Main

During the last trimester, the fetal adrenal cortex is functionally in a transitional phase. The outer definitive or adult zone of the adrenal cortex, being able to produce cortisol, is relatively thin, whereas the inner fetal zone, whose major secretory product is DHEAS, is predominant in both size and function(1). The biochemical basis for the different steroid secretory patterns of the two zones is the presence or absence of 3β-hydroxysteroid dehydrogenase expression in the adult and fetal zones, respectively(2–4). During the last weeks of normal pregnancy, the adult zone continues to develop. After birth, the fetal zone undergoes rapid involution.

Ill preterm neonates have been reported to have similar serum cortisol concentrations at birth and during the 1st wk of life as healthy preterm and full-term neonates(5–9). In most of these reports, the preterm neonates had a gestational age of 30 wk or more(5, 7, 8). Less is known of the adrenal function of the very preterm neonates. At birth, full-term newborns have lower concentrations of corticosteroid-binding globulin (CBG) than adults(10) but only few reports exist on CBG concentrations in preterm newborns(11, 12). CBG concentration influences the proportion of the functionally active free or unbound cortisol.

Since the early 1980s, several randomized studies have shown that DEX treatment has beneficial effects on pulmonary function of ventilator-dependent preterm infants with early or established chronic lung disease(13–16). Suppression of the pituitary-adrenal axis particularly after a long exposure to DEX therapy has caused concern.

The objectives of the present study were to assess the adrenocortical function of VLBW infants at risk for chronic lung disease before and after DEX therapy and to evaluate the impact of gestational age, postnatal age, and DEX therapy on serum concentrations of cortisol, DHEAS, and steroid-binding globulins.

METHODS

Patients

Of the 48 study infants, 23 participated in a randomized, multicenter trial of neonatal DEX therapy in infants at risk for chronic lung disease(17). All of these 23 VLBW infants were treated at the Children's Hospital, University of Helsinki. The control group consisted of 25 infants: 13 ill preterm infants of 23-28 wk of gestation and 12 healthy, moderately preterm infants of 30-34 wk of gestation or full-term infants. All of the 25 control infants were born in Departments I or II of Obstetrics and Gynecology, University of Helsinki. Preterm control infants were treated at the Children's Hospital, University of Helsinki. The study protocol was approved by the ethical committee of the hospital. Informed consent was obtained from the parents of neonates participating in this study.

The VLBW neonates were eligible for the neonatal DEX trial if they fulfilled the following criteria: 1) birth weight 1500 g or less,2) gestational age 24 wk or more, 3) ventilator-dependent at 10 d of age, and 4) no signs of major malformation, sepsis, or patent ductus arteriosus at entry(17). Infants were randomly allocated to receive a 1-wk course of either DEX sodium phosphate(Oradexon, Organon, Oss, The Netherlands) or placebo. Infants in the DEX group received 0.5 mg/kg/d DEX i.v., divided into two doses, and those in the placebo group received 0.9% saline. The treatment was started, on an average, at 15 d of age in the DEX and at 14 d of age in the placebo group. All preterm neonates of the DEX, placebo, and preterm control groups required ventilatory support from birth because of respiratory distress syndrome or immaturity. Control neonates were healthy or had mild prematurity-associated morbidity. Pulmonary outcome was evaluated at 28 d of age. Chronic lung disease was defined as the requirement of supplemental oxygen. Central nervous system abnormalities were identified by serial cranial ultrasonography in all preterm infants of the DEX, placebo, and preterm control groups.

Procedures

Collection of samples . Neonatal DEX trial. Before entry (at the mean age of 13 d), after the 1-wk treatment (at the mean age of 21 d), and at 30 d of age a 2-h ACTH test was performed on all neonates who participated in the neonatal DEX trial. Blood samples were obtained from an indwelling arterial line, by venipuncture, or by heel stick before (basal sample) and 2 h (stimulated sample) after an i.v. injection of 145μg/m2 tetracosactin (S-Cortrophin, Organon, Oss, The Netherlands). All tests were started between 0800 and 1000 h. The rise in the cortisol concentration, Δcortisol, was calculated by subtracting the basal from the stimulated value.

Control neonates. Umbilical cord samples (mixed arterial and venous sample) were collected from the preterm and full-term control neonates. In addition, basal samples were collected between 0800 and 1200 h from the very preterm control neonates who had an indwelling arterial line at the mean age of 2, 10, and 20 d. In the preterm control group, longitudinal samples were not available from each neonate on all time points. Because of limited volumes, all analyses could not be performed on all samples.

