The development of the hypothalamus-pituitary-adrenal axis during infancy may be affected by antenatal glucocorticoid therapy

Abstract

BACKGROUND:

Developmental changes in the hypothalamus-pituitary-adrenal (HPA) axis during infancy have been reported in term infants, but those in preterm infants have yet to be elucidated. If developmental changes in the HPA axis of preterm infants are modulated by any factors, it may affect their future health. Few studies have examined the lasting consequences of antenatal glucocorticoids on the development of the HPA axis.

METHODS:

We measured pre- and post-palivizumab vaccination salivary cortisol values in two conforming periods of three-months intervals during infancy, and compared cortisol values and the response of cortisol secretion between groups with and without antenatal glucocorticoid (AG) therapy.

RESULTS:

Although the strength of the response of cortisol secretion to palivizumab fell age-dependently (until late infancy) in the Non-AG group, the opposite pattern was exhibited in the AG group. The changes of the delta cortisol values between the 2 groups were significant.

CONCLUSIONS:

This study suggests that the HPA axis of preterm infants whose mothers receive AG therapy may be upregulated during infancy, possibly leading to long lasting health problems.

Keywords

Antenatal glucocorticoid cortisol hypothalamic-pituitary-adrenal axis infants preterm

1 Introduction

The morbidity and mortality rates of preterm infants have recently improved due to advances in perinatal medicine [1]. The use of antenatal glucocorticoid (AG) therapy for preterm labor is considered one of the most important factors for these improvements [2, 3]; however, we reported that AG may affect the HPA axis in offspring within a few weeks after birth [4]. Unfavorable long-lasting effects of AG therapy are also of concern [4 –6]. Larson et al. reported that in human infants, the responsiveness of the HPA axis falls markedly over the first few months of life [7]. In a later study, infants aged between 7 and 11 weeks exhibited increased salivary cortisol levels after undergoing a physical examination, whereas infants aged between 11 and 15 weeks did not. Although there are reports on term infants, there are none on preterm infants. If developmental changes in the HPA axis of preterm infants are modulated by any factors, it may affect their future health. It is important to clarify the characteristics of the developmental changes in the HPA axis of preterm infants. Several reports have suggested that AG therapy modulates the HPA axis for a while after birth [4 , 8–10]. Based on these findings, we suspected that AG therapy affects the development of the HPA axis. In order to confirm this hypothesis, we measured salivary cortisol secretion induced in response to noxious needle stimulation in two separate periods during infancy. We compared the basal and subsequent cortisol secretion levels in infants whose mothers were or were not administered AG therapy.

2 Materials and methods

2.1 Subjects

Preterm infants that received palivizumab vaccinations at the outpatient department of Kyoto University Hospital between September 2012 and April 2014 were included in this study. Infants who had major congenital anomalies were excluded. We divided the subjects into two groups by AG therapy. Infants whose mothers received AG therapy before birth were included in the AG group, and infants whose mothers did not receive AG were separated into the Non-AG group. Ethical approval was obtained from the ethics committee of the Kyoto University Graduate School Faculty of Medicine (No. E-581), and written informed consent was acquired from the parents of each infant.

2.2 Procedures

The subjects’ pre- and post-palivizumab vaccination salivary cortisol values were measured at two different periods that were more than 3 months apart between 3 and 9 months of corrected age (calculated by age from the expected date of birth). Then, their basal cortisol secretion levels and the cortisol responses were examined. We compared them with or without AG therapy.

2.3 Saliva sample collection

To minimize the influence of cortisol circadian rhythm, the timing of collecting the saliva sample was scheduled between 10:00 and 15:00. A Salivette tube containing a cotton wool swab (Sarstedt co.) was used to collect each saliva sample. The swab was rotated in the mouth for at least 2 minutes and then inserted back into the tube. The samples were centrifuged and stored at –30°C until the cortisol assay.

2.4 Salivary cortisol assay

The cortisol concentrations of the subjects were assayed in duplicate using a commercially available enzyme immunoassay (expanded range high sensitivity salivary cortisol enzyme immunoassay kit, Salimetrics LLC). According to the manufacturer’s instructions, the analytical detection range of the kit is 0.033–82.77 nmol/liter (0.012–3.0 μg/dl). The intra-assay precision of this method was reflected by the coefficients of variation (CV) of 3.35% at 0.033 μg/dl and 3.65% at 0.004 μg/dl, and the inter-assay precision was reflected by the CV of 3.75% at 0.038 μg/dl and 6.41% at 0.006 μg/dl.

2.5 Covariates

2.5.1 Antenatal glucocorticoid (AG) therapy

AG therapy was given to pregnant women who were considered to be at risk of preterm delivery or those at risk of delivering before 34 weeks of gestational age. Several women received a single course of muscular injections of betamethasone, but some mothers delivered their babies before such injections were administered. AG was administered 2 times with 12 mg of betamethasone 24 hours apart.

