Summary abstract During pregnancy, women experience major physiologic changes in glucose regulation that are regulated by the placenta. In some pregnant women, regulation between insulin sensitivity and secretion is imbalanced, leading to gestational diabetes mellitus (GDM). GDM is the most common metabolic disorder in pregnancy and leads to complications in both mothers and offspring during pregnancy and long afterwards. Better understanding of the placental role in gestational glucose regulation is necessary to identify early biomarkers that predict GDM, and for the development of novel therapeutic agents to influence insulin secretion and insulin sensitivity, the two major pathophysiologic determinants of diabetes inside and outside of pregnancy.. We propose to conduct placental genome-wide transcriptomic studies using the most recent methods for RNA- sequencing and cutting-edge technology for miRNA sequencing. Placenta samples (n=662) were carefully collected in a population-based prospective cohort of pregnant women called Gen3G. We have collected refined phenotypes and plasma samples in 809 Gen3G women across gestation, including oral glucose tolerance tests allowing us to characterize their insulin secretion, insulin sensitivity, and objectively diagnosed GDM. For identified miRNA associated with insulin secretion in vivo, we will test whether they can stimulate insulin secretion in in vitro validated ?-cell models. For identified miRNA associated with insulin sensitivity, we will test whether they can influence glucose-uptake in in vitro adipocytes models. After meticulous selection of the most promising transcripts from our GDM vs normoglycemic women, we will develop assays to detect secreted proteins in maternal plasma collected at 1st trimester. We will test associations between newly identified placenta-specific secreted proteins (ELISA or Mass Spectrometry) and miRNA (RT-qPCR) detectable in plasma at 1st trimester with GDM incidence. We will replicate our findings in two prospective multi-ethnic cohorts of pregnant women. This proposal leverages the richness of the unique Gen3G cohort, including placenta samples carefully collected and preserved for transcriptomic studies, refined phenotypes of glucose regulation and plasma samples across gestation. The team of investigators assembled for this application has substantial expertise and previous experience to conduct each step of the proposed study. Finally, our transcriptomic results will be rapidly available to the wider scientific community on the highly accessed GTEx portal, as we will follow all their standard procedures for our sequencing.