Hormone measurements. Serum cortisol was measured by RIA using reagents from Farmos Diagnostica, Turku, Finland. In the cortisol assay, the sensitivity was 3-5 nmol/L, and the intra- and interassay coefficients of variation were 1.5-2.8% and 4.5-7.1%, respectively. In this assay, cortisone had 20% cross-reactivity. DHEAS was determined by a direct RIA using tritiated label (DuPont NEN, Boston, MA) and commercial antiserum (Steranti; St. Albans, UK). Separation of free and antibody-bound DHEAS was performed by dextran-coated charcoal. In the DHEAS assay, the sensitivity was 0.06μmol/L, and the intra- and interassay coefficients of variation were 3.9-5.3% and 4.6-7.0%, respectively. CBG was measured by RIA (CBG-RIA-100, IRE-Medgenix, Fleurus, Belgium). In the CBG assay, the sensitivity was 4.5-5.1 nmol/L, and the intra- and interassay coefficients of variation were 3.3-7.7% and 4.5-5.4%, respectively. Free cortisol (U, μmol/L) was calculated according to the CBG RIA kit brochure as follows: U =(Z2 + 0.0122 × cortisol)1/2 - Z; Z = 0.0167 + 0.182 [CBG(μmol/L) - cortisol (μmol/L)]. SHBG was measured by an immunofluorometric assay (DELFIA SHBG, Wallac, Turku, Finland). In the SHBG assay, the sensitivity was 0.8 nmol/L, and the intra- and interassay coefficients of variation were 6.7-8.9% and 5.1-7.0%, respectively.

Statistics

The statistical analyses were performed using StatView software (Abacus Concepts Inc., 1987, Berkeley, CA). Twosided t test, χ2 test with continuity correction, and regression analysis were used. Ap value less than 0.05 was considered significant.

RESULTS

Clinical outcome. Some clinical characteristics of the 48 study neonates are presented in Table 1. All neonates in the DEX, placebo, and preterm control group required ventilatory support from birth for the treatment of respiratory distress syndrome or because of immaturity. During the initial hospitalization, five infants died; one in the DEX group (at the age of 125 d), two in the placebo group (at the ages of 22 and 28 d), and two in the preterm control group (at the ages of 3 and 21 d).

Table 1 Clinical characteristics of the study infants and their neonatal outcome [mean ± SD (range) and number of infants(%)]
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Pulmonary outcome of the study infants at 28 d of age is presented inTable 1. Ten infants developed intraventricular hemorrhage: one (9%) in the DEX group, three (25%) in the placebo group, and six (46%) in the preterm control group. Survival without chronic lung disease or intraventricular hemorrhage was studied at 28 d of age in the ill preterm infants (DEX group, placebo group, and preterm control group). At 28 d of age, only one infant in the DEX group survived without chronic lung disease or intraventricular hemorrhage.

Correlation between gestational age and umbilical cord concentrations of cortisol, CBG, DHEAS, and SHBG. Correlation between gestational age and the serum concentrations of total and free cortisol, CBG, DHEAS, SHBG, and the ratio of DHEAS to cortisol in umbilical cord samples is presented in Figure 1. Altogether 18 umbilical cord samples of the preterm and full-term control neonates were available. Both total and free cortisol correlated with gestational age (total cortisol:r = 0.702, p = 0.001; free cortisol: r = 0.489,p = 0.039). Similarly, the concentrations of DHEAS and SHBG correlated with gestational age (DHEAS: r = 0.608, p = 0.007; SHBG: r = 0.831, p = 0.0001), but no significant correlation was found between the concentration of CBG and gestational age(r = 0.428, p = 0.076). The ratio of DHEAS to cortisol correlated inversely with gestational age (r = -0.668, p = 0.002).

Figure 1
figure 1

Correlation between gestational age and concentration of total and free cortisol (nmol/L), CBG (nmol/L), DHEAS (μmol/L), SHBG(nmol/L), and ratio of DHEAS to cortisol (nmol × L-1/nmol × L-1) in umbilical cord samples of control neonates.

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Correlation between postnatal age and concentrations of cortisol, CBG, DHEAS, and SHBG. The correlation between postnatal age (from 1 to 30 d of age) and the concentrations of total cortisol, DHEAS, CBG, and SHBG is presented in Figure 2. Samples are of the preterm control neonates and of the placebo-treated neonates who participated in the neonatal DEX trial. None of the neonates received DEX therapy. Postnatal age correlated with the concentrations of DHEAS (r = -0.519, p = 0.002,n = 32), CBG (r = 0.477, p = 0.006, n= 32), and SHBG (r = 0.593, p = 0.0003, n = 33), whereas no correlation was found between postnatal age and the concentration of cortisol (r = -0.226, p = 0.131, n = 46). Neither did postnatal age correlate significantly with the concentration of free cortisol (r = -0.377, p = 0.053, n = 27) or the ratio of DHEAS to cortisol (r = -0.312, p = 0.082,n = 32).