2.5.2 SD score

Age- and gender-specific standard deviation scores (SDS) were calculated by the weight of the infants with calculation software for the body frame index at birth by gestational period (http://jspe.umin.jp/medical/keisan.html).

2.5.3 Postnatal glucocorticoid therapy

Some infants required glucocorticoid therapy to prevent acute or chronic circulatory failure, or bronchopulmonary dysplasia during hospitalization. Although hydrocortisone and/or dexamethasone were often employed for this purpose, the use of these drugs was terminated before the infant reached term (corrected age) in all cases.

2.5.4 Surgery

Surgical treatment may cause some preterm infants stress. Two infants included in this study underwent surgery, ligation of the patent ductus arteriosus and abdominal operation for necrotizing enterocolitis. None of these infants received stress dose steroids for surgical procedures.

2.5.5 Bronchopulmonary dysplasia (BPD)

As the criteria based on supplemental oxygen at 36 weeks postmenstrual age has gained wide acceptance, we followed this consensus [11]. In this study, 5 infants who were born before 27 weeks of gestational age diagnosed BPD (Table 1).

Table 1
Infant demographics

AG (n = 12) Non-AG (n = 10) p

Gestational age (w) 29.6 (26.6–33.4) 35.1 (30–35.7) 0.26

Birth weight (g) 1078 (695–1515) 2097 (1196–2328) 0.06

Gender (Males)^* 5 (41.7%) 4 (40.0%) 0.94

SD score –1.0 (–1.4–0) –0.3 (–0.6–0) 0.19

Apgar score (at 1 min) 6 (4–8) 8 (4–8) 0.45

Apgar score (at 5 min) 9 (7–9) 9 (7–9) 0.65

Postnatal glucocorticoid therapy^* 5 (41.7%) 2 (20.0%) 0.29

Tracheal intubation (days) 8 (0–17) 1 (0–6) 0.37

Surgery^* 2 (16.7%) 0 (0%) 0.17

Bronchopulmonary dysplasia^* 3 (25.0%) 2 (20.0%) 0.79

Rentinopathy of prematuritu^* 1 (8.3%) 2 (20.0%) 0.47

Developmental delay^* 3 (25.0%) 1 (10.0%) 0.37

	AG (n = 12)	Non-AG (n = 10)	p
Gestational age (w)	29.6 (26.6–33.4)	35.1 (30–35.7)	0.26
Birth weight (g)	1078 (695–1515)	2097 (1196–2328)	0.06
Gender (Males)^*	5 (41.7%)	4 (40.0%)	0.94
SD score	–1.0 (–1.4–0)	–0.3 (–0.6–0)	0.19
Apgar score (at 1 min)	6 (4–8)	8 (4–8)	0.45
Apgar score (at 5 min)	9 (7–9)	9 (7–9)	0.65
Postnatal glucocorticoid therapy^*	5 (41.7%)	2 (20.0%)	0.29
Tracheal intubation (days)	8 (0–17)	1 (0–6)	0.37
Surgery^*	2 (16.7%)	0 (0%)	0.17
Bronchopulmonary dysplasia^*	3 (25.0%)	2 (20.0%)	0.79
Rentinopathy of prematuritu^*	1 (8.3%)	2 (20.0%)	0.47
Developmental delay^*	3 (25.0%)	1 (10.0%)	0.37

Median (Interquartile range) *Number of infants (%). Abbreviations; AG: antenatal glucocorticoid, SD: standard deviation. Mann-Whitney U tests were used for comparisons of gestational age, birth weight, SD score, and Apgar score (at 1 minutes and 5 minutes, respectively), and the Chi-square test was used for comparison of gender, other perinatal factors, and development. P-values of <0.05 were considered significant. None of the parameters differed significantly between the 2 groups.

2.5.6 Retinopathy of prematurity (ROP)

Preterm infants are regularly examined by ophthalmologists, and sometimes receive laser therapy for retinopathy of prematurity (ROP). In this study, infants who received laser therapy for deterioration of retinopathy were evaluated upon ROP development.

2.5.7 Developmental delay

Developmental delay was defined as a total developmental quotient of less than 85 according to the Kyoto Scale of Psychological Development (2001) at a corrected age of 12 months [12].

2.6 Statistical analysis

Continuous variable data for background profiles, and perinatal and developmental factors were expressed as median values followed by the range values in parentheses. The cortisol values were presented as mean±standard deviation. P-values < 0.05 were considered significant.

