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

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

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

Steroid Screening Tools Differentiating Nonclassical Congenital Adrenal Hyperplasia and Polycystic Ovary Syndrome.

PURPOSE: To analyze the performance of basal 17OH-progesterone (17OHP) levels versus the basal 17OHP/cortisol ratio in nonclassical congenital adrenal hyperplasia (NCAH) and polycystic ovary syndrome (PCOS) differential diagnosis. Basal 17OHP levels >10 ng/mL have been used to confirm NCAH diagnosis without the adrenocorticotropic hormone (ACTH) test; however, the optimal cutoff value is a matter of debate. METHODS: A cross-sectional study was performed at the endocrinology and gynecological endocrinology outpatient clinics of a tertiary hospital. A total of 361 patients with PCOS (age 25.0 ± 5.3 years) and 113 (age 19.0 ± 13.6 years) patients with NCAH were enrolled. Basal and ACTH-17OHP levels were measured by radioimmunoassay, and CYP21A2 molecular analysis was performed to confirm hormonal NCAH diagnosis. Receiver operating characteristic curve analysis compared basal 17OHP levels and the 17OHP/cortisol ratio between NCAH and PCOS patients. RESULTS: Basal 17OHP levels were higher in NCAH patients than in those with PCOS (8.85 [4.20-17.30] vs 1.00 [0.70-1.50] ng/mL; P < 0.0001), along with 17OHP/cortisol ratio (0.86 [0.47-1.5]) vs 0.12 [0.07-0.19]; P < 0.0001, respectively). Basal 17OHP levels and the 17OHP/cortisol ratio were strongly correlated in both groups (rho = 0.82; P < 0.0001). Areas under the curves for basal 17OHP levels (0.9528) and the 17OHP/cortisol ratio (0.9455) were not different to discriminate NCAH and PCOS (P > 0.05). Basal 17OHP level >5.4 ng/mL and 17OHP/cortisol ratio >2.90 had 100% specificity to identify NCAH. MAIN CONCLUSIONS: Basal 17OHP levels >5.4 ng/mL can be used to perform differential diagnoses between NCAH and PCOS, dismissing the ACTH test. The basal 17OHP/cortisol ratio was not superior to basal 17OHP levels in this scenario.

Polycystic ovary syndrome associated with increased adiposity interferes with serum levels of TNF-alpha and IL-6 differently from leptin and adiponectin.

Objective The aim of this study was to investigate polycystic ovary syndrome (PCOS) and to explore the relationship between body fat percentage and metabolic markers. Subjects and methods Sedentary women were assigned to PCOS (N = 60) and CONTROL (N = 60) groups. Each group was subdivided into three subgroups according to body fat percentage (22-27%, 27-32% and 32-37%). The protocol consisted of assessments of glucose, insulin, androgens, follicle stimulating hormone (FSH), luteinizing hormone (LH), 17-hydroxyprogesterone (17-OHP), leptin, adiponectin, tumor necrosis factor (TNF-α) and interleukin-6 (IL-6). Results The PCOS subgroups showed higher concentrations of androgens, LH and 17-OHP. Leptin showed direct relationship with increased body fat percentage, whereas adiponectin showed the inverse effect. However, both were unaffected by PCOS. TNF-α and IL-6 were higher in PCOS women and showed a direct relationship with increased body fat percentage. Glucose showed direct relationship with body fat percentage, whereas insulin presented higher values in PCOS women and direct relationship with increased body fat percentage. Conclusions Our findings indicate that PCOS and body fat percentage directly influence concentrations of insulin, TNF-α and IL-6, whereas leptin and adiponectin are influenced only by the increase in body fat percentage in these women. Arch Endocrinol Metab. 2020;64(1):4-10.

Polycystic ovary syndrome associated with increased adiposity interferes with serum levels of TNF-alpha and IL-6 differently from leptin and adiponectin

ABSTRACT Objective The aim of this study was to investigate polycystic ovary syndrome (PCOS) and to explore the relationship between body fat percentage and metabolic markers. Subjects and methods Sedentary women were assigned to PCOS (N = 60) and CONTROL (N = 60) groups. Each group was subdivided into three subgroups according to body fat percentage (22-27%, 27-32% and 32-37%). The protocol consisted of assessments of glucose, insulin, androgens, follicle stimulating hormone (FSH), luteinizing hormone (LH), 17-hydroxyprogesterone (17-OHP), leptin, adiponectin, tumor necrosis factor (TNF-α) and interleukin-6 (IL-6). Results The PCOS subgroups showed higher concentrations of androgens, LH and 17-OHP. Leptin showed direct relationship with increased body fat percentage, whereas adiponectin showed the inverse effect. However, both were unaffected by PCOS. TNF-α and IL-6 were higher in PCOS women and showed a direct relationship with increased body fat percentage. Glucose showed direct relationship with body fat percentage, whereas insulin presented higher values in PCOS women and direct relationship with increased body fat percentage. Conclusions Our findings indicate that PCOS and body fat percentage directly influence concentrations of insulin, TNF-α and IL-6, whereas leptin and adiponectin are influenced only by the increase in body fat percentage in these women. Arch Endocrinol Metab. 2020;64(1):4-10