Figure 2
figure 2

Correlation between postnatal age and concentration of cortisol (nmol/L), DHEAS (μmol/L), CBG (nmol/L), and SHBG (nmol/L) in neonates of preterm control and placebo group.

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Effect of DEX treatment on cortisol, CBG, DHEAS, and SHBG concentrations. The effect of DEX treatment on the basal concentrations of free and total cortisol, CBG, DHEAS, and SHBG was studied in neonates participating in the neonatal DEX trial (Fig. 3). Before the onset of the treatment, at the mean age of 13 d, concentrations of these parameters were similar in the DEX and placebo groups.

Figure 3
figure 3

Concentration of total and free cortisol (nmol/L), CBG(nmol/L), and DHEAS (μmol/L) in preterm neonates who participated in the neonatal DEX trial. Samples were collected before the onset of DEX (□) or placebo (□)and after the 1-wk treatment. Mean ± SEM. Number of samples: total cortisol: before, 21; after, 21; free cortisol: before, 14; after, 12; DHEAS: before, 16; after, 16; and CBG: before, 16; after, 13.*p < 0.05 and †p < 0.01 for comparisons between the DEX and placebo group.

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After the 1-wk treatment, at the mean age of 21 d, the concentrations of cortisol (p = 0.008), CBG (p = 0.001), and DHEAS(p = 0.028) were significantly lower in the DEX- than in the placebo-treated neonates. Concentration of free cortisol was lower in the DEX- than in the placebo-treated neonates as well, but the difference was not statistically significant (p = 0.19). DEX therapy had no effect on the SHBG levels (before: DEX, 36 nmol/L; placebo, 29 nmol/L; after: DEX, 40 nmol/L; placebo, 48 nmol/L; all comparisons NS).

Effect of DEX treatment on cortisol and DHEAS responses to ACTH stimulation. An ACTH test was performed on all 23 infants participating in the neonatal DEX trial. Before the onset of DEX or placebo, at the mean age of 13 d, both basal and stimulated cortisol levels were similar in the DEX and placebo groups (Fig. 4). The rise in the cortisol level,Δcortisol, was also similar in both groups before the onset of the treatment (DEX, 482 nmol/L; placebo, 426 nmol/L; p = 0.60). After the 1-wk treatment, at the mean age of 21 d, DEX-treated infants had significantly lower basal (p = 0.008) and stimulated levels of cortisol (p = 0.011). The Δcortisol was also significantly lower in the DEX- than in the placebo-treated neonates after the 1-wk treatment (DEX, 377 nmol/L; placebo, 641 nmol/L; p = 0.038). At 30 d, the basal levels were similar in both groups (p = 0.32), but the stimulated cortisol levels (p = 0.006) as well as Δcortisol were still significantly lower in the DEX than in the placebo group(Δcortisol: DEX, 572 nmol/L; placebo, 946 nmol/L; p = 0.009).

Figure 4
figure 4

Concentration of basal cortisol (nmol/L) and response to ACTH stimulation in preterm neonates who participated in the neonatal DEX trial. Basal and stimulated samples were collected before the onset of DEX(□) or placebo (□), after the 1-wk treatment, and at 30 d of age as described in “Methods.” Mean ± SEM. Number of infants of whom samples were available: before treatment, 21 infants; after 1-wk treatment, 21 infants; and at 30 d of age, 19 infants. *p < 0.05 and †p < 0.01 for comparisons between the DEX and placebo group.

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Within the DEX group, stimulated cortisol levels after the treatment were significantly lower than before the onset of the treatment (before: 607 nmol/L; after: 458 nmol/L; p = 0.027). Within the placebo group, stimulated cortisol and Δcortisol before the treatment were significantly lower than the corresponding values after the 1-wk treatment(stimulated cortisol: before, 545 nmol/L; after, 817 nmol/L; p = 0.026; Δcortisol: before, 426 nmol/L; after, 641 nmol/L; p = 0.014) or at 30 d of age (stimulated cortisol: before, 545 nmol/L; at 30 d, 1127 nmol/L; p = 0.001; Δcortisol: before, 426 nmol/L; at 30 d, 946 nmol/L; p = 0.001).

DHEAS levels before the onset of treatment were as follows: DEX basal, 8.2μmol/L (n = 7); DEX stimulated, 8.6 μmol/L (n = 6); placebo basal, 10.5 μmol/L (n = 9); placebo stimulated, 12.2μmol/L (n = 10); all comparisons NS. DHEAS levels after the 1-wk treatment were as follows: DEX basal, 6.5 μmol/L (n = 6); DEX stimulated, 6.4 μmol/L (n = 6); placebo basal, 12.5 μmol/L(n = 8); placebo stimulated, 11.5 μmol/L (n = 9); both basal and stimulated levels were lower in the DEX- than in the placebo-treated neonates (p = 0.028 and 0.045, respectively).