3 Results

3.1 Infant profiles

In total, 142 preterm infants received palivizumab vaccinations at least once at the outpatient department of Kyoto University Hospital between September 2012 and April 2014. For 31 preterm infants pre- and post-palivizumab vaccination salivary cortisol values were measured at both conforming periods. Eight infants were excluded because one of their saliva samples was insufficient for measuring cortisol values. One infant whose mother developed systemic lupus erythematosus and received corticosteroid therapy throughout pregnancy was excluded due to concerns about the effects on the fetal adrenal gland. Thus, 22 preterm infants were analyzed. We divided the 22 infant subjects into two groups by AG therapy. Twelve infants were in the AG group and 10 were in the Non-AG group. The time period from delivery to the 2nd AG administration to the mothers ranged from 2 hours to 26 days (median 6 days) in the AG group (Fig. 1). The 1st period to measure cortisol values was at 3.9±1.4 months of corrected age, and the 2nd period was at 7.2±1.5 months of corrected age.

Fig.1

Flowchart of enrollment. For 31 preterm infants, pre- and post-palivizumab vaccination salivary cortisol values were measured at two conforming periods. Eight infants were excluded because at least one of their saliva samples was insufficient for measuring cortisol values. One infant whose mother developed systemic lupus erythematosus and received corticosteroid therapy throughout pregnancy was excluded due to concerns about the effects on the fetal adrenal gland. Thus, 22 preterm infants were analyzed. Twelve infants were in the AG group and 10 were in the Non-AG group.

3.2 Preliminary studies

In the preliminary experiment, we assessed the optimal time for evaluating the changes in cortisol secretion induced by palivizumab vaccinations in 8 infants at 3 months of corrected age (data not shown). Of 8 infants, 6 belonged to the AG group and 2 belonged to the Non-AG group. The following two results were obtained: First, the mean cortisol values recorded on arrival at the hospital did not differ significantly from those acquired just before the injection of palivizumab. Second, the mean cortisol values obtained 20 minutes after the vaccination were more than twice as high as those obtained just before the vaccination, and did not differ from those acquired 30 minutes after the vaccination. Based on these two findings, we decided the timing to collect saliva as the following 2 points; just before and 20 minutes after palivizumab injection.

3.3 Infant demographics

Table 1 shows the demographic data of the infants. There was no significant difference in gestational age, birth weight, gender, SD score, or Apgar score at 1 or 5 minutes between the two groups. Furthermore, we did not find any differences in perinatal factors or development at a corrected age of 12 months between the two groups.

3.4 Salivary cortisol levels in the AG and Non-AG groups (Fig. 2)

Fig.2

Salivary cortisol levels in the AG and Non-AG groups. The solid line indicates the post-cortisol levels and the dotted line indicates the pre-cortisol levels in each group. Delta (Δ) cortisol represents the difference between pre- and post- cortisol levels at both periods. * represents a significant difference in the cortisol levels between the AG group and the Non-AG group (p < 0.05).

(a) Comparison of the pre- and post-cortisol levels in the AG and Non-AG groups

We compared the pre- and post-palivizumab vaccination salivary cortisol levels of the two groups. In the Non-AG group, the post-cortisol levels were high in the 1st period and decreased in the 2nd period; however, they were relatively low in the 1st period and increased in the 2nd period in the AG group. Based on the comparison of cortisol levels in the AG and Non-AG groups, the differences in the post-cortisol levels in both periods and the difference between the periods were all significant (*; p < 0.05). On the other hand, the pre-cortisol levels did not exhibit any difference between the 2 groups.

(b) Comparison of the cortisol responses in the AG and Non-AG groups

We next compared the salivary cortisol responses (post-cortisol levels minus pre-cortisol levels, shown as Δ cortisol) in the two groups in the two periods. Regarding the cortisol response in the 1st period, the AG group demonstrated a significantly lower response than the Non-AG group (p = 0.023). Conversely in the 2nd period, the AG group had a significantly higher response than the Non-AG group (p = 0.024).

4 Discussion

As reported in the review article by Jansen et al., the cortisol response reflects age-related changes in infants, i.e., it weakens with age [13]. Lashansky also noted that the HPA axis undergoes developmental changes and found that HPA reactivity is decreased after 6 months of age [14]. Ramsay et al. reported data indicating reversal in the meaning of high adrenocortical reactivity between 2 and 6 months of age; high cortisol response may indicate optimal functioning at 2 months, but non-optimal functioning by 6 months of age [15]. They suggested that this reversal represents a developmental shift in adrenocortical functioning during that period. On the other hand, clear associations exist between a low birth weight and the development of several components of metabolic syndrome, such as hypertension and type 2 diabetes, and cardiovascular disease in adulthood [16]. HPA axis hypersensitivity is postulated to be one of the mechanisms responsible for these links.

