PROJECT SUMMARY Optimal fetal growth and development requires robust placental function, which is affected by blood glucose and lipid concentrations but also potentially chemical/drug exposures. Drugs may alter epigenetic homeostasis by direct or indirect mechanisms (transcription activity) that can persist long after exposure to the drug (i.e. pharmacoepigenomics). Our preliminary data has shown differentially methylated regions in DNA from trophoblasts in pregnancies complicated by gestational diabetes (GDM) compared to non-GDM trophoblasts. Moreover, metformin and GDM independently and significantly decreases mitochondrial respiration and placental hormone gene and protein expression in placental trophoblasts. Placental hormones, particularly β- hCG are necessary for normal fetal growth and development. Recent trials using metformin in pregnancy has demonstrated potential maternal benefits such as lower gestational weight gain but higher rates of small-for- gestational age infants and greater weight z-scores and adiposity in children. This highlights the critical gap in our knowledge of the effects of metformin on placental health and its role on potential beneficial and adverse perinatal outcomes. Our objectives are to (Aim 1) determine the effects of metformin on trophoblast epigenomic and transcriptomic profiles and (Aim 2) determine the degree to which metformin treatment suppresses cytotrophoblast maturation and hCG production in GDM and non-GDM placentas. Epigenomic and transcriptomic profiles from isolated trophoblasts in culture for 8-hrs (cytotrophoblasts) and 72-hrs (syncytiotrophoblasts) exposed to metformin will be compared to unexposed trophoblasts from GDM and non- GDM pregnancies. Live cell, time-lapsed imaging throughout trophoblast maturation (8, 24, 48, 72-hrs in vitro culture) will be done to visually correlate morphological maturation with gene markers of trophoblast differentiation and β-hCG production in the presence or absence of metformin cell exposure. Metformin and GDM must be studied together and concurrently because published trial data (i.e. obesity and polycystic ovarian syndrome) has shown they are independently associated with the growth and health of the fetus. We acknowledge metformin has been studied in many organ systems but how it effects the placenta and specifically the trophoblast cells is poorly understood. We believe that it is important to understand the degree to which metformin affects the maturation and metabolism of placental trophoblasts and whether epigenetic mechanisms underlie changes in gene expression patterns that regulate trophoblast function in GDM and non-GDM placentas. This study will offer new information that may caution the use of metformin in pregnancy and set the stage for more refined clinical trials.