DISCUSSION

In the present study, concentration of total and free cortisol in the umbilical cord samples correlated with gestational age. After 2-3 d of age, mean cortisol levels remained less than 200 nmol/L in most preterm neonates despite the continuing requirement of intensive care. During fetal life, the concentration of cortisol, produced by the adult or definitive zone of the adrenal cortex, correlates directly with gestational age(1). Ill preterm neonates have similar serum concentrations of cortisol during the 1st wk of life as healthy full-term neonates(6, 8, 11, 18, 19)(Table 2). However, some very ill preterm neonates have high cortisol levels(21, 22). In the present study, the basal cortisol concentrations were of similar magnitude as in most other reports(11, 12, 18, 19)(Table 2), but lower than in others(21, 22). Considering the requirement of intensive care and the poor outcome at 28 d of age, most preterm neonates in the present study had very low cortisol levels at birth and during the neonatal period. These inappropriately low cortisol levels in ill preterm neonates may be of clinical significance.

Table 2 Review of cortisol concentrations in preterm and full-term neonates
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Neonatal DEX therapy decreased both basal and ACTH-stimulated cortisol concentrations. At 30 d of age, 1 wk after discontinuation of treatment, basal cortisol levels were similar in the DEX and placebo group, but the stimulated cortisol levels were still lower in the DEX- than in the placebo-treated infants. DEX therapy in the early and established stage of chronic lung disease causes adrenal suppression(14, 23–26). After a short course of DEX, the suppression is transient(14, 23). Our results regarding the basal and ACTH-stimulated cortisol levels are in line with previous reports(14, 19, 23, 24). Although the basal cortisol levels were low, most of the neonates responded well to ACTH stimulation and the ACTH response.Δcortisol, increased as a function of postnatal age. Thus, ill VLBW neonates had low basal cortisol levels during the neonatal period but were able to respond well to exogenous ACTH. Low basal cortisol levels despite good response to high dose exogenous ACTH may indicate a poor ability of the preterm neonate to respond to stress by increasing the secretion of corticotropin-releasing hormone(18). On the other hand, it is not clear, if the condition of our preterm neonates was so stressful that they should have increased their endogenous corticotropin-releasing hormone and ACTH secretion. The metyrapone test(25) or recently introduced low dose ACTH test(27–29) might be better than the present high dose ACTH test for the evaluation of stress responsiveness.

In the present study, the CBG levels in umbilical cord blood did not correlate significantly with gestational age. However, during the neonatal period, the concentration of CBG correlated with the postnatal age. Neonatal DEX treatment prevented this increase. CBG, the major transport protein for cortisol, is synthesized in the liver. Its serum concentration increases during fetal life; at term, it is approximately half of that in adults(10). The CBG concentrations in the present study were of similar magnitude as reported previously in preterm neonates(11, 12, 30). Thus, both total and free cortisol levels were low in the very preterm neonates, which suggests that very immature neonates may suffer from mild hypocortisolism. The decrease in the CBG concentration after neonatal DEX therapy is in line with changes in adults on long-term glucocorticoid treatment(31).

At birth, DHEAS concentrations correlated with gestational age and the ratio of DHEAS to cortisol correlated inversely with gestational age. During the 1st wk of life, DHEAS levels tended to be higher than in other specimens. At birth, the concentration of DHEAS correlates directly with gestational age and is high compared with levels later in infancy(32). The high DHEAS to cortisol ratio in the very preterm newborns reflects the low expression of the 3β-hydroxysteroid dehydrogenase enzyme in the fetal adrenal(3). The high DHEAS levels during the 1st wk of life may reflect the sudden discontinuation at birth of the placental metabolism of circulating DHEAS into estrogens. Alternatively, the DHEAS peak may partly represent a stress response(8, 19, 33). Neonatal DEX therapy decreased the concentration of DHEAS, whereas ACTH stimulation had no effect on the DHEAS levels. The suppression of both DHEAS and cortisol secretion by DEX therapy indicates that both the fetal and adult zone of the adrenal cortex are suppressed similarly by DEX therapy during the neonatal period, as has been shown after prenatal steroid treatment(34).

The SHBG concentrations in umbilical samples correlated with gestational age. Similarly, in the preterm neonates the SHBG concentrations correlated with the postnatal age. DEX therapy had no effect on the concentrations of SHBG. Our results are in line with previous reports demonstrating that SHBG is low at birth and increases gradually during infancy(35).

Despite severe morbidity and continuing requirement of intensive care, very preterm neonates had low serum concentrations of total and free cortisol at birth and during the neonatal period, but they responded well to high dose exogenous ACTH. One-week DEX therapy suppressed only transiently basal and stimulated levels of total and free cortisol. Results of the present study suggest that very preterm neonates requiring intensive care may have a reduced ability to increase their serum cortisol concentrations and thus may suffer from mild hypocortisolism.