Clinical and molecular profile of newborns with confirmed or suspicious congenital adrenal hyperplasia detected after a public screening program implementation / Perfil clínico e molecular de recém-nascidos com suspeita ou confirmação de hiperplasia adrenal congênita após a implementação de um programa público de triagem neonatal

Abstract Objective: To describe the results obtained in a neonatal screening program after its implementation and to assess the clinical and molecular profiles of confirmed and suspicious congenital adrenal hyperplasia cases. Methods: A cross-sectional study was conducted. Newborns with suspected disease due to high 17-hydroxyprogesterone levels and adjusted for birth weight were selected. Classical congenital adrenal hyperplasia (salt-wasting and simple virilizing forms) was diagnosed by an increase in 17-hydroxyprogesterone levels as confirmed in the retest, clinical evaluation, and genotype determined by SNaPshot and multiplex ligation-dependent probe amplification. Results: After 24 months, 15 classic congenital adrenal hyperplasia cases were diagnosed in a total of 217,965 newborns, with an estimated incidence of 1:14,531. From 132 patients, seven non-classical and 14 heterozygous patients were screened for CYP21A2 mutations, and 96 patients presented false positives with wild type CYP21A2. On retest, increased 17-hydroxyprogesterone levels were found in classical congenital adrenal hyperplasia patients and showed significant correlation with genotype-related classical genital adrenal hyperplasia. The most frequent mutations were IVS2-13A/C>G followed by gene deletion or rearrangement events in the classical form. In non-classical and heterozygous diseases, p.Val282Leu was the most common mutation. Conclusions: The results underscore the effectiveness of congenital adrenal hyperplasia neonatal screening in the public health system and indicate that the adopted strategy was appropriate. The second sample collection along with genotyping of suspected cases helped to properly diagnose both severe and milder cases and delineate them from false positive patients.

Resumo Objetivo: Descrever os resultados obtidos em um programa de triagem neonatal após sua implementação e avaliar os perfis clínicos e moleculares de casos confirmados e suspeitos de hiperplasia adrenal congênita. Métodos: Foi feito um estudo transversal. Recém-nascidos com suspeita da doença devido aos altos níveis de 17-alfa-hidroxiprogesterona e ajustados pelo peso ao nascer foram selecionados. A hiperplasia adrenal congênita clássica (forma perdedora de sal e forma virilizante simples) foi diagnosticada por um aumento nos níveis de 17-alfa-hidroxiprogesterona confirmado no reteste, avaliação clínica e genótipo determinado com o uso do ensaio SNaPshot e amplificação multiplex de sondas dependente de ligação. Resultados: Após 24 meses, 15 casos clássicos de hiperplasia adrenal congênita foram diagnosticados em 217.965 recém-nascidos, com uma incidência estimada de 1:14.531. De 132 pacientes, sete não clássicos e 14 heterozigotos foram submetidos à triagem para mutações no gene CYP21A2 e 96 pacientes apresentaram resultados falso-positivos com CYP21A2 do tipo selvagem. No reteste, níveis aumentados de 17-alfa-hidroxiprogesterona foram encontrados em pacientes com hiperplasia adrenal congênita clássica e mostraram correlação significativa com HAC clássica relacionada ao genótipo. As mutações mais frequentes foram IVS2-13A/C>G, seguidas de deleção gênica ou eventos de rearranjo na forma clássica. Em casos de doenças não clássicas e heterozigose, a mutação p.Val282Leu foi a mais comum. Conclusões: Os resultados ressaltam a eficácia da triagem neonatal para a hiperplasia adrenal congênita no sistema público de saúde e indicam que a estratégia adotada foi adequada. A segunda coleta de amostras, juntamente com a genotipagem dos casos suspeitos, ajudou a diagnosticar adequadamente os casos graves e mais leves e diferenciá-los de pacientes com resultado falso-positivo.

High frequency of non-classical congenital adrenal hyperplasia form among children with persistently elevated levels of 17-hydroxyprogesterone after newborn screening.

