or published screening cuts-offs were evaluated for their impact on newborn screening performance.

Measurements: Precision, accuracy, linearity and recovery of the second tier LCMSMS method were evaluated. Second tier specimens were divided in 3 groups; newborn screening bloodspots from neonates with confirmed CAH (n=7) and 2 groups specimens from neonates with a birthweight (BW) ≤1500g (n=795) and with a BW>1500g (n=806) with a negative newborn screening test. Six protocols using four steroid ratio parameters were evaluated. The sensitivity, specificity, false positive rate and positive predictive value of screening was calculated for each protocol.

<strong class="sub-title"> Results: </strong> The LCMSMS method was sufficiently accurate and precise to be used as a second-tier test for CAH. Screening sensitivity remained at 100% for each protocol apart from (<em>17OHP</em>+androstenedione)/cortisol when the highest cut-off of 3.75 was applied. The false positive rate was significantly improved when (<em>17OHP</em>+androstenedione)/cortisol and (<em>17OHP</em>+21-deoxycortisol)/cortisol were evaluated with cut-offs of 2.5 and 1.5 respectively (p<0.01) and both with a positive predictive value of 64%.

Conclusions: A second tier LCMSMS newborn screening test for CAH offers significant improvements to screening specificity without any other changes to screening protocols.

Keywords: 21-hydroxylase; Congenital Adrenal Hyperplasia; Newborn Screening; Tandem mass spectrometry.

OBJECTIVE

We sought to determine if maternal weight or body mass index (BMI) modifies the effectiveness of 17-alpha hydroxyprogesterone caproate (<em>17OHP</em>-C).

METHODS

We performed a secondary analysis of the Maternal-Fetal Medicine Units Network Trial for the Prevention of Recurrent Preterm Delivery by 17-Alpha Hydroxyprogesterone Caproate. Binomial regression models were estimated to determine the relative risk (RR) of preterm birth (PTB) in women randomized to <em>17OHP</em>-C vs placebo according to BMI category and maternal weight. Adjusted models considered inclusion of potential confounders.

RESULTS

In all, 443 women with complete data were included. <em>17OHP</em>-C is effective in preventing PTB <37 weeks only in women with prepregnancy BMI <30 kg/m(2) (RR, 0.54; 95% confidence interval, 0.43-0.68). Above this BMI threshold there is a nonsignificant trend toward an increased risk of PTB (RR, 1.55; 95% confidence interval, 0.83-2.89) with <em>17OHP</em>-C treatment. When analyzing by maternal weight, a similar threshold is observed at 165 lb, above which <em>17OHP</em>-C is no longer effective.

CONCLUSIONS

The effectiveness of <em>17OHP</em>-C is modified by maternal weight and BMI, and treatment does not appear to reduce the rate of PTB in women who are obese or have a weight >165 lb. This finding may be due to subtherapeutic serum levels in women with increased BMI or weight. Studies of adjusted-dose <em>17OHP</em>-C in women who are obese or who weigh >165 lb are warranted, and current recommendations regarding the uniform use of <em>17OHP</em>-C regardless of maternal BMI and weight may deserve reassessment.

We have investigated a family with one child affected with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Prenatal determination of 17-alpha hydroxyprogesterone (<em>17OHP</em>) in amniotic fluid (AF) and HLA typing of amniotic fibroblasts from a pregnancy at risk showed that the fetus was not affected. A healthy cousin with HLA haplotypes identical to those of the proposita (only one being identical by descent) had a normal plasma level of <em>17OHP</em>. The prenatal diagnosis of a fetus affected with 21-hydroxylase deficiency CAH may be established by the determination of <em>17OHP</em> in AF. This is a relatively quick procedure that can be confirmed by the HLA genotype, and is mandatory in families with a parent homozygous for an HLA haplotype and in certain recombinant haplotypes in the fetus.

OBJECTIVE

The standard weekly dose of 17-alpha hydroxyprogesterone caproate (<em>17OHP</em>-C; 250 mg/wk) to reduce the risk of recurrent preterm birth was adopted without regard to patient characteristics. We examined the relationship between prepregnancy body mass index (BMI) and gestational age at birth after <em>17OHP</em>-C prophylaxis. We hypothesized that rates of births before 32, 35, and 37 weeks of gestation would be increased in women with a BMI of 25 kg/m(2) or greater.

