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Original Article |
Hospital for Children and Adolescents (E.K., L.D., S.A.), Laboratory (U.T.), and Departments of Clinical Chemistry (U.-H.S.) and Obstetrics and Gynecology (M.N., S.A.), Helsinki University Central Hospital, 00029 HUS, Helsinki, Finland; Endocrine Unit (P.J.W.), Southampton General Hospital, SO16 6YD Southampton, United Kingdom
Address all correspondence and requests for reprints to: Eero Kajantie, M.D., The Hospital for Children and Adolescents, Helsinki University Central Hospital, PL 280, 00029 HUS, Finland. E-mail: eero.kajantie{at}hus.fi.
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Abstract |
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Introduction |
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Of all infants, those born severely preterm are probably most likely to be exposed to excess glucocorticoids. Conditions associated with reduced placental 11ß-HSD2 function and subsequent glucocorticoid excess, such as intrauterine growth restriction (IUGR) (4, 6) and preeclampsia (13, 19), are frequent causes of iatrogenic prematurity when serious enough to necessitate immediate delivery. In addition, most small preterm infants become exposed to pharmacological glucocorticoids before and/or after birth. The most common indication for using glucocorticoids is maternal antenatal treatment, which is well established in enhancing fetal maturation and reducing neonatal mortality and morbidity (20).
Consequently, surprisingly little is known about the amount of physiological glucocorticoids small preterm infants with different clinical conditions have been exposed to in utero. The function of placental 11ß-HSD2 changes considerably during pregnancy (4, 6), but reports published so far have included relatively small numbers of small preterm infants. Therefore, we set out to study which clinical conditions are associated with altered placental 11ß-HSD2 function and other parameters of the F/E shuttle in infants born before 32 wk gestation.
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Subjects and Methods |
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The 110 preterm infants born before 32 wk gestation at the Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Helsinki, Finland, had their gestational age confirmed by ultrasound before 20 wk gestation. Infants from multiple pregnancies were included in the study only if they had clearly separate placentas. Infants of mothers receiving inhaled (n = 2) or systemic (n = 1) glucocorticoids, other than bethamethasone to enhance fetal maturation, were excluded. The mean gestational age at birth was 28.4 wk (range, 22.4–32.0 wk), mean birth weight 1067 g (395–2453 g) and mean relative birth weight -1.26 SD (-4.90–3.40 SD). Table 1
shows the clinical data.
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The study protocol was approved by the Ethics Committee of the Department of Obstetrics and Gynecology, Helsinki University Central Hospital.
Placental 11ß-HSD2 activity
Immediately after birth, a sample of placental tissue was dissected, snap frozen in liquid nitrogen, and stored at -70 C until analyzed. Placental 11ß-HSD2 activity was determined by a method modified from that described by Stewart et al. (1), with the F and E fractions measured by HPLC. Placental tissue carefully inspected not to contain any fetal membrane was thawed and homogenized by an Ultraturrax homogenizer, with the homogenate spun at 1000 g for 10 min at 4 C. After a protein assay (26), the supernatant was incubated in 1.0 ml phosphate buffer (0.1 mol/liter; pH 7.6) containing 0.5 g/liter protein, 0.8 nmol/liter nicotinamide adenine dinucleotide, and 0.2 µmol/liter F, in a shaking water bath at 37 C. After 30-min incubation, 1.0 ml methanol was added to stop the conversion of F to E. Samples in methanol were evaporated under nitrogen. Seven milliliters dichloromethane were added to the residues and tubes mixed by multitube Vortexer for 30 sec. The upper phase was removed, 2 ml water added, and tubes mixed as above. The upper phase was again removed and the lower phase dried under nitrogen. To the dry residue, 40 µl 40% methanol was added. Then 25 µl of the solution was injected into the HPLC. The chromatographic conditions were as described (27).
Cord vein F and E concentrations
Cord vein blood was drawn into EDTA tubes, with plasma separated immediately and stored at -20 C until analyzed. Plasma F concentrations were measured using a direct in-house method with antiserum HPS631/1G (antisera, code S020, Guildhay Ltd., Guildford, UK) and a cortisol-3CMO-histamine-[125-I] tracer as described by Moore et al. (28). Plasma E was extracted into chloroform, and the dried extracts were analyzed using a rabbit anticortisone antiserum N-137 and a 21-acetyl cortisone-3-CMO-histamine-[125-I] tracer (29). The cross-reactivity of E in the F assay was 1.2%, and of F in the E assay was less than 0.1%. Between-batch imprecision (percent coefficient of variation) for low-, medium-, and high-quality control samples ranged from 6.3% to 11.9% (F) and 6.1% to 10.0% (E).