Background Early diagnosis after newborn screening (NBS) for congenital adrenal hyperplasia (CAH) allows proper treatment, reducing mortality rates and preventing development of hyperandrogenic manifestations and incorrect sex assignment at birth. Despite the high NBS sensitivity to detect CAH classical forms, one of the main issues is identifying asymptomatic children who remained with increased 17-hydroxyprogesterone (17-OHP) levels. In this study, we aimed to contribute to understanding the diagnosis of these children. Methods Children with increased serum 17-OHP levels, and without disease-related clinical features during follow-up, underwent the entire CYP21A2 gene sequencing and multiplex ligation-dependent probe amplification (MLPA) analysis (SALSA MLPA P050B CAH). Patients' genotypes were subsequently sorted as compatible with CAH disease, and children were evaluated to determine the clinical status. Results During the study period, 106,476 newborns underwent CAH NBS. During follow-up, 328 children (0.3%) were identified as having false-positive tests and 295 were discharged after presenting with 17-OHP levels within reference values. Thirty-three remained asymptomatic and with increased serum 17-OHP levels after a mean follow-up of 3.4 years, and were subjected to molecular analysis. Seventeen out of the 33 children carried mutations: seven in the heterozygous state, nine carried non-classical genotypes and the remaining child carried a classical genotype. Conclusions We found a high frequency of non-classical CAH (NCCAH) diagnosis among children with persistent elevation of 17-OHP levels. Our findings support molecular study as decisive for elucidating diagnosis in these asymptomatic children. Molecular analysis as a confirmatory test is relevant to guide their follow-up, allows genetic counseling and avoids over treating NCCAH form.

Cortisol, progesterone, 17αOHprogesterone, and pregnenolone in foals born from mare's hormone-treated for experimentally induced ascending placentitis.

This study aimed to evaluate steroid hormones in foals born from mares treated for ascending placentitis with different combinations of trimethoprim-sulfamethoxazole (TMS), flunixin meglumine (FM), long-acting altrenogest (ALT) and estradiol cypionate (ECP) for ten consecutive days, starting two days after experimental induction of placentitis with Streptococcus zooepidemicus. Fourty-six pregnant mares and respective foals were assigned as healthy group (Control, n = 8) or treated groups as follows: TMS+FM (n = 8), TMS+FM+ALT (n = 8), TMS+FM+ALT+ECP (n = 6), TMS+FM+ECP (n = 6) and no treatment (NO TREAT n = 10). At delivery, foals were classified as high-risk or low-risk based on clinical and hematologic findings, and survival rates were recorded during the first week of life for comparisons across groups. Cortisol, progesterone, 17αOHprogesterone, and pregnenolone concentrations were determined via immunoassays in 31 of the 46 foals immediately after foaling (0 h), at 12, 24, 48 h, and seven days post-partum (168h). At birth, serum cortisol concentrations were higher in Control and TMS+FM+ECP foals than in remaining groups (p < 0.05). Foals in TMS+FM+ALT and TMS+FM groups had higher 17αOHprogesterone concentrations at 24 h and 48 h, respectively (p < 0.05). Pregnenolone concentrations were higher in TMS+FM than TMS+FM+ALT+ECP foals at 7 days (p < 0.05). High-risk and non-surviving foals had decreased concentrations of cortisol at parturition, but increased concentrations of progesterone from 0 h to 48 h. Pregnenolone and 17αOHprogesterone concentrations were increased and pregnenolone after 12 h in high-risk and non-surviving foals (p < 0.05). In conclusion, adding ECP to the treatment of experimentally-induced placentitis appears to improve foal viability and endocrine response. Cortisol and progestogen profiles were abnormal in high-risk and non-surviving foals, and those treated with ALT or TMS+FM only.

Clinical and molecular profile of newborns with confirmed or suspicious congenital adrenal hyperplasia detected after a public screening program implementation.

OBJECTIVE: To describe the results obtained in a neonatal screening program after its implementation and to assess the clinical and molecular profiles of confirmed and suspicious congenital adrenal hyperplasia cases. METHODS: A cross-sectional study was conducted. Newborns with suspected disease due to high 17-hydroxyprogesterone levels and adjusted for birth weight were selected. Classical congenital adrenal hyperplasia (salt-wasting and simple virilizing forms) was diagnosed by an increase in 17-hydroxyprogesterone levels as confirmed in the retest, clinical evaluation, and genotype determined by SNaPshot and multiplex ligation-dependent probe amplification. RESULTS: After 24 months, 15 classic congenital adrenal hyperplasia cases were diagnosed in a total of 217,965 newborns, with an estimated incidence of 1:14,531. From 132 patients, seven non-classical and 14 heterozygous patients were screened for CYP21A2 mutations, and 96 patients presented false positives with wild type CYP21A2. On retest, increased 17-hydroxyprogesterone levels were found in classical congenital adrenal hyperplasia patients and showed significant correlation with genotype-related classical genital adrenal hyperplasia. The most frequent mutations were IVS2-13A/C>G followed by gene deletion or rearrangement events in the classical form. In non-classical and heterozygous diseases, p.Val282Leu was the most common mutation. CONCLUSIONS: The results underscore the effectiveness of congenital adrenal hyperplasia neonatal screening in the public health system and indicate that the adopted strategy was appropriate. The second sample collection along with genotyping of suspected cases helped to properly diagnose both severe and milder cases and delineate them from false positive patients.

