ABSTRACT Pregnant and lactating people remain therapeutic orphans as they are excluded from the vast majority of clinical drug development and therapeutic trials. Moreover, current practices in drug evaluation in pregnancy have been hindered by the lack of effective biomarkers and innovative study designs. There is a need to develop novel placental-specific biomarkers in order to assess placental function and response to therapeutics, as a way to inform on their safety and efficacy. An example of these novel biomarkers is placental (fetal) specific extracellular vesicles (EVs). Recent advances in characterizing the cargo content of these EVs demonstrated their potential to be used as placental biomarkers. We have shown using funding support from the MPRINT that fourteen proteins found in EVs significantly correlated with aspirin use (FDR<0.1) in at-risk people, but that more power is needed to confidently assess the relationship with aspirin dosage and pregnancy outcomes. In addition, prior studies showed that aspirin affects endothelial and trophoblast cells, thus potentially modulating exosome derived from these cells and their cargo contents. Leveraging our prior proof of principle success, previously collected, and ongoing collection of maternal plasma from people at-risk of preeclampsia (PE) receiving 81mg or 162mg of aspirin daily and low-risk people at the Ohio State University, we are submitting this application with the overarching goal to develop a novel platform to augment the MPRINT resources using exosome profiling, as novel biomarkers, to monitor placental mediated adverse pregnancy outcomes and response to therapeutics. For this, we will use PE as a hallmark of these outcomes and aspirin as the therapeutic agent to show case the utility of the platform. In this study, we will test the hypothesis that differential expression of EVs proteome cargo is associated with placental and pregnancy health in response to aspirin treatment at different gestational periods. We will test the following specific aims: 1) Validate our preliminary proteome data and model using larger cohort from additional biobanked samples of at-risk people receiving 81mg or 162 mg aspirin, characterize fetal specific EVs (placental alkaline phosphatase [PLAP] +ve) isolated from maternal plasma, and determine the differences in between the maternal vs. fetal EV cargo difference with aspirin treatment; and 2) Determine the changes associated with EV proteome profile (maternal and fetal) in at-risk people receiving aspirin prospectively and correlate with clinical outcomes (development of PE) and angiogenic and inflammatory biomarkers associated with PE. This project has the potential to play a seminal role in the development of a novel platform for therapeutics research in pregnancy. This translational research platform will substantially add to the MPRINT Network repertoire and be available for scientific community through the already established relationship of the...