Multi-omic profiling defines subtype-specific extracellular vesicle signatures in early human pregnancy.

Early human pregnancy (11-15 weeks) is a critical period when placental dysfunction begins but remains clinically undetectable, preventing early intervention for pregnancy complications. This highlights a pressing need for circulating biomarkers that can signal the onset of a complication. Circulating extracellular vesicles (EVs) carry bioactive cargo that reflect originating cell activity in real-time, positioning them as early biomarkers of pregnancy complications. While pregnancy studies have identified plasma EV cargo alterations across complications, studying small EVs alone-the field standard-or bulk EVs together obscures pathological changes that may manifest preferentially in one population. To address this, we performed the first multi-omic comparison of small and large plasma EVs from healthy pregnancies at 11-13 weeks (n=10) to establish subtype-specific cargo differences and provide a reference for normal pregnancy EV biology. We quantified mitochondrial DNA (mtDNA), performed transcriptomics (mRNAs, miRNAs) and proteomics to define small and large EV signatures. Large EVs were preferentially enriched for mitochondrial cargo including DNA, transcripts, and proteins as well as hypoxia response, endocrine signaling, and VEGF signaling related transcripts and proteins. In contrast, small EVs were enriched for canonical EV markers and basement membrane proteins. Additionally, we found that EV miRNA composition was similar across EV subtypes with only 7 miRNAs differentially abundant between small and large EVs. These distinct small and large EV cargo signatures underscore the importance of profiling both populations for liquid biopsy potential. Together these data provide a baseline for future studies linking EV signature alterations to placental dysfunction and adverse pregnancy outcomes.