Generation of monoclonal antibodies against 17α-hydroxyprogesterone for newborn screening of congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by the deficiency of one of the five enzymes involved in the biosynthesis of corticosteroids. The most common form of the disease is the lack of 21-hydroxylase which provokes an accumulation of high levels of 17α-hydroxyprogesterone (17-OHP), the main biochemical marker for illness detection. Given the significance of neonatal diagnosis for ensuring a timely treatment to patients suffering from CAH, newborn screening is worldwide performed for the determination of 17-OHP from dried blood spots on filter paper. The non-specificity of antisera employed in immunoassays and the cross-reaction with fetal adrenal hormones produce an overestimation in the 17-OHP quantification. Immunization of mice with 17-OHP-3-(O-carboxymethyl) oxime-bovine serum albumin led to the generation of 15 anti-17-OHP IgG1-and-IgG2b-secreting hybridomas. The 6E2G9 monoclonal antibody presents cross-reactivity values similar to those achieved by rabbit antibodies employed in the solid phase of UMELISA® 17-OH Progesterona Neonatal, assay for the newborn screening of CAH in Cuba. Additionally, the use of 6E2G9 in the evaluation of dried blood spots samples from newborns on filter paper showed a decrease in the mean 17-OHP levels, thus demonstrating it can replace the conventional rabbit antisera.

17-hydroxiprogesterone values in healthy preterm infants / Valores de 17-hidroxiprogesterona en recién nacidos prematuros sanos

Abstract Introduction: In preterm newborn, problems with the interpretation of 17-OHP may occur. Objective: Evaluate 17-OHP values in healthy preterm newborns until they reach the corrected gestational age. Methods: Longitudinal study of 36 preterm infants with 17-OHP evaluation using ELISA from heel blood from 3 to 5 days and thereafter every 2 weeks until the corrected gestational age. Values adjusting multiple variables such as gestational age, birth weight and sex, among others were compared. The results were analyzed against 82 healthy full-term infants. Results: In the first week of life, early term infants born within less than 34 months of gestational age show 17-OHP values that are much higher than the full term neonates. After a week, the values decrease and stabilize, but are still higher than those of full term neonates and remain so even at the corrected gestational age. (average difference of 63.0%, CI 95%: 11.8%-115.5%). 33.6% (41 samples) of a total of 122 samples taken from preterm infants were higher than 30 ng/mL. Conclusions: 17-OHP values in early term infants are higher than those in full term neonates and can be related to postnatal adaptive processes. It is suggested that a second screening at the 37th week of corrected age be performed.

Resumen Introducción: En recién nacidos pretérmino se presentan problemas para interpretar la 17-OHP. Objetivo: Evaluar los valores de 17-OHP en recién nacidos sanos pretérmino hasta cuando alcanzan el término de edad gestacional corregida. Métodos: Estudio longitudinal de 36 prematuros con evaluación de la 17-OHP por ELISA en sangre de talón desde los 3-5 días de vida y luego cada dos semanas hasta la edad gestacional de término corregida. Se comparó los valores ajustando múltiples variables como edad gestacional, peso al nacer y sexo, entre otras. Se analizaron los resultados frente a los de 82 recién nacidos a término sanos. Resultados: En la primera semana de vida, los prematuros menores de 34 semanas de edad gestacional tienen valores de 17-OHP muy superiores a los neonatos de término. Al alcanzar la semana 34 de edad gestacional corregida, los valores descienden y se mantienen estables, siempre mayores a los de término, incluso al llegar a edad a término corregida (diferencia promedio de 63.0%, IC 95%: 11.8%-115.5%). El 33.6% (41 muestras) de un total de 122 muestras hechas en los prematuros eran mayores de 30 ng/mL. Conclusiones: Los valores de 17-OHP en recién nacidos pretérmino son más altos que en neonatos a término, pudiendo ser relacionado con los procesos adaptativos postnatales. Se sugiere realizar un segundo tamizaje al llegar a la semana 37 de edad corregida.

Neonatal screening for congenital adrenal hyperplasia in Southern Brazil: a population based study with 108,409 infants.

