Genetic Background and Sex Modulate Androgen Responses in Human Brain Microphysiological System

Sex steroids shape human brain development, yet the cellular and molecular consequences of androgen addition need further exploration. Here, we used a human neural organoid model - brain microphysiological system (bMPS), derived from nine induced pluripotent stem cell (iPSC) lines to model the impact of dihydrotestosterone (DHT), a potent non-aromatizable androgen. All lines differentiated reproducibly into electrically active, neuron–glia–oligodendrocyte organoids expressing robust androgen-signaling components. DHT was bioavailable, elicited nuclear androgen receptor (AR) translocation and increased organoid size in most cell lines, consistent with androgen-responsive mTOR and metabolic pathway activation. Bulk RNA-seq across nine lines revealed that transcriptional responses varied across donor backgrounds, but DHT-responsive genes converged on mitochondrial energetics, lysosomal function, glycoprotein processing, apoptosis, and mTOR signaling. Cell-type expression profiling showed an androgen-driven shift primarily in male lines toward astrocytic profiles with reductions in oligodendrocyte, oligodendrocyte progenitor (OPC), excitatory, and inhibitory neuronal signatures, supported by immunohistochemistry and AR enrichment in astrocytes and OPCs. DHT also altered neurodevelopmental pathways, increasing variation in synaptic pruning and decreasing variation in neuronal migration, with autism spectrum disorder (ASD) diagnosis and seizure status of donors moderating these effects more strongly than sex. Baseline transcriptional differences distinguished iPSC lines which responded more strongly to DHT from weak responders: responders displayed enhanced synaptic maturity and reduced ECM gene expression. Using isogenic XX/XY lines, we found that differences in sex-chromosome expression exceeded DHT-induced changes and that DHT decreased expression of inhibitory neuron genes in males and increased it in females. Finally, DHT induced extensive DNA methylation changes, targeting HOX genes, patterning, and synaptic genes. Collectively, these findings reveal that androgen signaling shapes transcription, cell populations, and epigenetic landscapes in a genetic background-dependent manner. This work contributes to understanding how androgens influence human brain development and highlights how in vitro models can contribute to representing inter-individual variability in neurodevelopment and neurodevelopmental disorders.