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Division of Paediatric Endocrinology, Department of Paediatrics, Christian-Albrechts-University Kiel, 24105 Kiel, Germany
Address all correspondence and requests for reprints to: Carl-Joachim Partsch, M.D., Division of Pediatric Endocrinology, Department of Pediatrics, Christian-Albrechts-University of Kiel, Schwanenweg 20, D-24105 Kiel, Germany. E-mail: . partsch{at}pediatrics.uni-kiel.de
Abstract
Pregnenolone (Preg) and 17-hydroxypregnenolone (17OH-Preg) are marker steroids that are elevated in the 3ß-hydroxysteroid-dehydrogenase-II deficiency form of congenital adrenal hyperplasia. The aim of this study was to establish normative data for both steroids in healthy preterm (28–33 and 34–37 wk gestation) and full-term infants, because reference values for the early neonatal period do not exist.
At delivery, the main source of Preg is the placenta, because the highest levels were found in the retroplacental space (median, 141.31 nmol/liter), with no significant difference between preterm and full-term pregnancies. The fetal adrenals provide most of the circulating 17OH-Preg in full-term neonates, as demonstrated by a marked arteriovenous gradient in cord blood (40.96 nmol/liter vs. 10.77 nmol/liter). 17OH-Preg levels in the umbilical arteries were significantly lower in premature infants than in full-term infants (8.06 nmol/liter vs. 40.96 nmol/liter). During the first 2 postnatal weeks, Preg concentrations showed a rapid and significant fall in early preterm infants [95.78 nmol/liter (0 h) to 36.69 nmol/liter (d 14)] as well as in full-term infants [66.62 nmol/liter (0 h) to 14.81 nmol/liter (d 6)]. In addition, a significant fall in 17OH-Preg levels was found in full-term neonates [40.96 nmol/liter (0 h) to 11.32 nmol/liter (d 6)]. After 12 h, significantly higher levels for Preg and 17OH-Preg were found in early preterm infants (98.01 nmol/liter and 69.13 nmol/liter), compared with full-term neonates (36.29 nmol/liter and 28.55 nmol/liter, P < 0.05), reflecting the increased fetocortical activity as a response to the stress of delivery in the prematures. With these longitudinal data, it is now possible to confirm or exclude the diagnosis of 3ß-hydroxysteroid-dehydrogenase-II deficiency within the first postnatal week.
THE 
5-STEROIDS PREGNENOLONE (3ß-hydroxypregn-5-en-20-one) (Preg) and 17-hydroxypregnenolone (3ß, 17-dihydroxypregn-5-en-20-one) (17OH-Preg) are substrates for the enzyme 3-ß-hydroxysteroid dehydrogenase (3ß-HSD) (EC 1.1.1.145), which is necessary for the biosynthesis of all steroid hormones, i.e. of adrenal glucocorticoids and mineralocorticoids, and of gonadal sex hormones. The two 3ß-HSD isoenzymes, designated type 1 and 2, are encoded by the two genes HSDB1 and HSDB2 on chromosome 1p13.1 (1, 2, 3, 4). The type 1 isoform is expressed in the placenta and other peripheral tissues, whereas the type 2 isoform is expressed in the adrenal gland, the ovary, and the testis (3, 4). The 3ß-HSD-II-deficiency form of congenital adrenogenital hyperplasia (CAH) results in incomplete sexual differentiation in male children or in virilization of female infants at birth. The complete block of 3ß-HSD-II is associated with salt and water loss (5). 3ß-HSD-II-deficiency is the third most common form of CAH (6, 7). The plasma steroid precursors Preg and 17OH-Preg are the best markers for a rapid early diagnosis of CAH because of 3ß-HSD-II-deficiency (5, 6, 7, 8, 9, 10). There are few data on Preg and 17OH-Preg in normal full-term infants (10, 11, 12, 13, 14) and older children (15, 16, 17, 18). We were able to set up reference data for prepubertal children and adults (19). This reference data allowed the biochemical diagnosis of 3ß-HSD-II deficiency in a child originating from Sri Lanka. The diagnosis was confirmed by direct sequencing of the HSD3B2 gene (20). It is believed that plasma concentrations of Preg and 17OH-Preg decline during the first days of life in full-term infants (6). There is only little cross-sectional data available on 17OH-Preg plasma concentrations in preterm children (21, 22); and, to our knowledge, there are no longitudinal reference values for Preg and 17OH-Preg concentrations in preterm children during the early neonatal period. Such data are of importance because affected infants may be seen at an early age in the case of an affected sibling or because of the possibility of prenatal diagnosis based on molecular findings (20, 23). We therefore longitudinally studied the age-related changes of Preg and 17OH-Preg concentrations in a small plasma sample of 200 µl in full-term and preterm infants, from birth to 2 wk of age, by means of a newly developed simultaneous RIA, after extraction and thorough separation from other steroid hormones.
