Prenatal Acetaminophen Exposure Does Not Disrupt Human Fetal Brain Development in Cortical Organoid Models

Acetaminophen (APAP) is the most widely used analgesic during pregnancy, yet epidemiological studies have reported conflicting associations between prenatal exposure and neurodevelopmental disorders (NDDs) such as autism spectrum disorder (ASD) and Attention Deficit Hyperactivity Disorder (ADHD). Direct mechanistic evidence from human-relevant models remains limited. To address this gap, we exposed human induced pluripotent stem cell-derived cortical organoids (COs; six independent lines) to physiologically relevant APAP concentrations (25, 50, and 100 M) for 5 days starting at day 21, a developmental stage corresponding to the late first trimester. We assessed organoid growth, apoptosis, neuronal/progenitor ratios, transcriptomics (RNA-seq), and functional maturation using multielectrode arrays (2-4 months). APAP exposure did not alter growth dynamics, cytoarchitecture, or viability, with apoptosis rates remaining below 1% in all conditions. Neuronal and progenitor cell proportions were unchanged. Bulk RNA-seq revealed subtle, dose-dependent transcriptional changes only at higher doses (0 differentially expressed genes at 25 M, 9 at 50 M, and 16 at 100 M), enriched for neurodevelopmental pathways. Despite these slight molecular perturbations, electrophysiological recordings showed normal firing rates, burst frequency, and network synchronicity across exposure groups. Taken together, therapeutic APAP concentrations produced only modest transcriptional effects without structural or functional consequences in developing cortical organoids. These results align with recent sibling-controlled epidemiological studies that attribute reported associations to confounding and provide mechanistic reassurance that recommended APAP use during pregnancy is unlikely to disrupt fetal brain development.