AG therapy for threatened labor improves the prognosis of premature infants. For example, it prevents respiratory distress syndrome and neonatal mortality [17, 18]. However, some reports have suggested that AG therapy affects development not only for several weeks, but also for a long period, and causes endocrinological problems [19, 20]. Exposure to AG was found to be associated with higher blood pressure in adulthood in several animal species [21 –23]. Seckl et al. reported that prenatal glucocorticoid exposure permanently increases basal plasma corticosterone levels because it induces permanent reductions in the hippocampal density of both glucocorticoid receptors and mineralocorticoid receptors, which attenuate the feedback sensitivity of the HPA axis [10, 24]. In addition, they suggested that “HPA programming” occurs relatively frequently after prenatal environmental challenges and may act in part via alterations in placental 11βHSD2 activity [8, 25]. Similar findings have been obtained in human studies. Reynolds et al. reported that fetal glucocorticoid exposure alters the set-point of the HPA axis and activates the HPA axis. Persistent activation of the HPA axis, which is associated with a low birth weight, is also correlated with cardiovascular disease [26, 27]. In the Auckland Steroid Trial, which examined 30-year-old individuals whose mothers had participated in a randomized trial of AG therapy, the only potentially detrimental outcome of AG therapy noted was a small but significant increase in insulin resistance, which is a precursor of type 2 diabetes [19]. Therefore, AG therapy should be reduced to a minimum to avoid possible long-term adverse effects.

Taking these findings into consideration, we hypothesized that AG therapy affects the developmental changes in the HPA axis that occur during infancy. Thus, we designed this study to clarify whether the response of cortisol secretion to the noxious needle stimulation, i.e., injection of palivizumab, during infancy differs between patients whose mothers did or did not receive AG therapy.

Palivizumab (Synagis^®) is a monoclonal antibody that acts as an inhibitor of the respiratory syncytial virus (RSV) F protein and is indicated for the prevention of serious lower respiratory tract diseases caused by RSV in children that are at high risk of RSV infection. It is typically given as a shot, usually into the thigh muscle, during each month of the RSV season. Therefore, as many preterm infants receive multiple palivizumab vaccinations during infancy, we considered it possible to perform a longitudinal study of cortisol responses in which palivizumab was used as the stimulus.

In this study we made several important observations. First, the cortisol response slightly weakened towards late infancy in the Non-AG group, as was reported in previous studies [28, 29]. On the contrary, no such reduction in the strength of the cortisol response was noted in the AG group. Second, the activity of the HPA axis may not be suppressed and hyperactivity was maintained in the AG group, which suggests that AG therapy disrupts the normal development of the HPA axis [30]. Careful follow-up of infants whose mothers receive AG therapy is necessary because AG therapy may have long-term adverse effects, such as the risk of metabolic syndrome, via upregulation of the HPA axis.

This study has several limitations. First, the number of subjects was small. The main aim of this study was to assess the response of cortisol secretion to a single noxious stimulus in a longitudinal manner. To achieve this, the subjects were restricted to infants who received at least four noxious needle stimulations, which made it possible to employ an inter-injection interval of 3 months or longer. As such, only a few infants met this criterion. The second limitation was that 7 of 22 subgroup infants had received postnatal glucocorticoids. Similar with AG, the adverse effects on the HPA axis from postnatal glucocorticoid were of concern. Thus, we compared the salivary cortisol data between the AG group (n = 7) and the Non-AG group (n = 8) without postnatal glucocorticoid therapy, but the tendency for both cortisol levels and the cortisol responses of these subjects was similar to those of all subjects. Based on this, we considered that the effects of AG on the HPA axis did not depend on postnatal glucocorticoid therapy. The third limitation was that we were unable to find any differences in clinical manifestations, e.g., psychomotor development or physical findings between the AG and non-AG groups even though we detected prolonged HPA axis hyperactivity in infants who had been exposed to AG. Preterm infants experience several kinds of stress during their stay in the neonatal intensive care unit, such as breathing disorders and painful procedures, and such conditions may affect their subsequent development [31, 32]. Thus, long-term careful follow-up may be necessary to clarify the clinical implications of our findings.

This study provides new evidence that the strength of the cortisol response to noxious stimulation increases age-dependently in preterm infants whose mothers received AG therapy. This suggests that AG modulates the HPA axis in infants long after birth and may need to be evaluated in further studies. As HPA hyperactivity can affect life-long health careful follow-up of infants who are exposed to AG may be necessary.

Funding

This work was supported by funding from the Japan Science and Technology Agency, Exploratory Research for Advanced Technology, and the Okanoya Emotional Information Project.

Disclosure statement

The authors declare that they have no conflicts of interest.

Footnotes

Acknowledgments

We thank all of the infants who participated in this study and their parents, and Yuta Shinya for helping with the sample collection and data analysis.

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