Materials and Methods
Fourteen full-term neonates (full-term group) (birth weight 2560–4150 g; 38–41 wk gestation) of both sexes (9 males, 5 females), 11 premature neonates born at 28–33 wk gestation (group 1) (birth weight, 1050–2300 g) of both sexes (4 males, 7 females), and 9 premature neonates born at 34–37 wk gestation (group 2) (birth weight, 1670–3200 g) of both sexes (4 males, 5 females) were studied longitudinally, from birth to d 7 (full-term neonates) and d 14 (preterm neonates), respectively. The length of gestation was determined by the last menstrual period and by antenatal ultrasound. The mothers (20–37 yr old) were healthy; and, except for the preterm delivery, their pregnancies were uneventful. None of the mothers received glucocorticoids for the prevention of respiratory distress syndrome. None of the included neonates received glucocorticoids during the study. Rupture of membranes was between 2 and 41 h before delivery. There were no laboratory or clinical signs of amnionitis.
In the full-term group, five infants were born by cesarean section. In groups 1 and 2, eight and two infants, respectively, were born by cesarean section. The median APGAR score, at 5 min, was 9 (range, 8–10) in the full-term neonates, 7 (range, 6–9) in group 1, and 8 (range, 7–9) in group 2, respectively. Because there was no significant difference in Preg and 17OH-Preg levels between vaginally born infants and children born by cesarean section, they were treated as one group, according to the gestational age (ANOVA on ranks, P < 0.05 considered as significant).
We excluded all severely ill and stressed infants from this study. One neonate in group 1 and two children in group 2 developed a mild respiratory distress syndrome (stage 1–2); however, no mechanical ventilation was necessary.
Initially, all preterm infants were treated with iv nutrition fluids of increasing quantity (50–150 ml/kg·d, from d 1–7) containing glucose (15 g/kg·d), amino acids (2 g/kg·d), and fat (3 g/kg·d). The iv fluids also contained 2–4 mmol sodium/kg·d, 2 mmol potassium/kg·d, 1 mmol calcium gluconate/kg·d (except on d 1, when no electrolytes were given), trace elements, and vitamins in the recommended dosages. This regimen of parenteral nutrition was gradually replaced by feeding of breast milk or adapted formula. All full-term infants were either breast-fed or received a commercial adapted formula.
Blood was drawn from the mothers‘ antecubital vein, from the retroplacental (RP) space, and from the umbilical artery (UA) and umbilical vein (UV) immediately after delivery and after cutting the umbilical cord. Peripheral venous blood samples from the neonates were drawn by venipuncture from the infants’ peripheral veins 2–4 h, 12 h, and 24 h after delivery. After the first 24 h, venous blood samples were drawn between 0800 and 1000 h, in connection with routine laboratory examinations at d 3–4 and 6–7. A blood sample at d 14 was taken from the premature neonates only. The study was approved by the Medical Faculty’s ethics committee, and informed consent was obtained from the parents.
The blood was collected in heparinized tubes and centrifuged immediately. The plasma was stored at -20 C until assay. Plasma concentrations of Preg and 17OH-Preg were measured by a simultaneous RIA after extraction and chromatography from the same plasma sample of 200 µl. Our micromethod of simultaneous steroid analysis has been previously described and evaluated (19). In brief, tritium-labeled amounts of both steroids were added to 200 µl plasma as internal standard. After extraction with methylene chloride, the plasma samples were submitted to an automated Sephadex LH-20 high performance chromatography system using 10 columns in parallel (24). The appropriate steroid fraction was quantified by RIA. The lower detection limit of the assay was 0.15 nmol/liter and 0.28 nmol/liter for Preg and 17OH-Preg, respectively. The intra- and interassay coefficients of variation were 8.4% and 11.6% for Preg and 7.0% and 10.9% for 17OH-Preg, respectively.
Comparisons of samples, i.e. full-term and preterm infants, were performed using Kruskal-Wallis ANOVA on ranks; comparisons of more than two paired samples, i.e. analyzing the time course of levels of Preg or 17OH-Preg during the first weeks of life, were made by ranked ANOVA according to Friedman’s comparisons of repeated measures. Newman Keul’s test was used to test for significant differences within the group post hoc. P value less than 0.05 was considered significant.
Results
Preg
There was no difference in the median of plasma Preg level in the maternal vein at delivery depending on premature or full-term delivery (Table 1
). The Preg levels in group 1 in the RP space were significantly higher than in the maternal vein or UA or UV, respectively. In group 2, median Preg was also significantly higher in the RP space than in the maternal vein.
