Objective
We sought to evaluate the interaction between repeated-course antenatal corticosteroids and inflammation gene polymorphisms with neurodevelopmental outcomes at age 2 years.
Study Design
We conducted nested case-control analysis of a randomized controlled trial of single- vs repeated-course antenatal corticosteroids. Cases had mental and/or psychomotor delay at age 2 years. Controls had normal neurodevelopment. Previous analyses of 125 cases and 147 controls identified 4 inflammation gene polymorphisms associated with neurodevelopmental delay at age 2 years.
Results
The interaction between repeated-course corticosteroids and the interleukin (IL)-6 –174 genotype with neurodevelopmental delay was significant ( P = .046). The IL-6 –174 GG genotype was associated with neurodevelopmental delay at age 2 years in the single-course corticosteroid group (odds ratio, 6.47; 95% confidence interval, 1.86–22.50). Exposure to repeated-course antenatal corticosteroids abrogated this genotype effect (odds ratio, 1.30; 95% confidence interval, 0.48–3.54). Results were unchanged after controlling for potential confounders.
Conclusion
Repeated-course antenatal steroids may reduce the increased risk of neurodevelopmental delay at age 2 years associated with IL-6 –174 GG genotype.
Single-course antenatal corticosteroid treatment is recommended for women at risk for preterm delivery, and is associated with a reduction in neonatal mortality and morbidity. The administration of repeated-course antenatal corticosteroids has been the subject of recent clinical trials, and has been found to further reduce neonatal respiratory morbidity, relative to single-course regimens. However, these studies have also supported concerns regarding the impact of repeated-course antenatal corticosteroids on fetal growth, head circumference, and neurodevelopment.
The clinical effects of antenatal corticosteroids may be mediated, in part, by their antiinflammatory effects. Corticosteroids affect inflammation signal transduction pathways by several different mechanisms: (1) increased synthesis of antiinflammatory proteins, (2) inactivation of inflammatory genes, and (3) postgenomic effects leading to a reduction in inflammatory protein expression.
The predominant effect of corticosteroids appears to be inhibition of inflammatory protein synthesis (eg, proinflammatory cytokines) through gene suppression. Inflammatory stimuli result in activation of the transcription factor nuclear factor-κβ, which then translocates to the nucleus and binds to specific DNA recognition sequences, resulting in histone acetylation and increased expression of genes encoding proinflammatory cytokines (eg, interleukin [IL]-6 and IL-1β). There is persuasive evidence that corticosteroids inhibit the downstream effects of nuclear factor-κβ. The corticosteroid/glucocorticoid receptor complex translocates to the nucleus and binds to coactivators, leading to reversal of histone acetylation, decreased gene transcription, and decreased proinflammatory cytokine production.
We have recently shown that single nucleotide polymorphisms (SNPs) in IL-1β and IL-6 genes may be associated with neurodevelopmental delay at age 2 years. This is consistent with the reported associations among proinflammatory cytokines, perinatal white matter injury, and subsequent neurodevelopmental abnormalities. We hypothesized that the antiinflammatory effects of repeated-course antenatal corticosteroids would reduce the risk of neurodevelopmental delay at age 2 years associated with proinflammatory gene SNPs.
Materials and Methods
Subjects
Our subjects were the children of women enrolled in the Eunice Kennedy Shriver National Institute of Child Health and Human Development Maternal Fetal Medicine Units (MFMU) Network randomized, double-masked, placebo-controlled, multicenter clinical trial of single vs repeated courses of antenatal corticosteroids. The primary aim of the randomized trial was to assess the clinical efficacy and safety of repeated courses of antenatal corticosteroids in pregnancies at risk for preterm birth. All women in the trial received 1 course of either betamethasone or dexamethasone 6-10 days prior to enrollment. Consenting women were then randomly assigned to either weekly courses of betamethasone or placebo. The study protocol and results of the trial, which was conducted from 2000 through 2003, have been previously published. Placental samples and fetal cord serum were collected in a subset of subjects enrolled in the MFMU Network antenatal corticosteroid trial per study protocol.
A secondary aim of the MFMU Network antenatal corticosteroid trial was the correlation of steroid regimen with neurodevelopmental outcomes as assessed by the Bayley Scales of Infant Development at age 2 years. The Bayley Scales consist of mental development index (MDI) and psychomotor developmental index (PDI). A Bayley score of 85 is 1 SD below the mean and consistent with mild neurodevelopmental delay. A Bayley score of 70 is 2 SD below the mean and consistent with more significant delay. Overall, there were no significant differences between single- vs repeated-course treatment groups in Bayley scores.
This secondary analysis aimed to evaluate whether there was an association between repeated-course antenatal steroids and inflammation gene SNPs with neurodevelopmental outcomes at age 2 years. Inclusion criteria consisted of: (1) Bayley scores at age 2 years, and (2) available DNA for genotyping, from fresh placental tissue, formalin-fixed paraffin-imbedded placental tissue, or fetal cord serum collected at the time of delivery. Cases were children with mental and/or neurodevelopmental delay, defined by a Bayley MDI and/or PDI <85 at age 2 years. Cerebral palsy (CP) cases (as defined in the primary trial, n = 7) were excluded from the analysis to test our hypothesis of an interaction between corticosteroid regimen and genotype on neurodevelopmental outcomes in the absence of CP. Controls were children with normal neurodevelopment, defined by Bayley MDI and PDI ≥85.
Previously reported univariate analyses compared allele and genotype frequencies between cases and controls for 48 SNPs in inflammation and coagulation genes. Significant genotype or allele differences were reported for IL-1β –511, IL-6 –174 and –176, and IL-4R 148. Of particular interest were the results for IL-1β –511 and IL-6 –174, as these SNPs are in the promoter region of these genes and have been associated with altered gene expression and resultant functional levels of IL-1β and IL-6, respectively. The interaction between each of these inflammation gene SNPs and antenatal corticosteroid exposure with neurodevelopmental delay at age 2 years was assessed in the current analysis.
The study protocol was reviewed by the institutional review board at the University of Utah and was determined to be exempt from institutional review board approval procedures secondary to deidentification of data and study samples prior to analysis. DNA extraction, genotyping, and quality control methods for this sample set have been previously reported.
Statistics
Demographic and clinical characteristics of cases and controls were compared using χ 2 or Fisher’s exact test, to compare categorical variables, and the Wilcoxon rank sum test, to compare continuous variables. SNPs found to be significantly related to MDI and/or PDI <85 in prior univariate analyses ( P < .05) were further analyzed for interaction with antenatal corticosteroid exposure using the Breslow-Day test for homogeneity of the odds ratio (OR) and multivariable logistic regression modeling. The genotype model for each SNP was the best model determined in previous logistic regression analyses. Logistic regression model covariates included gestational age at delivery, sex, small for gestational age, and steroid treatment group, as well as maternal race, smoking, and level of education. The sample size and the rarity of some genotypes and phenotypes limited further modeling of interactions. Tests for Hardy-Weinberg equilibrium (χ 2 or Fisher’s exact test) were performed on control subjects for each SNP significant in the univariate analysis. A nominal P value of < .05 was considered to be statistically significant. All calculations were performed using SAS software (SAS Institute Inc, Cary, NC).
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