METHODS

A retrospective cohort study was conducted from a deidentified database of women treated with <em>17OHP</em>-C for prior spontaneous preterm birth. The frequency of recurrent preterm delivery before 32, 35, and 37 weeks of gestation was investigated for women with a BMI less than 25 kg/m(2) compared with women with a BMI of 25 kg/m(2) or greater. The adjusted relative risk of preterm delivery was estimated through a modified Poisson regression approach.

RESULTS

Of 390 women who met inclusion criteria, 60 (15.4%) delivered before 32 weeks, 89 (22.8%) before 35 weeks, and 156 (40.0%) before 37 weeks. A total of 174 women had a BMI less than 25 kg/m(2) (mean [SD], 21.2 [2.5]) and 216 had a BMI of 25 kg/m(2) or greater (mean [SD], 33.5 [6.7]). Risk of birth before 32 weeks was 1.7 times higher on average (adjusted relative risk, 1.7; 95% confidence interval, 1.05-2.77) in overweight women than in women with a BMI less than 25 kg/m(2), adjusting for age, race, smoking, and short cervix. There was no difference in the risk of preterm birth before 35 or 37 weeks.

CONCLUSIONS

Among pregnant women receiving <em>17OHP</em>-C prophylaxis for a prior preterm birth, recurrent preterm birth before 32 weeks was significantly more common in those women whose prepregnancy BMI was 25 kg/m(2) or greater than in women with BMI less than 25 kg/m(2). This observation is consistent with pharmacological studies suggesting that dosing regimens of <em>17OHP</em>-C may affect efficacy.

OBJECTIVE

It is not known whether 17-alpha-hydroxyprogesterone caproate (<em>17OHP</em>-C) is effective for preventing preterm delivery with an episode of preterm labor (PTL) with or without intra-amniotic inflammation/infection.

METHODS

This was a retrospective cohort study. One hundred and seven PTL patients were selected and divided into a <em>17OHP</em>-C group (use of <em>17OHP</em>-C: n = 53) and a no-treatment group (no use of <em>17OHP</em>-C: n = 54). Moreover, the patients were divided into three subgroups (subgroup A: without intra-amniotic inflammation, B: with mild intra-amniotic inflammation, and C: with severe intra-amniotic inflammation) according to their level of amniotic interleukin (IL)-8, and perinatal prognosis was analyzed.

RESULTS

Interval from admission to delivery (days) in the <em>17OHP</em>-C group (76 [13-126], n = 34) was significantly longer than that in the no-treatment group (50 [8-104], n = 33; P = .012) in subgroup B. In cases without intra-amniotic microbes in subgroup B, a significant prolongation of gestational days was associated with the <em>17OHP</em>-C group (79 [13-126], n = 25) compared with the no-treatment group (50 [8-104], n = 29; P = .029). However, there were no significant differences in subgroups A or C.

CONCLUSIONS

<em>17OHP</em>-C could prolong gestational period in limited PTL cases with sterile mild intra-amniotic inflammation.