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). In contrast, the first postnatal Preg level was comparable with UA in group 2 and markedly below UA in full-term neonates. Thereafter, median plasma Preg levels decreased exponentially in all three groups until d 6 and 14, respectively. The median plasma Preg level was well below UA at 12 h in group 2 and in full-term neonates, whereas a level below UA in group 1 was reached only after 24 h. Median Preg values in both groups of premature infants were constantly higher than in full-term infants throughout the entire early neonatal period, with a significant difference at 12 h between group 1 and full-term infants. In group 1, the median plasma Preg level at d 14 was one third of the 2-h level (36.69 vs. 95.78 nmol/liter, P < 0.05). Although not significant, the same postnatal decrease of plasma Preg was observed in group 2. In full-term infants, the median Preg level at d 6 was only about one fifth of the UA level (14.81 vs. 66.62 nmol/liter).
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The median plasma 17OH-Preg level showed no significant differences between maternal vein and RP space in all three groups. Similarly, no significant differences were observed between UA and UV in all groups. However, there was a trend to a positive umbilical arteriovenous difference in all three groups. Full-term infants had significantly higher UA 17OH-Preg levels than the preterms in group 1 (Table 1
).
There was a significant, progressive 8.6-fold rise of median plasma 17OH-Preg in the most premature infants (group 1) from UA to 12 h after delivery. In contrast to this, the 17OH-Preg levels of group 2 showed only a short-lived rise, peaking at 2 h, followed by an exponential decrease to levels below UA at 12 h. No peaking was seen in the full-term group. 17OH-Preg levels at 12 h were significantly lower in group 2 and full-term infants than in group 1. Thereafter, median plasma 17OH-Preg levels remained higher in both premature groups than in the full-term infants (not significant).
For comparative use as reference data, numerical results of steroid measurements are compiled in Table 2.
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Preg
The main source for Preg at delivery is the placenta (25). This is also evident from our present study, where the highest amount of the unconjugated steroid was found in the RP space. There were no significant differences in RP Preg levels between full-term and preterm deliveries, although the median Preg level was lower in the full-term group. Preg originates mainly from maternal cholesterol (25, 26). Because of the absence of 3ß-HSD-II within the fetocortex of the adrenals, the placental Preg is mostly conjugated to Preg-sulfate (27, 28). Preg-sulfate from the fetal adrenal serves as a precursor for dehydroepiandrosterone sulfate, the major substrate for placental estriol production during pregnancy. A minor amount of unconjugated Preg is synthesized de novo from cholesterol in the neocortex of the fetal adrenal. Because this was seen as early as midgestation by Telegdy et al. (26), it is not surprising that we found no differences in Preg levels in the fetal circulation between the various gestational ages. Our results for Preg levels in the maternal venous circulation and in UA and UV agree with the findings of Laatikainen et al. (29), although our levels are somewhat higher. A possible reason for the slightly higher Preg levels observed in our study may be the presence of signs of labor before cesarean section in all cases in our study. Similar to Laatikainen et al. (29), we also found no differences between the different sexes.
At birth, the adrenal cortex consists primarily of the fetal zone (fetocortex), which involutes rapidly after birth (30). In the undisturbed pregnancy, the definitive zone (neocortex) produces significant amounts of 4-corticosteroids, from about the 32nd week of gestation. In contrast, the fetal zone secretes high levels of 5–3ß-hydroxysteroids, such as Preg, 17OH-Preg and dehydroepiandrosterone sulfate. Whereas some longitudinal data for Preg sulfate and dehydroepiandrosterone sulfate are available (12, 31, 32, 33), no such data exist for the postnatal course of plasma Preg and 17OH-Preg in full-term and preterm neonates. In our earlier work, we showed a rapid postnatal decline for the 4-steroid progesterone levels in full-term and preterm infants (34, 35, 36), which, in preterm infants, was prolonged over several days (36). A similar time course of plasma levels of the 5-steroid Preg during the early postnatal period was observed in the present study. Preg levels in preterm infants remained slightly higher than in full-term infants throughout the observation period. Most probably, the postnatal decline of Preg levels in premature children is prolonged because of a slower involution of the fetal adrenal zone and/or because of the immaturity of hepatic and renal elimination. Interestingly, we noticed a significant early postnatal increase in plasma Preg in group 1, from 0 h to 2 h after birth. This rise was limited to a high plateau-like phase in group 2. In full-term infants, on the other hand, no increase or plateau of plasma Preg was found. The early postnatal increase of plasma Preg in the most immature neonates may reflect a higher response to increased postnatal stress in contrast to the more mature infants. The data should be handled with special care in severely stressed and ill infants, because Preg levels may be significantly higher than in healthy newborns. Repeated measurements should be obtained in such cases, to observe the fall in steroid levels. The upper limit of the range for each time point can be used as the cutoff level for the diagnosis. Blood sampling should be obtained after 24 h of postnatal life or later, because the postnatally increased steroid level has dropped to its plateau by then.