BACKGROUND: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder associated with inborn errors of steroid metabolism. 21-hydroxylase enzyme deficiency occurs in 90 to 95% of all cases of CAH, with accumulation of 17 hydroxyprogesterone (17-OHP). Early diagnosis of CAH based on newborn screening is possible before the development of symptoms and allows proper treatment, correct sex assignment, and reduced mortality rates. This study describes the results obtained in the first year of a public CAH screening program in the state of Rio Grande do Sul, Brazil. METHODS: We reviewed the screening database in search of babies with suspected CAH, that is, altered birth-weight adjusted 17-OHP values at screening. The following data were analyzed for this population: screening 17-OHP values, retest 17-OHP values, serum 17-OHP values for those with confirmed CAH on retest, maternal and newborn data, and family history of CAH. For the screening program, 17-OHP levels are determined on dried blood spots obtained in filter paper with GSP solid phase time-resolved immunofluorescence. RESULTS: Of 108,409 newborns screened, eight were diagnosed with CAH (four males, four females). The incidence of CAH in the state was 1:13,551. Six cases were identified as classic salt-wasting CAH and two were cases of virilizing CAH. The positive predictive value (PPV) of the initial screening (before diagnostic confirmation) was 1.6%. The overall rate of false positive results was 0.47%. The number of false positive results was higher among newborns with birth weight < 2000 g. CONCLUSION: The present results support the need for CAH screening by the public health care system in the state, and show that the strategy adopted is adequate. PPV and false positive results were similar to those reported for other states of Brazil with similar ethnic backgrounds.

Assessment of the body composition of patients with polycystic ovary syndrome using dual-energy X-ray absorptiometry.

OBJECTIVE: To compare the body composition among patients with polycystic ovary syndrome (PCOS) and patients without PCOS. METHODS: A cross-sectional study enrolled patients aged 12-39 years, with body mass index (BMI, calculated as weight in kilograms divided by the square of height in meters) at least 18.5 but below 25, who attended the Endocrine Gynecology Clinic of Santa Casa de Sao Paulo School of Medical Sciences, Brazil, between January 1, 2014, and July 31, 2015. Anthropometric measurements, metabolic and androgenic profiles, and dual-energy X-ray absorptiometry measurements were compared between patients with PCOS and those without PCOS. RESULTS: In total, 102 eligible patients attended the study clinical during the study period; 43 were excluded owing to not meeting the inclusion criteria or declining to undergo complete study testing, and 15 withdrew from the study. Of the 44 participants, 28 had PCOS and 16 were included in the control group. Serum 17-hydroxyprogesterone concentration (P=0.046), leg-fat (P=0.031), and truncal-fat (P=0.001) were all higher among patients with PCOS. CONCLUSION: The present study demonstrated increased truncal and leg fat among women with PCOS. The study did not detect any difference in insulin parameters but larger studies could be more suitably powered to investigate this. CLINICALTRIALS.GOV: NCT02467751.

Neonatal 17-hydroxyprogesterone levels adjusted according to age at sample collection and birthweight improve the efficacy of congenital adrenal hyperplasia newborn screening.

INTRODUCTION: The primary concern related to congenital adrenal hyperplasia (CAH) newborn screening (NBS) is the high rate of false-positive results (FPR) associated with prematurity; false-negative results (FNR) can also occur due to precocious sample collection. OBJECTIVE: To determine the neonatal 17-hydroxyprogesterone (N17OHP) normal range in newborns in Sao Paulo using different references according to age and birthweight (BW) and to establish the optimal NBS cut-off levels. METHODS: Neonatal 17-hydroxyprogesterone levels from 271 810 newborns (NBs) according to sample collection time (G1: 48-<72 h and G2: ≥72 h) and BW (≤1500 g, 1501-2000 g, 2001-2500 and >2500 g) were evaluated. N17OHP was measured by an fluoroimmunoassay, and serum 17OHP was measured by liquid chromatography-mass spectrometry. Affected and asymptomatic NBs with persistently increased 17OHP levels were submitted to CYP21A2-sequencing. RESULTS: Neonatal 17-hydroxyprogesterone levels in G1 were lower than G2 in all BW groups (P < 0·001). The FPR rate in G1/G2 was 0·2% using the 99·8th and 0·5% using the 99·5th percentile. The 99·8th percentile N17OHP value was the best cut-off for distinguishing between unaffected and affected NBs. Forty-four salt wasters, and five simple virilisers were diagnosed; N17OHP levels ranged from 93·3 to 2209·8 nmol/l, and no affected neonates with FNR were identified. The positive predictive value in G1 and G2 using the 99·8th percentile was 5·6% and 14·1%, respectively, and 2·3% and 7%, respectively, using the 99·5th percentile. Molecular tests identified two NBs with the nonclassical form among the 29 FPR. CONCLUSION: Neonatal 17-hydroxyprogesterone levels adjusted to sample collection age and birthweight reduced the FPR, and the use of N17OHP values based upon the 99·8th percentile improved the NBS efficacy.

17-hydroxiprogesterone values in healthy preterm infants.