Indicators of fetal F/E shuttle
Five different variables were used to illustrate different aspects of the fetal F/E shuttle. Placental 11ß-HSD2 activity rate per unit placental weight was expressed as picomoles E per minute per milligram protein (1, 3). Total placental 11ß-HSD2 activity was expressed as micromole E per min (1, 3). Cord vein F and E concentrations were as well used, and an E/(E + F) ratio was calculated to express the fraction of E of the total (E + F) pool (2). This ratio served as an indicator of the balance between 11ß-HSD1 and 11ß-HSD2 activity in those maternal and fetal tissues that contributed to the circulating concentrations.
Data analysis
Cord vein F and E concentrations were log transformed to normality. Partial correlation was used to assess relationships between variables. All correlation analyses including comparisons between two groups were adjusted for gestational age at birth, number of antenatal betamethasone doses, and time between the last betamethasone dose and birth. Adjustment for the time between last betamethasone dose and birth was performed by dividing the infants into three groups: 1) less than 24 h; 2) 24–72 h; and 3) over 72 h since last betamethasone or no betamethasone. Dummy variables were created for these groups to allow for possible nonlinear effects. The time limits were based on estimated proportion of glucocorticoid bioactivity caused by betamethasone during each period (30).
To assess which variables independently predict each index of placental 11ß-HSD2 activity, multiple regression models were created by use of forward stepwise method starting from all variables with a significant univariate correlation with any of the indices. However, because of strong intercorrelations among different birth measurements, no other birth measurement than relative birth weight was included in the model.
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Results |
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presents the clinical characteristics of the study population as well as indicators of fetal F/E shuttle: placental 11ß-HSD2 activity rate, total placental 11ß-HSD2 activity, cord vein F and E concentrations, and the E/(E + F) ratio. All these indicators were similar in male and female infants and infants from singleton or multiple pregnancies. Placental 11ß-HSD2 activity rate or total activity was not correlated with cord vein F or E concentrations or E/(E + F) ratio. Disorders of pregnancy
Figure 1 shows that the 35 infants from pregnancies complicated by increased umbilical artery resistance had reduced total placental 11ß-HSD2 activity, cord vein E/(E + F) ratio, and E concentration. These infants also had lower relative birth weight (-2.28 ± 0.23 SD vs. -0.75 ± 0.16 SD, P < 0.0001) and smaller placentas (233 ± 24 g vs. 292 ± 12 g, P = 0.001). However, there was no difference either in placental 11ß-HSD2 activity rate calculated per milligram placental protein or in F concentration. Correspondingly, the 29 infants of mothers with preeclampsia had reduced total placental 11ß-HSD2 activity (1.03 ± 0.12 vs. 1.40 ± 0.10 µmol E per minute, P = 0.01) and cord vein E concentration (119 ± 24 nmol/liter vs. 190 ± 22 nmol/liter, P = 0.03) but no difference in placental 11ß-HSD2 activity rate (P = 0.5), cord vein E/(E + F) ratio, or F concentration. Comparison between the infants from all hypertensive (n = 49) and nonhypertensive pregnancies gave a similar result: lower total 11ß-HSD2 activity (P = 0.001) and cord vein E concentration (P = 0.02) but no other difference.
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Gestational age and size at birth
Placental 11ß-HSD2 activity rate, as assessed per milligram placental protein, decreased with increasing gestational age (P = 0.009; Fig. 2). This was, however, not accompanied by any association with total placental 11ß-HSD2 activity or the cord vein E/(E + F) ratio (Table 2). Low relative birth weight (Fig. 2) was associated with reduced 11ß-HSD2 activity rate (P = 0.002) and total activity (P < 0.0001) as well as lower cord vein E/(E + F) ratio (P = 0.01) and E concentration (P = 0.003). Further analysis of body proportions showed comparable associations with relative length at birth (Table 2). By contrast, relative head circumference and ponderal index at birth were associated only with total placental 11ß-HSD2 activity (P = 0.002 and P = 0.05, respectively) but not with any other index of fetal F/E shuttle. Moreover, placental weight showed no correlation with 11ß-HSD2 activity rate (P = 0.9) but, apart from the obvious association with total 11ß-HSD2 activity, was also weakly associated with cord vein E/(E + F) ratio (r = 0.19; P = 0.09) and E concentration (r = 0.25; P = 0.03).
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The total number of antenatal betamethasone doses given was inversely associated with cord vein F (r = -0.38; P = 0.001) and E (r= -0.28; P = 0.009) concentrations but not with E/(E + F) ratio or placental 11ß-HSD2 activity rate or total activity. To assess the possible effect of the time between last glucocorticoid dose and birth, infants unexposed to betamethasone or with at least 72 h between last betamethasone dose and birth were selected to establish a reference group (30). Compared with this group, infants whose mothers had received betamethasone 24–72 h before birth had lower cord vein F (P = 0.05) and E (P = 0.001) concentrations and higher placental 11ß-HSD2 activity rate (P = 0.03). By contrast, no difference in any index of fetal F/E shuttle was seen in infants exposed to betamethasone less than 24 h before birth.