17OH-Preg
Data concerning 17OH-Preg levels in various compartments at delivery are rare. We found no significant differences between 17OH-Preg levels in the maternal circulation and the RP space, regardless of gestational age, because the maternal adrenals provide most of the circulating maternal 17OH-Preg, as shown by Belisle et al. (25). Sources other than the maternal adrenals are unlikely to contribute significantly to maternal 17OH-Preg production (37). In the fetus, the adrenals provide the bulk of circulating 17OH-Preg. This is suggested by a positive umbilical arteriovenous gradient seen also in our present study, as well as by the significant suppression after dexamethasone administration observed by Belisle et al. (25). In the most immature infants (group 1), we found significantly lower 17OH-Preg levels in the UA than in full-term infants. This is possibly attributable to a relatively increased synthesis of dehydroepiandrosterone and estriol, because there is less or lagging activity of 3ß-HSD-II in the predominating fetocortical zone of the adrenals and in the definitive adrenal zone in the very immature infants (27).
Postnatally, there is a rapid fall of plasma 17OH-Preg in full-term infants. This reflects the involution of the fetal adrenal zone, the predominant source of 17OH-Preg in the fetal circulation. As for Preg, we found a marked increase of 17OH-Preg after delivery in the preterm groups. The peak was reached after 2 h in the more mature preterm neonates (group 2) and only after 12 h in the most immature neonates (group 1). This might reflect a more prolonged stress response in the immature neonates and a slower clearance of these plasma 5-steroids correlating with gestational age. There is, depending on gestational age, a trend to 2-fold higher plasma 17OH-Preg levels at 7 and 14 d, most likely because of a slower steroid clearance in the premature newborns. Our longitudinal data in the full-term group are comparable with the cross-sectional data of Lee et al. (21) and Wiener et al. (13) obtained on d 2–5 of extrauterine life. In contrast to our results in healthy preterm infants, Hingre et al. (22) found still higher 17OH-Preg levels in sick preterm infants with a gestational age less than 30 wk. These higher values may be attributable to severely stressed and ill infants and possibly also to less specific assays. Our reference data do not necessarily apply in the case of severely stressed and ill infants. Repeated measurements should be obtained in such cases to observe a possible fall in steroid levels. As for Preg, the upper limit of the range for each time point can be used as the cut off level for the diagnosis of 3ß-HSD-II deficiency. Blood sampling should be obtained after 24 h of postnatal life or later, because the postnatally increased steroid level has dropped to its plateau by then.
Substantial changes occur in adrenocortical function during the transition from intrauterine to extrauterine life. To our knowledge, the present study shows, for the first time, the longitudinal course of plasma levels of Preg and 17OH-Preg in healthy full-term and healthy preterm infants during the early neonatal period. A major pitfall for the correct diagnosis of the various forms of CAH in the early postnatal period is the use of inadequate reference values for the respective marker steroid. We therefore collected such reference data for plasma Preg and 17OH-Preg, both for various gestational ages and at different stages of early postnatal life, using our newly developed simultaneous assay for both 5-steroids (19). With this tool, it is now possible to confirm or rule out the diagnosis of CAH caused by 3ß-HSD-II deficiency, as soon as a neonate shows clinical symptoms, such as ambiguous genitalia and/or salt wasting, in early postnatal life.
Acknowledgments
We thank Gaby Ferrazini for valuable help with the blood sampling. We are grateful to Susanne Olin and Sabine Stein for expert technical assistance. We also thank Joanna Voerste for linguistic help with the manuscript.
Footnotes
Abbreviations: 3ß-HSD, 3ß-Hydroxysteroid dehydrogenase (EC 1.1.1.145); CAH, congenital adrenal hyperplasia; Preg, pregnenolone (3ß-hydroxypregn-5-en-20-one); 17OH-Preg, 17-hydroxypregnenolone (3ß, 17-dihydroxypregn-5-en-20-one); RP, retroplacental; UA, umbilical artery; UV, umbilical vein.
Received March 22, 2002.
Accepted May 30, 2002.
References
-dihydrotestosterone in different types of congenital adrenal hyperplasia. Clin Endocrinol (Oxf) 4:39–48[Medline]
-hydroxypregnenolone and its sulfate in human pregnancy. J Clin Endocrinol Metab 46:721–7285–4 isomerase in the human fetal adrenal gland. Endocr Res 21:69–80[Medline]
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