Objectives
Exposure to glucocorticoid levels inappropriately high for current maturation alters fetal hypothalamo-pituitary-adrenal axis (HPAA) development. In an established fetal sheep model, we determined whether clinical betamethasone doses used to accelerate fetal lung maturation have persistent effects on fetal HPAA hypotensive-stress responses.
Study Design
Pregnant ewes received saline (n = 6) or betamethasone (n = 6); 2 × 110 μg/kg body weight doses injected 24 hours apart (106/107 and 112/113 days’ gestational age, term 150 days). Basal adrenocorticotropin (ACTH) and cortisol and responses to fetal hypotension were measured before and 5 days after the first course and 14 days after the second course.
Results
Basal ACTH and cortisol were similar with treatment. HPAA responses to hypotension increased after the second but not first course and ACTH/cortisol ratio increased indicating central HPAA effects.
Conclusions
Results demonstrate latency in the emergence of fetal HPAA hyperresponsiveness following betamethasone exposure that may explain hyperresponsiveness in full-term but not preterm neonates.
The incidence of preterm delivery is rising in the developed world. Currently, about 10% of pregnancies are delivered preterm, and most mothers in North America, Europe, and Australia who threaten premature labor receive synthetic glucocorticoids in accordance with National Institutes of Health consensus conference recommendations to decrease neonatal mortality and morbidity by accelerating fetal lung maturation.
The window of 24-34 weeks of gestation when prenatal glucocorticoid administration to pregnant women is recommended parallels a critical phase of development of many fetal systems including the hypothalamo-pituitary adrenal axis (HPAA). To avoid potential side effects, National Institutes of Health guidelines recommend only a single course of prenatal glucocorticoid administration. Nevertheless, in the last 2 decades, many pregnant women have received multiple courses because the original findings of Liggins and Howie indicated that benefits of maternal glucocorticoid administration may persist for only 7-10 days.
The therapeutic benefits of accelerated lung maturation are accompanied by exposure of the fetus to an inappropriate level of fetal glucocorticoids for the current stage of fetal maturation. Animal studies show that fetal exposure to levels of glucocorticoids higher than those appropriate for the current stage of maturation can reset the HPAA’s feedback set point with sustained consequences for the stress response in offspring that potentially mediate later life neuropsychological and cardiovascular disorders.
Many pregnancies in which this treatment is administered eventually go to term, and these fetuses will have experienced a very different intrauterine developmental history. One report shows that 76% of pregnancies in which the antenatal glucocorticoid therapy is given do not deliver before 32 weeks and 37% deliver after 37 weeks. Thus, it is important to determine any treatment effects on fetuses remaining in utero to permit the effects of glucocorticoids themselves without any effects of prematurity. Human studies have shown several outcomes: a transient suppression, no effects of prenatal betamethasone treatment on baseline adrenocorticotropin (ACTH) and cortisol levels, or a blunted response to stress in preterm newborns (ie, when the time between treatment and delivery was short).
Similarly, fetal exposure to betamethasone or dexamethasone resulted in a dose-dependent (transient) reduction in basal plasma cortisol concentrations in guinea pig and nonhuman primate fetuses.
On the other hand, cortisol response to stress was greater in full-term newborns when the time between treatment and delivery was longer. In agreement with those results, Sloboda et al have shown in sheep that 3 single weekly intramuscular maternal injections of 500 μg/kg –1 betamethasone starting at 104 days’ gestational age (dGA; term 150 days) lead to increased basal cord levels of ACTH at term. However, this dose was higher than the clinically 2 × 8-12 mg betamethasone 24 hours apart (equivalent to 2 × 110-170 μg/kg weight adjusted to a 70 kg woman).
Generally, the potential of prenatal synthetic glucocorticoid exposure to alter the HPAA responsiveness permanently appears to vary as a function of dose and timing as has been shown in rats, guinea pigs, and sheep. Better appreciation of these relationships is necessary to understand the programming effects of prenatal glucocorticoids on HPAA function.
Although modulation of fetal HPAA function by various glucocorticoid interventions has been examined in response to hypoxemia and corticotropin-releasing hormone (CRH) stimulation before, we know of no studies in which fetal HPAA response to hypotension modulated by a clinically relevant glucocorticoid administration has been examined. We hypothesized that prior exposure to a single course of betamethasone at the time, dose, and route of clinical administration would increase fetal HPAA sensitivity to a hypotensive challenge, and repetition of betamethasone exposure enhances the effects. Fetal exposure to hypotension is a clinically relevant challenge to fetuses of high-risk pregnancies likely to affect fetuses that experienced prior exposure to betamethasone. We studied fetal sheep because they have a neuroendocrine developmental profile similar to humans. Importantly, maternal administration of betamethasone to accelerate fetal lung maturation was developed in this animal model.