Multiple regression models
Multiple regression models shown in Table 3 demonstrate that placental 11ß-HSD2 activity rate was independently associated with both relative birth weight and, inversely, with gestational age at birth, whereas low relative birth weight remained the sole factor to explain reduced total placental 11ß-HSD2 activity. Antenatal betamethasone treatment was excluded from models explaining both placental 11ß-HSD2 activity rate and total activity. However, treatment with betamethasone 24–72 h before delivery was, together with lower relative birth weight, associated with lower cord vein E/(E + F) ratio. It also predicted low E and F concentrations, which were also related to the total number of betamethasone doses given. Additional predictors for low E concentrations were low gestational age and impaired cord artery flow.
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Discussion |
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Despite the impressive capacity of placental 11ß-HSD2, there are other regulators of the fetal F/E balance. 11ß-HSD2 is expressed in a number of maternal and fetal tissues including the kidney and brain, in which this cytoplasmic enzyme regulates the access of F to mineralocorticoid or glucocorticoid receptors (7, 8). In several tissues, including adipose tissue, liver, and the fetal membranes but placenta only to a minor extent (31), the low-affinity enzyme 11ß-HSD1 performs the inverse function: cytoplasmic conversion of E to F (9). To what extent different tissues contribute to F and E concentrations in fetal circulation is not known, but our finding of no correlation between placental 11ß-HSD2 activity and the E and F concentrations or their ratio contrasts to a positive correlation observed in term infants (4). This suggests that between 22 and 32 wk gestation, the role of other fetal or maternal tissues in the fetal F/E interconversion is perhaps more significant than later during pregnancy.
Our finding of a clear correlation between relative birth weight and both placental 11ß-HSD2 activity rate and cord vein E/(E + F) ratio is in agreement with similar findings in term or near-term pregnancies (1, 13) and studies reporting reduced 11ß-HSD2 activity (4) and mRNA expression (6) in severe IUGR. The difference in placental 11ß-HSD2 activity and expression between IUGR and normal pregnancies is actually accentuating with increasing gestational age (4, 6). Consequently, the solidity of the relationship with relative birth weight in the present study is perhaps surprising. It implies that even in small preterm infants, placental 11ß-HSD2 activity and other determinants of the fetal F/E shuttle are significant contributors to size at birth. In an observational study, we could not assess whether small size at birth is a direct consequence of fetal glucocorticoid excess, and the possibility of an unknown cause affecting both placental 11ß-HSD2 function and fetal growth through some other mechanism cannot be strictly excluded. However, fetal growth restriction is a plausible consequence of intrauterine glucocorticoid excess, and the finding that the strongest correlation of E/(E + F) ratio was seen with relative length at birth is consistent with the known effect of excess glucocorticoids in inhibiting longitudinal growth (32).
The magnitude of the association between relative birth weight and determinants of the fetal F/E shuttle can be deduced from the r values in Table 2. Consistent with the r2 values of the models in Table 3, the r values indicate that slightly less than 10% of the variation in relative birth weight is explained by variation in 11ß-HSD2 activity rate or the E/(E + F) ratio. Correspondingly, about 30% of this variation is explained by variation in total placental 11ß-HSD2 activity, which includes the effect of variation in placental size. Obviously, there are other contributors to fetal size, including the fetal and maternal genome and effects of nutrition and oxygen supply through other hormonal systems such as the insulin-IGF axis (33). An important question is whether different mechanisms of fetal growth retardation are operative in different disorders associated with prematurity and/or IUGR.
A major cause of prematurity is fetal distress severe enough to necessitate immediate delivery. Such fetal distress is commonly related to placental insufficiency associated with preeclampsia or nonpreeclamptic (i.e. without proteinuria) hypertension. These are overlapping conditions, many of the nonproteinuric hypertensive mothers representing an early stage of frank preeclampsia (34). Both are frequent causes of IUGR. Increased umbilical artery resistance is an end-stage feature of these and other disorders of impaired placental function and as such probably the most specific marker of severe fetal distress. Our finding of an association between increased umbilical artery resistance and reduced total placental 11ß-HSD2 activity as well as cord vein E/(E + F) ratio and E concentration is consistent with the hypothesis that these conditions are associated with increased fetal glucocorticoid exposure. Moreover, we found reduced total placental 11ß-HSD2 activity and E concentration in preeclampsia and hypertension, although contrasting to studies in term and near term-infants (13, 19), there was no difference in the 11ß-HSD2 activity rate per se. It is therefore likely that between 22 and 32 wk gestation, the decreased overall F-to-E conversion in these conditions is attributable to smaller size of the placenta and possibly altered contribution of other fetal or maternal tissues, such as the maternal kidney (8). An intriguing explanation for the grossly reduced E concentrations could be an increased local conversion of E to F in fetal tissues. This would constitute a biologically meaningful way of increasing glucocorticoid effect during fetal distress despite relatively low F concentrations. However, there is so far little experimental evidence to substantiate this hypothesis.
