The placenta is essential for fetal development and growth, maternal homeostasis, and broadly, pregnancy health. Yet, our ability to non-invasively probe placental health during human pregnancy is hampered by its deep intrauterine location and its highly vascular composition, rendering the placenta largely inaccessibly for safe and dynamic investigation. Whereas placental research has been advanced by cell culture, ex vivo systems, animal models, and postpartum analyses, these indirect approaches provide ex post facto information about placental health. Placental imaging has revolutionized the field of placental medicine, but resolution at the molecular, cellular, or metabolic level remains limited. To address these challenges, we and others have focused on the release of extracellular vesicles (EVs) from placental trophoblasts, which, in humans, are directly bathed in maternal blood. We focused on exosomes (now termed small EVs or sEVs), microvesicles, and apoptotic blebs, which are continuously and abundantly released from trophoblasts into the maternal circulation and are accessible throughout pregnancy by peripheral blood tests. Among these EVs, we focus mainly on placental sEVs, which harbor messages that are seldom expressed by any other cell types and execute unique placental biological functions, such as an antiviral response. While informative, recent data indicate that sEVs are not a uniform population of vesicles, but comprise several subgroups, defined as large sEVs, small sEVs, and exomeres. In addition to their size, these sEV subtypes are characterized by distinctive cargo. Although the recent discovery of sEV subpopulations has excited researchers due to their potential to revolutionize the field of non-invasive diagnostics, sEV subpopulations have yet to be utilized in clinical settings. This is largely due to the difficulties associated with separation and isolation the nano-sized sEV subpopulations. Our group has now developed advanced acoustofluidic technologies designed to effectively, reproducibly, and rapidly isolate sEVs from blood. We show that we can separate placental sEVs into their specific subpopulations, which has not been previously accomplished. Our proposed investigation therefore focuses on the production of human placental sEV subpopulations, along with their RNA and proteome cargo. We posit that, by profiling these analytes from sEV subpopulations, we can illuminate a unique landscape of bioactive molecules that are relevant to placental health. To reduce data complexity, we propose a machine learning pipeline that will be used to probe the sub-sEV spectra during normal and pathological pregnancies. Further, we will improve our ability to purify sEV subpopulations from lipoproteins, and generate a single, integrated device that can reliably separate vesicles in real time across human gestation. We believe that our automated acoustofluidic approach to separating sEV subpopulations in a high-yield, biocompatible manner is critical to unlocking the clinical utility of sEVs. Insights gained from our investigation will improve non-invasive diagnostics during pregnancy and may uncover new targets for personalized placental therapeutics.
