AIMS To determine normal concentrations of 17α-hydroxyprogesterone (17OHP) for premature infants.
METHODS 17OHP was measured in 66 consecutive premature infants once a week during the first month, and once every two weeks thereafter, until the age of 3 months. The 17OHP values in 100 full term healthy neonates on the third day of life served as controls. Blood was sampled on filter paper using a neonatal radioimmunoassay kit. Findings were correlated with gestational age, birthweight, mode of delivery, Apgar scores, presence of respiratory distress syndrome and intake of maternal steroids.
RESULTS Mean 17OHP was raised at 7 days of age (138.9, 46.3, 53.3, 29.9 nmol/l, respectively, for infants whose gestational age was under 29 weeks, 29 to 30 weeks, 31 to 32 weeks, and 33 weeks and above). It fell sharply in the first two weeks after which it gradually decreased further, reaching 32.7, 23.6, 16.9, and 13.0 nmol/l, respectively, by the age of 90 days. The mean (SEM) 17OHP concentration in full term infants on day 3 of life was 17.8 (8.9) nmol/l. These values were independent of the presence and severity of respiratory distress syndrome and of prenatal maternal steroids.
CONCLUSIONS The increased 17OHP concentrations found at birth fell to those found in term infants during the first three months of life in infants over 31 weeks of gestation. Postconceptional age is the most important factor determining 17OHP concentration.
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Congenital adrenal hyperplasia (CAH) is a family of recessive inherited disorders of adrenal steroid hormone origin, caused in 90–95% of cases, by 21-hydroxylase deficiency.1 CAH is suspected in newborn girls with ambiguous genitalia, phenotypic boys with bilateral cryptorchidism, older boys with progressive virilisation and in neonates with acute adrenal insufficiency.2-4 The diagnosis is readily confirmed by increased 17α-hydroxyprogesterone (17OHP) concentrations.
Over the past two decades neonatal (age 2–5 days) screening programmes for high 17OHP values using heel blood spots have been instituted in several regions and countries.1 5-7 In Israel a nationwide random screening programme revealed a high incidence of CAH in the Arab population, which prompted a second programme for neonates in northern Israel.8 One of the major problems encountered in our programme and in others is the high incidence of false positive results in preterm infants.2-5 8-13
This study aimed to determine normal concentrations of 17OHP for premature infants during the first three months of life and to correlate these with postconceptional age, birthweight, incidence and severity of respiratory distress syndrome, and antenatal use of steroids.
The study was conducted in the neonatal intensive care unit of the Chaim Sheba Medical Center. Sixty six consecutive premature infants (gestational age <35 weeks, birthweight <2000 g), born between January and August 1994, were included. One hundred term healthy infants (gestational age >37 weeks, birthweight >2500 g) served as controls. The study was approved by the hospital’s human rights committee, and informed consent was obtained from the parents of each participant. The clinical characteristics of the patients are shown in table 1. 17OHP was correlated with sex, Apgar score, mode of delivery (vaginal or caesarean) the need for blood transfusions, presence of neonatal sepsis, incidence and severity of respiratory distress syndrome—mild, need for oxygen; moderate, need for mechanical ventilation for up to 5 days; severe, need for mechanical ventilation > 5 days—or prenatal treatment with dexamethasone for fetal lung immaturity.
In the preterm group blood was sampled on filter paper during the first 24 hours, then once a week during the first 28 days, and thereafter every two weeks until 60 days of age, and again at 90 days. In the term group blood samples were obtained on the third day of life. 17OHP concentrations were measured by radioimmunoassay (Diagnostic Products Corporation, Los Angeles, CA) in the National Unit for Congenital Hypothyroidism and Screening Program for 21-hydroxylase deficiency.8
Blood spot 17-OHP concentrations were determined using a solid phase125I radioimmunoassay with the Coat-A-Count Neonatal 17-alpha-OH Progesterone kit (Diagnostic Products Corporation, Los Angeles, CA).
The procedure is based on a polyclonal (rabbit) antibody and designed for use with blood spot samples. 125I-labelled 17OHP competes for a fixed time with 17OHP in the patient sample for antibody sites. As the antibody is immobilised to the wall of a tube, decanting suffices to terminate the competition and to isolate the antibody bound fraction of the radiolabelled 17OHP. Counting the tube in a gamma counter then yields a number which converts, by way of a calibration curve, to a measure of the 17OHP in the patient sample.
The sensitivity of the 17OHP assay is 0.8 nmol/l and the intra- and interassay coefficients of variation (CV) are 6% and 0%, respectively. Cross reactivity with other naturally occurring steroids or therapeutic drugs is extremely low, maximally to 17OHP (3.5%) and to 11-deoxycortisol (0.8%).
