The first trimester of pregnancy is a critical period for placental development. We have shown that diabetes-associated environmental perturbations affect trophoblast proliferation (GD, MG). Inflammation and oxidative stress can induce DNA damage, which may result in reduced proliferation. The specific effects depend on the pregnancy week. We hypothesize that obesity-associated enhanced inflammation early in pregnancy leads to DNA damage of the trophoblasts, with ensuing cell cycle arrest until DNA damage has been repaired. We further hypothesize that GADD45α, which we found upregulated in placentas from obese women at week 7 of pregnancy, plays a crucial role in this process by integrating various input signals and orchestrating the trophoblast response. We will dissect the sequence of these events and take into account the sex of the fetus, which may affect stress susceptibility and/or response to environmental stimuli. The overall objective is to delineate molecular mechanisms underlying the stress response to maternal obesity of first trimester trophoblasts, using cell cycle regulation and DNA damage/repair as readout, and to test for male vs female differences. First the key pro-inflammatory cytokines in early obese male/female pregnancies will be identified with a focus on three early pregnancy periods. Second, the molecular mechanisms involved in trophoblast growth regulation modified by maternal inflammatory stress will be identified in vitro in these three periods focusing on FOXO3A-GADD45α/ß-DNA damage/repair pathways. Third, key targets will be analysed for sex-specific changes in trophoblast DNA methylation associated with the pro-inflammatory environment of maternal obesity. The project will capitalize on an existing (n=216) and ongoing first trimester (weeks 5-12) placental tissue and serum bank spanning, a wide range of maternal pre-pregnancy BMI (19-44 kg/m2). In vitro experiments will be performed at 3 different low oxygen tensions. The results will allow conclusions on the period in early pregnancy, in which the placenta/trophoblast is most susceptible to adverse effects of the obesogenic environment. This will add molecular evidence to the growing body of clinical information about the relevance of the first trimester for pregnancy outcome in maternal obesity and add the dimension of fetal sex influences and timing in first trimester. We will establish whether the molecular changes induced by the obesogenic environment are paralleled by DNA methylation changes, which may confer lasting effects on placental growth.