Material and Methods
Animal care and surgical instrumentation
All procedures were approved by the Cornell University Animal Use and Care Committee. Rambouillet-Colombia ewes of known gestational age were acclimated to the animal facilities for at least 5 days before surgery and kept in rooms with controlled light/dark cycles (14 h light/10 h dark: lights off at 9:00 p.m. and lights on at 7:00 a.m.) and fasted 24 hours before surgery. At 101 ± 1 (mean ± SEM) dGA, ewes were sedated with intramuscular ketamine (Ketaflo; Abbott, Abbott Park, IL). General anesthesia was provided with 1-2.5% isoflurane (Isoflo; Abbott).
Ewes were instrumented with a polyvinyl carotid arterial catheter to record fetal blood pressure (FBP) and jugular vein catheter for administration of drugs. Following laparotomy and hysterotomy, fetuses were instrumented with catheters in the left carotid artery and jugular vein, with their tips in the ascending aorta and superior vena cava, respectively. An amniotic cavity catheter was placed to correct FBP. The abdomen was closed and catheters maintained patent via infusion of heparinized saline (12.5 IU/mL to 0.5 mL/h). Ewes received 0.5 g ampicillin (AMP-Equine; Pfizer Animal Health, New York, NY) intravenously and 0.5 g ampicillin into the amniotic cavity every 12 hours for 3 days and 0.5 g phenylbutazone orally (Phenylzone paste; Schering-Plough, Kenilworth, NJ) twice daily for 3 days for postoperative analgesia.
Experimental protocol
Pregnant sheep were randomized to saline (n = 6) and betamethasone treated (n = 6) groups following at least 3 days of recovery. They received 2 betamethasone courses or equivalent volume of saline intramuscularly at 106 and 107 dGA and 112 and 113 dGA. Each course of betamethasone consisted of 2 doses of 110 μg/kg betamethasone phosphate (Celestan solubile; Essex, Munich, Germany) 24 hours apart or an equal volume saline.
The average maternal weights in the control group were 52.3 ± 2.23 kg and in the betamethasone group 54.5 ± 2.0 kg. The average betamethasone dose was 6.0 ± 0.22 g. Fetal HPAA activity was examined in response to 3 hypotensive challenges: 105 dGA (ie, before the first course of treatment), at 111 dGA (ie, 4 days after the first and before the second course of treatment), and at 127 dGA (ie, 14 days after the second course of treatment). FBP and fetal amniotic pressure were recorded continuously. Pressures were monitored using calibrated pressure transducers (Cobe, Lakewood, CO). FBP was corrected for the amniotic pressure and mean FBP was calculated.
Hypotensive challenge
The hypotensive challenge was induced by fetal jugular vein infusion of sodium nitroprusside (SNP;10 μg/mL; Sigma-Aldrich, Deisenhofen, Germany) beginning at 0.1 mL/min. The SNP infusion rate was increased stepwise from 0.2 mL/min to 3.2 mL/min by doubling the infusion rate every 2 minutes. Fetal and maternal arterial blood samples were taken prior to the stress responses for measurement of blood gases, hemoglobin concentration, and oxygen saturation using a blood gas analyzer (ABL600; Radiometer, Copenhagen, Denmark; measurements corrected to 39°C) and a hemoximeter (OSM2; Radiometer). Plasma ACTH and cortisol were determined 30 minutes before, at the end (0 minutes), and 15, 60, and 120 minutes after the end of the SNP infusion. One milliliter of blood was collected in chilled EDTA tubes, plasma separated by centrifugation at 4°C for 10 minutes at 3000 × g , flash frozen, and stored at –80°C.
Hormone analyses
Fetal plasma cortisol was measured using the Coat-A-Count radioimmunoassay kit (Diagnostic Products Inc, Los Angeles, CA) with a sensitivity of 5.5 nmol/L. For plasma pools measuring 27.60 nmol/L and 138 nmol/L, the intraassay coefficients of variation (CVs) were 4.14% and 3.3%, respectively. Fetal ACTH was measured by chemiluminescence enzyme immunometric assay (DPC Immulite assay; Diagnostic Products). Assay sensitivity was 1.98 pmol/L. All samples were measured in 1 assay. For samples measuring 4.38 pmol/L and 46.48 pmol/L, the intraassay CVs were 4.75% and 3.74%, respectively. The ACTH/cortisol ratio was calculated to examine relative pituitary and adrenal responses to hypotension.
Data acquisition and statistical analysis
FBP and amniotic pressure were amplified and digitized using a 16 bit analog-digital interface card and a data acquisition system (Windaq; DATAQ Instruments, Akron, OH) at a sample rate of 256 Hz and stored on a hard disc of a personal computer. Baseline FBP was averaged over 10 minutes before the hypotensive challenge.
The integrated FBP decrease (FBPint) was used as the index of the challenge to the fetal HPAA and estimated using the area under the curve with the formula:
BP int = ∑ t = 1 n − 1 1 2 ( B P t + B P t + 1 ) Δ t
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