PROJECT SUMMARY Experimental models and human studies have repeatedly linked persistent organic pollutants (POPs) such as perfluorinated alkylate substances (PFASs) and polychlorinated biphenyls (PCBs) to abnormal fat distribution, body mass index, and glucose and lipid metabolic abnormalities during early life. These are pervasive chemicals to which nearly all children are exposed during early life: PFASs are used in several consumer and industrial applications and PCBs accumulate in food chains. Evidence suggests that these abnormal cardiometabolic trajectories during early childhood may predict insulin resistance, metabolic syndrome, obesity, and even cardiovascular events in adulthood. However, no biomarkers are available to identify in the immediate postnatal period children who are at risk for abnormal cardiometabolic development, thus curbing opportunities for effective, targeted prevention. To address this gap, our long-term goal is to identify novel mechanistic biomarkers in breast milk that reflect environmental influences and predict the risk of abnormal cardiometabolic programming and childhood obesity. We will leverage groundbreaking evidence on the roles of breast milk extracellular vesicles (BMEVs) in metabolic programming. BMEVs are small vesicles that are released by luminal epithelial cells in breast milk. BMEVs have been proposed as conveyors of molecular signals from the mother to the child. In particular, BMEVs transport a cargo of microRNAs (miRNAs) that, once ingested by the child, integrate themselves in recipient cells in the child's body and can remotely affect the expression and translation of child's messenger RNAs. This process has been shown to be key to the child's metabolic programming in early life. To date, no studies have been conducted to identify the potential roles of BMEVs as part of the paths linking PFASs and PCBs exposure to its adverse effects on cardiometabolic trajectories and obesity during childhood. To achieve this goal, we will leverage the unique resources of the longitudinal Faroe Islands birth cohort, a prenatal cohort with biobanked breast milk (N=300, all from nursing mothers), extensive exposure data, and repeated postnatal cardiometabolic measures over 13 years of follow up. We hypothesize that BMEV number, BMEV size, and BMEV-encapsulated miRNAs are modified in response to prenatal (third-trimester of pregnancy) exposure to PFASs and PCBs (Aim 1) and that BMEV number, BMEV size, and BMEV- encapsulated miRNA predict cardiometabolic outcomes over 13 years of follow up (Aim 2). We will use advanced statistical modeling to integrate BMEV biomarkers in the paths linking exposure and abnormal cardiometabolic trajectories (Exploratory Aim 3). This study is a high-risk/high-reward and cost-effective project that will provide new noninvasive tools to identify and reduce the burden of abnormal cardiometabolic programming during childhood.