INTRODUCTION: In preterm newborn, problems with the interpretation of 17-OHP may occur. OBJECTIVE: Evaluate 17-OHP values in healthy preterm newborns until they reach the corrected gestational age. METHODS: Longitudinal study of 36 preterm infants with 17-OHP evaluation using ELISA from heel blood from 3 to 5 days and thereafter every 2 weeks until the corrected gestational age. Values adjusting multiple variables such as gestational age, birth weight and sex, among others were compared. The results were analyzed against 82 healthy full-term infants. RESULTS: In the first week of life, early term infants born within less than 34 months of gestational age show 17-OHP values that are much higher than the full term neonates. After a week, the values decrease and stabilize, but are still higher than those of full term neonates and remain so even at the corrected gestational age. (average difference of 63.0%, CI 95%: 11.8%-115.5%). 33.6% (41 samples) of a total of 122 samples taken from preterm infants were higher than 30 ng/mL. CONCLUSIONS: 17-OHP values in early term infants are higher than those in full term neonates and can be related to postnatal adaptive processes. It is suggested that a second screening at the 37th week of corrected age be performed.

INTRODUCCIÓN: En recién nacidos pretérmino se presentan problemas para interpretar la 17-OHP. OBJETIVO: Evaluar los valores de 17-OHP en recién nacidos sanos pretérmino hasta cuando alcanzan el término de edad gestacional corregida. MÉTODOS: Estudio longitudinal de 36 prematuros con evaluación de la 17-OHP por ELISA en sangre de talón desde los 3-5 días de vida y luego cada dos semanas hasta la edad gestacional de término corregida. Se comparó los valores ajustando múltiples variables como edad gestacional, peso al nacer y sexo, entre otras. Se analizaron los resultados frente a los de 82 recién nacidos a término sanos. RESULTADOS: En la primera semana de vida, los prematuros menores de 34 semanas de edad gestacional tienen valores de 17-OHP muy superiores a los neonatos de término. Al alcanzar la semana 34 de edad gestacional corregida, los valores descienden y se mantienen estables, siempre mayores a los de término, incluso al llegar a edad a término corregida (diferencia promedio de 63.0%, IC 95%: 11.8%-115.5%). El 33.6% (41 muestras) de un total de 122 muestras hechas en los prematuros eran mayores de 30 ng/mL. CONCLUSIONES: Los valores de 17-OHP en recién nacidos pretérmino son más altos que en neonatos a término, pudiendo ser relacionado con los procesos adaptativos postnatales. Se sugiere realizar un segundo tamizaje al llegar a la semana 37 de edad corregida.

The involvement of gonadotropins and gonadal steroids in the ovulatory dysfunction of the potamodromous Salminus hilarii (Teleostei: Characidae) in captivity.

Potamodromous teleosts that require migration to reproduce show dysfunctions that block ovulation and spawning while in captivity. To understand the physiological basis of these reproductive dysfunctions, follicle-stimulating hormone b subunit (fshb) and luteinizing hormone b subunit (lhb) gene expression analyses by real-time quantitative PCR, together with measurements of estradiol (E 2), 17α-hydroxyprogesterone (17α-OHP) and 17α,20ß-dihydroxy-4-pregnen-3-one (17α,20ß-DHP) levels, were carried out throughout the reproductive cycle of the potamodromous Salminus hilarii. The following reproductive stages were evaluated in captive and wild females: previtellogenic (PV), advanced maturation/mature (AM) and regression/spent (REG/SPENT). In the wild females, fshb expression decreased from the PV to the AM stage, and the opposite pattern was detected for E 2, which increased from the PV to the AM stage. fshb was expressed at lower levels in captive than in wild females, and this difference did not change during the reproductive cycle. lhb expression also increased from the PV to the AM stage in both groups, but the wild females at the AM and REG/SPENT stages showed higher lhb expression levels than the captive females. The concentrations of 17α-OHP did not change during the reproductive cycle, and the levels were higher in the captive than in the wild females at all reproductive stages. 17α,20ß-DHP levels did not change between wild and captive females. However, in captive females, the transition from PV to AM stage was followed by an increase in 17α,20ß-DHP levels. These data indicate that dysfunctions in the gonadotropins and steroids synthesis pathways cause the ovulation failure in captive S. hilarii.

Characterization of persistent follicles induced by prolonged treatment with progesterone in dairy cows: an experimental model for the study of ovarian follicular cysts.

