Project Summary We are submitting this proposal in response to PAR-20-147 (Extracellular RNA carrier subclasses in processes relevant to Substance Use Disorders or HIV infection). The opioid epidemic in the United States has far reaching consequences for public health. In recent decades, the rate of newborns born with neonatal abstinence syndrome (NAS) has increased dramatically. However, not all neonates exposed to opioids during gestation develop NAS or require treatment. NAS is a complex and idiosyncratic condition, and it is currently not possible to predict even at birth which infants will require pharmacotherapy or be susceptible to its worst outcomes. Accessible biomarkers for fetal brain and CNS development during opioid exposure would be highly valuable for deciding which infants will need opioid therapy for NAS and making personalized risk assessments for opioid exposed infants. They could also shed light on the biological pathways that lead to the worst effects of opioid exposure during gestation. Extracellular vesicles (EVs) derived from the Fetal brain and central Nervous system (FNEV) can cross the fetal/placental barrier and can be isolated from maternal blood. They represent a powerful accessible tool to provide insight on fetal neurodevelopment and damage. However, EVs are highly heterogeneous, with a diverse set of surface proteins and intra-vesicular cargo, such as RNA. Current approaches to the isolation and study of EVs lack the necessary sensitivity and precision to characterize EV subpopulations. To address the current limitations of EV analyses, we developed a high-throughput single-EV flow analyzer and sorter capable of detecting single dye molecules. This new technical capability allows us to exquisitely define the profiles of plasma FNEV during pregnancy. Our studies could lead to assays that define NAS risk pre-birth and bring new insights into the mechanistic pathways of fetal brain injury.