Associations of maternal blood biomarkers of prenatal APAP exposure with placental gene expression and child attention deficit hyperactivity disorder

Abstract

Despite evidence linking prenatal acetaminophen (APAP) exposure and adverse neurodevelopment in humans and animals, over half of pregnant women in most populations use APAP. Prior studies could be biased by inaccurate self-reported APAP use, and the molecular mechanisms linking prenatal APAP with adverse neurodevelopment are unknown. We estimated associations between maternal plasma biomarkers of APAP exposure, child attention deficit hyperactivity disorder (ADHD), and placental gene expression in 307 African-American mother-child pairs. Overall, detection of APAP in 2^nd trimester plasma was associated with higher odds for child ADHD diagnosis (Odds Ratio (OR)=3.15 [95%CI: 1.20-8.29]). Prenatal APAP exposure and ADHD were associated with placental upregulation of immune system pathways in females, and downregulation of oxidative phosphorylation in both sexes. In females only, prenatal APAP was associated with 5.22% higher odds (0.0456%-13.1%) of ADHD statistically mediated through increased immunoglobulin heavy constant gamma 1 (IGHG1) expression. These results highlight placental molecular mechanisms that may underlie developmental toxicity of prenatal APAP exposure.

Figure 1:. Study Inclusion Flowchart

Study flowchart for inclusion of participants shows analytic sample sizes remaining from the implementation of each exclusion criterion. Participants come from Conditions Affecting Neurocognitive Development and Learning in Early Childhood (CANDLE) birth cohort.

Figure 2:. Conceptual Model

Conceptual model of confounders, mediators, precision variables, and effect modifiers in the relationship between prenatal APAP exposure and child ADHD-related outcomes (A). Primary models adjusted for confounders and precision variables, while sensitivity models additionally adjusted for birthweight and gestational age. Effect modification was tested by including APAP by child sex interaction terms. Prenatal NSAID and antibiotics, which were included as covariates in the primary models, were conceptualized as instrumental variables for APAP indications (B). ¹ Potential mediators were only included as adjustment variables in a sensitivity analysis.

Figure 3:. Associations of Prenatal APAP Exposure with Child ADHD

Associations of prenatal APAP with maternal report of child ADHD diagnosis and ADHD prescription medications (N = 307) (A), CBCL DSM5-oriented ADHD scale and externalizing scale (N = 305) (B), and task-based NIH Flanker (N = 281) and Digit Span (N = 251) scores (C). Non-interaction models (indicated as “Both” in legend) included child sex as a covariate with no sex interaction terms. Sex-specific model estimates were obtained by adding prenatal APAP by child sex interaction product terms to the models. Models were re-run with male and female sex set as the reference level to obtain sex specific estimates for prenatal APAP. All models adjusted for maternal age, education, ethnicity, pre-pregnancy BMI, prenatal alcohol, prenatal tobacco, prenatal stressful life events, gravidity, delivery method, labor type, household income, neighborhood deprivation index, child age at the study visit, mental health disorders in the child’s immediate family, and maternal NSAID and antibiotic use (as instruments for APAP indications). Points indicate odds ratios from logistic regressions and beta coefficients from linear regressions, while error bars depict 95% confidence intervals.

Figure 4:. Placental IGHG1 Expression Links Prenatal APAP with Child ADHD in Females but not Males

Boxplots depict median (center line), 25^th percentile (minima), and 75^th percentile (maxima), while whiskers extend to the highest and lowest values that are within 1.5 times the inter-quartile range of the box. One point per individual (N = 174) depicts placental IGHG1 expression according to child ADHD diagnosis (A) and prenatal APAP exposure (B) among females. Diamonds indicate mean gene expression. Unadjusted and false discovery rate (FDR) adjusted two-sided p-values from moderated t-tests from limma-voom differential expression analysis shown. Mediation by placental IGHG1 expression of the association between prenatal APAP exposure and child ADHD among females shows mediation effect estimates and 95% confidence intervals from causal mediation analysis (C). IGHG1 expression according to child ADHD diagnosis (D) and prenatal APAP exposure (E), and mediation analysis (F) in males.

Figure 5:. KEGG Pathways Associated with Prenatal APAP Exposure and Child ADHD

T-statistics (Log2FCs divided by standard error) from the differential expression analyses were input into gene set enrichment analyses for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using the generally applicable gene set enrichment (GAGE) and Correlation Adjusted MEan RAnk (CAMERA) methods. Gene set enrichment P-Values (larger among the two methods) and mean gene T-statistics shown for pathways with FDR<0.05 in both CAMERA and GAGE methods (N = 174).