We found placental 11ß-HSD2 activity rate to decrease with increasing gestational age, although the effect on total placental activity was counterbalanced by increasing placental size. This finding is at variance with others that show an increase in 11ß-HSD2 activity (4) and mRNA expression (6) throughout pregnancy. However, Shams et al. (4) observed a similar negative correlation with gestational age within the IUGR group, possibly because of different pathophysiology of IUGR at different gestational ages. It is thus possible that the relatively large proportion of growth-restricted infants in our study may have contributed to this finding.
Antenatal glucocorticoid treatment is currently much debated. Despite its unequivocal benefit in reducing neonatal mortality and morbidity (20), administering repeated doses may instead increase mortality, reduce birth weight, and suppress the HPA axis (20, 35). We found the number of betamethasone treatments to be associated with a decrease in both F and E concentration in cord vein. Because maternal administration of betamethasone has been shown to result in similar betamethasone concentrations in maternal and fetal serum (25), this finding is likely to reflect both maternal and fetal HPA axis suppression. Otherwise the F/E shuttle appeared unaffected by the number of betamethasone doses. Whether betamethasone has any direct effect on placental 11ß-HSD2 expression or function is poorly known, and our study with betamethasone administered on clinical indications was not designed to assess such direct effects. Instead, the significance of our findings lies in the notable capacity of 11ß-HSD2 we found to exist also between 22 and 32 wk gestation and the large interindividual variation in this capacity as well as other determinants of the F/E shuttle. In fact, the side effects of betamethasone are observed already after relatively small doses, which result in a peak glucocorticoid activity of about 250 nmol/liter F equivalents and return to normal within 2–3 d (30). This underlines the potential importance of alterations in the fetal F/E shuttle, which could be expected to cause a similar or greater increase in overall glucocorticoid exposure.
The hypocortisolic fetal milieu created by placental 11ß-HSD2 has been suggested to be crucial for the maturation of fetal HPA axis (1). With this in mind, the relationship we found between low relative birth weight and reduced placental 11ß-HSD2 function in small preterm infants gives rise to putative long- and short-term health consequences.
A series of elegant studies in rodents link impaired placental 11ß-HSD2 function with reduced birth weight and persistent alterations in tissue 11ß-HSD2 and glucocorticoid receptor expression (16, 36), resulting in e.g. hypertension (16) and altered behavior (5). Although there are species differences, recent studies in humans point to a link between HPA axis function in adulthood, cardiovascular risk factors, and size at birth (17, 18). Although these associations are evident already within the range of normal term birth, small preterm infants have frequently suffered from grave IUGR and may receive large doses of glucocorticoids. They thus have much greater potential for adverse effects of early HPA axis programing, warranting thorough follow-up.
Many sick very low-birth-weight infants exhibit disproportionately low postnatal F concentrations, which are associated with e.g. subsequent chronic lung disease (37, 38, 39, 40). This is a known phenomenon as well in adult intensive care, in which corticosteroid supplementation reduces the mortality of hypocortisolemic patients (41). These observations have introduced the concept of early adrenal insufficiency, and in preliminary trials some infants indeed seem to benefit from low-dose early glucocorticoid replacement (42). However, at least high-dose glucocorticoids in small preterm infants are associated with major adverse effects including cerebral palsy (43) and cause concern because of possible lifelong HPA axis programming. It would therefore be crucial to identify infants with early adrenal insufficiency who might benefit from glucocorticoid replacement during the immediate postnatal period. Our finding of reduced 11ß-HSD2 activity in infants with IUGR and/or severe fetal distress suggests that these infants constitute a susceptible group worth focusing on in prospective studies.
We conclude that infants born between 22 and 32 wk gestation exhibit large interindividual variation in placental 11ß-HSD2 function and other determinants of the F/E shuttle. Within these infants, those born small for gestational age as well as those suffering from severe fetal distress have lower placental 11ß-HSD2 function and reduced overall conversion of F to E. Possible health consequences to be addressed in further studies range from early postnatal adrenal insufficiency in small preterm infants to adverse HPA axis programing with increased adulthood cardiovascular risk.
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Acknowledgments |
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Footnotes |
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Abbreviations: E, cortisone; F, cortisol; HPA, hypothalamo-pituitary-adrenal; 11ß-HSD, 11ß-hydroxysteroid dehydrogenase; IUGR, intrauterine growth restriction.
Received August 29, 2002.
Accepted October 14, 2002.
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