Cystic ovarian disease (COD) is a major factor contributing to poor reproductive efficiency of lactating dairy cows. The objective of the present study was to analyze the endocrine profile, growth dynamics, and histologic characteristics of persistent ovarian follicles-cysts developing in response to long-term administration of intermediate levels of progesterone. To this end, after synchronization of cows, a low dose of progesterone was administered for 5, 10, and 15 days after the expected day of ovulation in treated cows (groups P5, P10, and P15, respectively), using an intravaginal progesterone-releasing device. A significant increase in diameter was detected on Day 11 of progesterone treatment and thereafter (P < 0.05), and at Day 15 of persistence, the diameter of the persistent follicle reached a mean of 23 ± 0.6 mm. Microscopically, the persistent follicles had a complete granulosa, an intensely vascularized theca interna, and a collagenous theca externa layer. Temporal changes in the serum concentrations of estradiol, progesterone, and FSH were detected (effects of time, P < 0.01). Progesterone treatment completely inhibited the LH preovulatory surge in treated cows and affected the basal concentration of LH. The pulse frequency remained high at 5 and 10 days of persistence and declined (P < 0.05) after 15 days of persistence. The LH pulse concentration and pulse amplitude had a significant reduction (P < 0.05) during follicular persistence. Changes in the serum levels of estradiol, progesterone, 17-hydroxyprogesterone, and testosterone in serum and follicular fluid were also observed. In serum, estradiol increased gradually from proestrus to Day 10 of follicular persistence (P < 0.05), progesterone showed an increase (P < 0.05) at Day 5 of follicular persistence, 17-hydroxyprogesterone showed a significant decrease at 5 days of follicular persistence in relation to proestrus, and testosterone showed a significant increase (P < 0.05) from proestrus and Day 5 of persistence through Day 15 of follicular persistence. Correlation between serum and follicular fluid steroid concentrations was significant for testosterone (P < 0.0001) and not significant for estradiol and progesterone. These findings indicate that ovarian cysts in COD are similar in many ways to the persistent follicles induced by progesterone, with an analogous hormonal and morphologic context, thus confirming a local role of subluteal levels of progesterone in COD pathogenesis and in the regulatory mechanisms of the ovarian function.

Leuprolide acetate-stimulated androgen response during female puberty.

BACKGROUND: A physiological increase in androgen levels occurs during adolescence. Measuring androgen concentrations is the best method to distinguish normal evolution processes from hyperandrogenic disorders. HYPOTHESIS: The increase in circulating androgens during puberty is inversely associated with insulin sensitivity in normal weight girls. OBJECTIVE: To assess circulating levels of ovarian androgens and anti-Müllerian hormone (AMH) at baseline and after GnRH analogue (GnRH-a) stimulation in normal pubertal girls across different Tanner stages. We also studied the association between this response and insulin sensitivity. DESIGN: Prospective study of healthy girls (6-12 years) from the local community (n = 63). METHODS: Tanner I (n = 23) subjects were assessed cross-sectionally, and Tanner II girls (n = 40) were evaluated every 6 months until they reached Tanner V. Early morning dehydroepiandrosterone sulphate (DHEA-S), AMH, sex hormone-binding globulin (SHBG), androstenedione, glucose and insulin levels were measured. A GnRH-a test (500 µg/m(2) ; sc) and oral glucose intolerance test (OGTT) were performed. Differences throughout puberty were evaluated. RESULTS: Basal and/or stimulated Testosterone DHEA-S and 17-hydroxyprogesterone (17OHP) were inversely associated with insulin sensitivity (WIBSI) from the beginning of puberty, whereas androstenedione was directly associated with gonadotrophins. AMH was inversely associated with basal and stimulated gonadotrophins and directly with insulin area under the curve (AUC) only in the early stages of puberty. 17OHP and testosterone responsiveness increased significantly during puberty in all subjects, whereas testosterone levels changed less consistently. This pattern of ovarian-steroidogenic response was most evident during mid- and late puberty. Moreover, during late puberty only, basal 17OHP, testosterone and DHEA-S were positively associated with gonadotrophins. CONCLUSION: In normal nonobese girls born appropriate for gestational age, androgen synthesis was associated with insulin sensitivity in early puberty and with LH only in late puberty.

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

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

Puntos de corte de 17-hidroxiprogesterona en la detección de Hiperplasia Suprarrenal Congénita / Cut offs levels of 17-hydroxyprogesterone in screening of Congenital Adrenal Hyperplasia / Pontos de corte da 17-hidroxiprogesterona na detecção de Hiperplasia Adrenal Congênita

El presente estudio investiga la utilidad de determinar puntos de corte ajustados según la edad gestacional y el peso al nacer de neonatos (2-100 días) en la cuantificación de 17-hidroxiprogesterona en muestras de sangre seca en papel de filtro. Se analizaron los resultados de 6.266 determinaciones realizadas en el marco del Programa Nacional de Fortalecimiento de la Detección Precoz de Enfermedades Congénitas. Los datos se dividieron en cuatro grupos; Grupo 1: recién nacido pretérmino con bajo peso; Grupo 2: recién nacido pretérmino con peso normal; Grupo 3: recién nacido a término con bajo peso y Grupo 4: recién nacido a término con peso normal. Se establecieron puntos de corte diferentes a partir del cálculo del percentilo 99 de la distribución de frecuencias. Basado en este análisis se realizó la comparación de la tasa de resultados falsos positivos que se obtuvieron según el punto de corte establecido por el fabricante y los obtenidos en el estudio. Los nuevos puntos de corte obtenidos fueron: 217,72 nmol/L, 102,14 nmol/L, 61,62 nmol/L y 82,38 nmol/L para los grupos 1, 2, 3 y 4 respectivamente. Se evidenció una tasa total de falsos positivos del 1% con los nuevos puntos de corte, significativamente menor a la tasa del 6,2% obtenida al utilizar el punto de corte del fabricante. Esto puso en evidencia que el uso de puntos de corte adecuadamente establecidos para la población en estudio reduce significativamente las complicaciones derivadas de las repeticiones de análisis y eventualmente la tasa de recitaciones, lo cual es una importante contribución a la Salud Pública.

