Proteomic Analysis of Placental Extracellular Vesicles Isolated from Maternal Blood by High Resolution Flow Cytometry

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The ability to monitor the placenta in vivo from early gestation to term is limited and there is immense interest in developing new methods to perform “liquid biopsies” of maternal blood to test for placental dysfunction. Biomarker studies using plasma have been largely unsuccessful to predict adverse outcomes before mid-gestation; and, it is clear that a more sensitive approach to enrich placental signals is needed if we are to understand the early pathophysiology of placental dysfunction and better predict serious complications. Fragments of the placenta are released into the maternal blood stream as early as 6 weeks' gestation. The concentration of these extracellular vesicles (EVs) appears to be related to placental size and pregnancy outcomes. The term EV includes a range of submicron particles, including small exosomes derived from endosomal biogenesis, larger vesicles budding directly from the cell membrane, and fragments of cells undergoing apoptosis. These lipid encapsulated particles contain protein, RNA, and DNA, which are suitable for –omics. There is considerable interest in the potential utility of EVs for biomarker studies, because they provide an in vivo window into placental function. Progress in the field has been limited, however, by the lack of placental-specific EV isolation methods. To address this need, our group will employ a new high resolution flow cytometry (HRFC) sorting method that can reliably identify, quantitate, and purify cell-specific submicron- sized EVs. HRFC provides 10-fold better submicron resolution than currently available flow sorting technology, enabling cell-specific EV isolation and purification down to the exosome range (100nm). We hypothesize that isolating placental EVs from early pregnancy plasma will enrich biomarker signals and provide a new means to reveal novel markers that are otherwise undetectable by conventional proteomic analysis of maternal plasma. We will first validate the HRFC sorting method using placental explant-derived EVs diluted in non-pregnant plasma. We will then test whether enriching placental signals from early (12-14 weeks') and late mid-gestation (26-28 weeks') correlate with clinical outcomes. Our highly novel approach will combine this new placental-specific EV isolation method with state-of-the-art magnetic resonance imaging (MRI) technology funded by the NIH to measure placental size and uteroplacental blood flow in a prospective cohort (U01-HD087182-01). Plasma, total EVs (differential centrifugation), and purified placental EVs isolated from early and late mid-gestation will be banked for proteomic, transcriptomic, and genomic studies. Biomarker concentrations will be compared in plasma, total EVs, and placental-specific EVs. Linear regression modeling will analyze potential relationships between biomarker levels, placental size, and perfusion measurements made by MRI after adjusting for covariates (e.g. age, parity, race, body mass index, and gestational age at the time of blood collection). Proteomic analysis of placental EVs collected at 12-14 and 26- 28 weeks' will compare 12 women who have severe placental dysfunction with 12 closely matched controls.