Consensus-Based Minimum Requirements for the Adoption of a Computational Pathology Algorithm for Tumor Budding in Colorectal Cancer: An Early Health Technology Assessment.

Biological factors influencing maternal health during pregnancy and the postpartum period are understudied, even as rates of maternal morbidity and mortality remain high (). Pregnancy and postpartum are biologically unique periods in the lifespan, with dynamic and significant changes occurring in maternal physiology and a high demand for mechanisms important to systemic homeostasis, especially those involved in glucose and immune regulation. Adverse health during pregnancy, including preterm birth, gestational diabetes, and preeclampsia are strong predictors of poor postpartum health that may last for years or decades after birth (–). While early identification of elevated risk can ameliorate or prevent maternal pathophysiology, we still lack a sufficient understanding of underlying mechanisms or validated biomarkers that significantly contribute to improving maternal health. Extracellular vesicles (EVs) are of increasing focus across a number of disease areas for their key role in systemic communication and coordination, especially with the immune system and have been identified as having high potential to serve as predictive biomarkers (–). EVs are biological nanoparticles secreted by all tissues tasked with transporting biological cargo including small RNAs, proteins, and lipids throughout the body, and are a heterogeneous population comprised of several subtypes, including smaller sized exosomes and larger sized microvesicles. In pregnancy, EVs are one of the most dynamic and abundant biological signals in circulation. Maternal levels increase 3-4 fold, in part due to EVs secretion from the placenta (,,). The increased EV concentration during pregnancy supports proposed critical roles for these nanoparticles in diverse functions including embryo implantation, immune regulation, angiogenesis, glucose regulation, and the onset of parturition (,–). While much of this work in this area has contributed to our understanding of roles for EVs important to maternal health, the precise mechanisms for temporal changes in EV biogenesis and composition remain less clear, in part due to insufficient tools with which we can ask such questions (). The functional dynamics of placental-derived EVs is of particular interest because it could identify key processes for sensing and regulating the maternal environment, a valuable step toward biomarker confirmation and potential novel therapeutic targets. Importantly, although not yet examined, removal of the placenta at birth likely results in a rapid reduction in EV concentration in maternal circulation. Thus, EV signaling during the transition from pregnancy to the postpartum period may play a similar role in regulating the maternal environment after birth. Previous studies by our group and others using and models identified an important role for circulating EVs in maternal glucose homeostasis. In mice, we previously isolated EVs from pregnant females and injected them into nonpregnant mice, producing a significant change in glucose dynamics (). However, a limitation of this method is an inability to temporally increase circulating levels of EVs in the pregnancy environment due to an already profoundly high EV concentration. In the current studies, we first examined temporal changes in key EV characteristics and using an unbiased approach also assessed changes in EV protein cargo between nonpregnant, pregnant, and postpartum mice, focusing on the functional interpretation of protein pathways and tissues involved to define what EVs are likely doing at that time point. We hypothesized EVs would increase in concentration during pregnancy and decrease in postpartum to similar levels as nonpregnant mice. Further, we hypothesized EVs would exhibit unique proteomic signatures at each time reflecting dynamic changes in signaling required for maintaining homeostasis. Next, to specifically probe circulating EV contribution to maternal homeostasis, we ‘highjacked’ the trophoblast cell secretory pathway using a chemogenetic approach to drive dynamic EV secretion. Extracellular vesicles are released through diverse intracellular signals with calcium-based signal transduction via GPCRs constituting a common signaling pathway (). Utilizing a transgenic mouse model to express the metabotropic DREADD receptor specifically in placental cells of trophoblast origin, we activated the secretory pathway of trophoblast cells to elicit an increase in extracellular vesicles in maternal circulation. Measuring changes in glucose homeostasis as a proxy for control of maternal physiology we investigated the effect of this acute increase in EV concentration and in real-time in the pregnancy state. We hypothesized chemogenetic increases in EVs secreted from the placenta would impact maternal glucose regulation.