The present work studies the usefulness of determining adjusted cut-offs for the quantification of 17-hydroxyprogesterone in dried blood samples on filter paper, taking into account the gestational age and weight of the neonates. The results of 6266 determinations made within the framework of the National Program of Strengthening Early Detection of Congenital Disease were analysed. Data were divided into groups, Group 1: early established from the calculation of the 99 percentiles of the frequency distribution. New cutoff points were: 217.72 nmol/L, 102.14 nmol/L, 61.62 nmol/L and 82.38 nmol/L for groups 1, 2, 3 and 4 respectively. It showed a total rate of 1% false positives with the new cut-off points, which was significantly lower than the rate of 6.2% obtained using the manufacturer's cutoff. This revealed that the use of properly established cut-offs for the study of population reduces significantly the complications derived fromn analysis repetitions and eventually the recitation rate, which is an important contribution to Public Health.

O presente estudo investiga a utilidade de determinar pontos de corte estabelecidos conforme a idade gestacional e o peso ao nascer de neonatos (2-100 dias) na quantificação da 17-hidroxiprogesterona em amostras de sangue seco em papel filtro. Foram analisados os resultados de 6.266 determinações feitas no âmbito do Programa Nacional de Fortalecimento da Detecção Precoce de Doenças Congênitas. Os dados foram divididos em quatro grupos; Grupo 1: recém-nascido pré-termo com baixo peso, Grupo 2: recém-nascido pré-termo com peso normal, Grupo 3: recém-nascido a termo com baixo peso e Grupo 4: recém-nascido a termo com peso normal e foram estabelecidos pontos de corte diferentes a partir do cálculo do percentil 99 da distribuição de frequências. Com base nesta análise foi realizada a comparação da taxa de resultados falsos positivos obtidos conforme o ponto de corte estabelecido pelo fabricante e os obtidos no estudo. Os novos pontos de corte obtidos foram: 217,72 nmol/L, 102,14 nmol/L, 61,62 nmol/L e 82,38 nmol/L para os grupos 1, 2, 3 e 4, respectivamente. Tornou-se evidente uma taxa total de 1% de falsos positivos, com os novos pontos de corte significativamente menor do que a taxa de 6,2% obtida utilizando o ponto de corte do fabricante. Isto revelou que o uso de pontos de corte de forma adequada estabelecidos para a população em estudo reduz significativamente as complicações decorrentes das repetições de análises e eventualmente a taxa de repetição de novos encontros, o que é uma importante contribuição para a saúde pública.

A three-year follow-up of congenital adrenal hyperplasia newborn screening.

OBJECTIVE: congenital adrenal hyperplasia (CAH) newborn screening can prevent neonatal mortality in children with the salt-wasting form of the disease and prevent incorrect gender assignments, which can occur in females. However, the occurrence of false-positive results in preterm or low-birth-weight newborns creates some diagnostic difficulties, with consequent therapeutic implications. This study aimed to report the results of a pilot project for neonatal CAH screening conducted in the state of Minas Gerais, Brazil from 09/2007 to 05/2008 with a three-year follow-up. METHODS: dried blood specimens were collected on filter paper cards three to seven days after birth of all newborns in the period. Samples were analyzed for 17-hydroxyprogesterone using an enzyme-linked immunosorbent assay (ELISA). RESULTS: a total of 159,415 children were screened. The apparent incidence of the classic variant of the disease was 1:9,963, based on initial diagnoses following newborn screening. During the follow-up period, eight of 16 children initially diagnosed with CAH were reclassified as unaffected, resulting in a revised incidence of 1:19,927. The false-positive rate was 0.31%, and the positive predictive value was 2.1%. Sensitivity and specificity were 100% and 99.7%, respectively. CONCLUSIONS: newborn screening is an important public health policy in developing countries such as Brazil, where CAH remains underdiagnosed. It has great potential to identify children with the disease who otherwise cannot be diagnosed earlier. Long-term follow-up and monitoring of all children with positive screening results are crucial to ensure a correct diagnosis and to calculate a reliable incidence ratio of the disease.