Statistical analysis was performed using BMDP Statistical Software.14 One and two way analysis of variance and covariance with repeated measures, Fisher’s exact test, Pearson’s χ2 test, paired t test and regression analysis were used, as appropriate. Correlations were performed using Student’s linear regression analysis.
Mean 17OHP concentrations by day 7 of life were higher in the premature infants than in full term infants on day 3 of life (p<0.01). Concentrations sharply declined in the first two weeks, and then continued to decrease more gradually. By the age of 3 months, the average 17OHP concentrations for preterm infants over 29 weeks decreased to those of full term infants at the age of 3 days. However, for infants below 29 weeks, the concentration at 3 months was almost double that of full term infants (p<0.05). Only those who were born at more than 33 weeks of gestation achieved term values by 60 days of age. During the first two weeks of life, 17OHP concentration was inversely correlated with postconceptional age and birthweight. Analysis of variance and covariance was significant for both factors, but stronger for postconceptional age (p<0.0001) (fig 1) than for birthweight (table 2) (p<0.004). The normalisation of 17OHP concentrations was slower in the smaller and younger infants (500–1000 g; 26–28 weeks, respectively). Analysis of variance and covariance showed no relation between 17OHP concentrations and sex, Apgar score, mode of delivery, need for blood transfusions, presence of neonatal sepsis, incidence and severity of respiratory distress syndrome, or prenatal treatment with dexamethasone for fetal lung immaturity. Regression analysis revealed a significant influence for both birthweight and postconceptional age on 17OHP concentrations (R2=0.058, F=11.9, p<0.00001).
The higher rate of respiratory distress syndrome in the young and low birthweight premature infants gave the impression that these infants have higher 17OHP concentrations. However, analysis of variance and covariance showed that it was gestational age rather than the lung disorder which was the significant factor.
Although pilot programmes for infant screening for high 17OHP concentrations began more than 15 years ago, only 23 are used today in 10 countries. This is partly due to the high financial outlay required, but also to the psychological cost of the high rate of false positive results,15 particularly in preterm, small for gestational age, and sick infants under stress.5 11 The latter may be attributed to the following: differences in the activity of enzymes in the glucocorticoid pathway which may be developmentally regulated to increase with advancing postconceptional age; decreased metabolic clearance of 17OHP due to immature hepatic function; physiological pituitary adrenal stress response; and cross reaction with conjugated steroids in the premature neonate’s serum.4 10 12 For these reasons, higher recall values for preterm infants have since been established in the various screening programmes (table3).
Contrary to Thompson et al,3who reported that age at the time of specimen collection had no effect on 17OHP concentrations in infants weighing under 2500 g at birth, we found that they sharply declined in the first two weeks of life and thereafter gradually reached full term values within the first three months of life. This phenomenon was accentuated in very young and low birthweight infants.
Al Saedi et al 4 were the first to describe sequential sampling of 17OHP concentrations in preterm infants, performed weekly until 37 weeks of age. However, their mean concentrations were lower than those found in this study. Normal 170HP concentrations vary considerably, as noted in the various screening programmes; this reflects differences in the assay used (RIA, ELISA, FIA), antibody specificity, and the thickness of the blood spot.5 Mean 17OHP concentrations were not affected by the incidence of caesarean section, low Apgar scores, incidence and severity of respiratory distress syndrome, or maternal use of steroids. Gestational age had a greater effect on 17OHP concentrations than birthweight. This contrasts with the findings of Cacciariet al,16 who found that birthweight was more significant than gestational age in determining 17OHP concentration in infants weighing under 2500 g.
Recently, Allen et al 15 suggested a four tiered weight adjusted threshold for 17OHP. This reduced the number of false positive results, particularly among low birthweight infants, without diminishing the sensitivity of the test. Birthweight was chosen for categorisation because it was thought that the birthweight data supplied on the newborn screening form were more reliable and accurate than those on gestational age. Their criteria, however, did not take into account the day of specimen collection, an important variable in low birthweight babies.
In conclusion, increased 17OHP concentrations in premature infants may lead to a misdiagnosis of congenital adrenal hyperplasia. Notably, 17OHP concentrations in premature infants are independent of maternal glucocorticoid treatment, and the incidence and severity of respiratory distress syndrome, and will normally reach full term values within the first three months of life.
We are grateful to Mrs Ilsa Katz for her support, Mrs Pearl Lilos of the Department of Statistics, Tel Aviv University, for performing the data analysis, and Mrs Ora Amado and Mrs Judith Bruck of the Central Endocrinology Laboratory for their excellent technical assistance.
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