ABSTRACT Prenatal opioid exposure can influence the integrity and health of the placenta and the developing fetal brain. These modifications at critical time points can have lasting effects on pregnancy outcomes and subsequent infant neurodevelopment. The mechanisms by which opioids modify the placenta and fetal brain, critical time points for these modifications, and whether there are useful biomarkers that can be tracked throughout the pregnancy are unknown. Two hypothesis driven mechanisms are 1) gene expression changes, and 2) intrauterine inflammation, both known to occur after exposure to opioids. These can be measured using biomarkers from maternal plasma and placental tissue, specifically microRNAs and maternal-infant inflammatory cytokine profiles. We propose a prospective cohort study comparing 100 opioid-exposed to 50 non-exposed control pregnancies to fill these gaps in knowledge. The specific aims of this proposal are: 1) Compare serial miRNA signatures in opioid-exposed and control pregnancies. In 100 opioid and 50 control participants, we will collect serial maternal plasma samples in the 2nd trimester, 3rd trimester, and delivery, as well as placental tissue at delivery. Maternal exosomal miRNA expression levels will be compared between groups with a focus on placenta-derived miRNA. Top miRNA associations with prenatal opioid exposure will be identified. 2) Compare serial inflammatory cytokines in opioid- exposed and control pregnancies. In the 100 opioid and 50 control participants, we will collect serial maternal plasma samples, placental tissue, and cord blood for analysis with a multiplex inflammatory cytokine panel. Cytokine levels will be compared between groups at each time point, and top associations identified. 3) Neurodevelopment: Association of top miRNAs and cytokines with placental health, infant neuroimaging and developmental outcomes in opioid-exposed pregnancies. Top miRNAs and cytokines identified through Aims 1 and 2 will be analyzed for association with infant neurodevelopment in the opioid cohort. 3A) Placental health: We will evaluate associations of top biomarkers with placental health measures (IUGR, pre-eclampsia, preterm birth, placental efficiency scores). 3B) Neuroimaging: Ultrasound imaging will be obtained in the 2nd trimester, 3rd trimester, and 1-month corrected gestational age, and associations evaluated for top biomarkers. 3C) Neurodevelopment: Infants will undergo a 12-month Bayley development examination. In our final regression model, we will examine associations between top miRNAs and cytokines with neurodevelopment with consideration of placental health (3A) and brain measurements (3B) as intermediates in the pathway. This study will help us to gain insight into mechanisms and developmental timing of how prenatal opioid exposure shapes both placental and fetal brain development, and ultimately infant neurodevelopmental outcomes.

PROJECT NARRATIVE In-utero opioid exposure can lead to alterations in placental function and neonatal brain development, contributing to long-term neurodevelopmental outcomes for affected infants. This application aims to gain insight into the mechanisms and developmental timing of how prenatal opioid exposure shapes both placental and fetal brain development through the examination of the non-invasive biomarkers of microRNAs and inflammatory cytokines.