Maternal Pregnancy-Associated Circulating MicroRNA predictors of 1 infant birth outcomes are Determinants of Placental Maturation 2

45 We previously identified 11 miRNAs which were significantly elevated in blood plasma of 46 pregnant mothers who subsequently gave birth to infants affected by prenatal alcohol 47 exposure (PAE, Heavily Exposed Affected: HEa) compared to those with infants who were 48 exposed but apparently unaffected (Heavily Exposed Unaffected: HEua) or unexposed (UE). 49 These maternal HEa miRNAs were predicted to originate in part, as a paracrine placental signal to 50 influence and coordinate placental epithelial-mesenchymal transition (EMT), a pathway 51 essential for endometrial invasion and development. We now report that PAE inhibits 52 expression of placental EMT pathway members in rodent and primate voluntary alcohol 53 consumption models, with HEa miRNAs collectively mediating placental EMT inhibition. To 54 directly investigate the interaction between HEa miRNAs and ethanol on placenta, we assessed 55 their effects on human placental trophoblast cell lines. When administered together, HEa miRNAs 56 retarded trophoblast cell cycle, significantly impaired expression of core EMT pathway 57 members, and reduced invasiveness, pointing to their collective role in modulating placental 58 growth and invasion deficits seen with PAE. HEa miRNAs additionally interfered with maturation- 59 dependent intracellular calcium dynamics, while promoting syncytialization-dependent 60 increases in placental hormone expression. Finally, a single systemic administration of the 61 pooled murine-expressed HEa miRNA subpopulation, to pregnant mice, decreased fetal and 62 placental growth and inhibited expression of EMT pathway mRNA transcripts in placenta. Taken 63 together, our data suggests that, following PAE, HEa miRNAs interfere with placental 64 development and may contribute to the pathology of Fetal Alcohol Spectrum Disorders. placental placental chorionic gonadotropin Our is the to report that PAE interferes with expression of core placental EMT pathway members. We also provide evidence that a group of circulating maternal miRNAs, HEa miRNAs, which predict adverse infant outcomes due to PAE, mediate some of PAE’s effects and interfere with EMT and cytotrophoblast maturation.