Preterm birth is a substantial public health concern. In 2019, the United States preterm birth rate was 10.23%, which is the fifth straight year of increase in this rate. Moreover, preterm birth accounts for approximately one-in-six infant deaths, and surviving children often suffer developmental delay or long-term neurologic impairment. While the burden of preterm birth is clear, identifying strategies to reduce preterm birth has been challenging. On October 29, 2019, a United States Food and Drug Administration Advisory Committee voted 9 to 7 to withdraw interim accelerated approval of 17-alpha hydroxyprogesterone caproate for prevention of recurrent preterm birth because the called for confirmatory trial, known as the PROLONG trial, was not confirmatory. The PROLONG trial included subjects enrolled in the United States and Canada in an effort to ensure that at least 10% of patients would be from North America, however, this trial took nine years to complete and did not demonstrate significant treatment effects in the two primary outcomes of interest. Delivery before 35 weeks occurred in 11% (N=122/1130) of women given 17-alpha hydroxyprogesterone caproate compared to 11.5% (N=66/578) given placebo (RR 0.95; 95%CI: 0.71-1.26, P=0.72).9 Similarly, the co-primary outcome neonatal composite index occurred in 5.6% (N=61/1093) of women given 17-alpha hydroxyprogesterone caproate compared to 5.0% (N=28/559) given placebo (RR 1.12; 95%CI: 0.68-1.61, P=0.73). There was also a lack of efficacy for 17-alpha hydroxyprogesterone caproate treatment in the analysis of a variety of secondary outcomes. Like the Maternal-Fetal Medicine Units Network trial, the PROLONG trial was also flawed. Importantly, the Maternal-Fetal Medicine Unit Network trial was the sole justification for treating women in the United States with 17-alpha hydroxyprogesterone caproate for nearly two decades. And now, despite more than half a century, 17-alpha hydroxyprogesterone caproate still has not been shown to be clearly effective. So, in this context, how does the advising physician dependent upon scientific evidence advise a patient that 17-alpha hydroxyprogesterone caproate is effective when the evidence to support this advice has repeatedly been found to be inadequate? This Clinical Opinion is a critical appraisal of the two randomized trials examining the efficacy of <em>17OHP</em>-C to prevent recurrent preterm birth and a chronicle of events in the regulatory process of drug approval to help answer this question. With this examination, these events illustrate the complexity of pharmaceutical regulation in the era of accelerated FDA approval and characterize the financial impact and influence in medicine. In this report, we also emphasize the value of observational studies in contemporary practice as well as identify other examples in medicine wherein FDA accelerated approval has been withdrawn. Importantly, the themes of the <em>17OHP</em>-C story are not limited to obstetrics but also serve as a microcosm of issues within the United States healthcare system that ultimately contribute to the high cost of healthcare. In our opinion, the answer to the question is clear-the facts speak for themselves-and we believe 17-alpha hydroxyprogesterone caproate should not be endorsed for use to prevent recurrent preterm birth in the United States.

<strong class="sub-title"> Keywords: </strong> 17-alpha hydroxyprogesterone caproate; <em>17OHP</em>-C; Food and Drug Administration; accelerated approval; evidence based medicine; healthcare cost; pharmaceutical industry; pregnancy; preterm labor; progesterone; progestin; progestogen; randomized clinical trial; real world evidence; regulatory process; subgroup analysis; withdrawal.

The positive predictive value of newborn screening for congenital adrenal hyperplasia due to 21-hydroxylase deficiency was <2% in New Zealand. This is despite a bloodspot second-tier immunoassay method for 17-hydroxyprogesterone measurement with an additional solvent extract step to reduce the number of false positive screening tests. We developed a liquid chromatography tandem mass spectrometry (LCMSMS) method to measure 17-hydroxyprogesterone in bloodspots to replace our current second-tier immunoassay method. The method was assessed using reference material and residual samples with a positive newborn screening result. Correlation with the second-tier immunoassay was determined and the method was implemented. Newborn screening performance was assessed by comparing screening metrics 2 years before and 2 years after LCMSMS implementation. Screening data analysis demonstrated the number of false positive screening tests was reduced from 172 to 40 in the 2 years after LCMSMS implementation. The positive predictive value of screening significantly increased from 1.71% to 11.1% (X² test, <i>p</i> < 0.0001). LCMSMS analysis of <em>17OHP</em> as a second-tier test significantly improves screening specificity for CAH due to 21-hydroxylase deficiency in New Zealand.

Keywords: 17-hydroxyprogesterone; congenital adrenal hyperplasia; liquid chromatography tandem mass spectrometry; newborn screening.

<strong class="sub-title"> Objectives: </strong> Accurate measurements of serum 17-hydroxyprogesterone (<em>17OHP</em>) are essential for diagnosis and treatment monitoring for congenital adrenal hyperplasia patients. The performance of serum <em>17OHP</em> routine methods remains highly variable that calls for a candidate reference measurement procedure (cRMP) to improve the standardization of serum <em>17OHP</em> measurements.

<strong class="sub-title"> Methods: </strong> Serum samples spiked with internal standards were extracted with a combination of solid-phase extraction and liquid-liquid extraction. The <em>17OHP</em> 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 <em>17OHP</em> 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.

<strong class="sub-title"> Results: </strong> No apparent interferences were found with the <em>17OHP</em> 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 (2.46-102.72 nmol/L), respectively. The recoveries of added <em>17OHP</em> 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.

<strong class="sub-title"> Conclusions: </strong> We developed and validated a reliable serum <em>17OHP</em> 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 <em>17OHP</em> routine measurements.

Keywords: congenital adrenal hyperplasia; liquid chromatography/tandem mass spectrometry; method evaluation; reference measurement procedure; serum 17-hydroxyprogesterone.