The Minichromosome Maintainance Complex is Up-regulated in the Placentas of Low-Insulin Sensitive Mothers in the First Trimester of Pregnancy

INTRODUCTION: Obesity in women at reproductive age is increasing worldwide and is a risk factor for pregnancy complications. In the first trimester of pregnancy the human placenta is rapidly growing making it sensitive to environmental influences. Therefore, we hypothesized that the altered intrauterine milieu associated with maternal obesity modifies placental proteome and function. METHODS: Maternal BMI was calculated and leptin, glucose, C-peptide and insulin sensitivity (ISHOMA) quantified in maternal serum. To identify obesity-associated changes in the placental proteome we run untargeted proteomics (LC-MS/MS) in placentas from lean (n=10) and obese (n=10) women (gestational age 5+0- 6+6 weeks). The results were validated using westernblotting and immunohistochemistry. The first trimester trophoblast cell line ACH-3P was used to perform functional assays in vitro.The cells were challenged with insulin (10nM) or palmitoleoyl ethanolamide (100nM), a metabolite found in higher concentrations in women with low insulin sensitivity (untargeted metabolomics) for 24h at physiological oxygen tension (6.5% O2) in a hypoxia bench. Changes in the concentration of the top identified proteins under exposure to the different stimuli was quantified by qPCR and westernblotting. RESULTS: Placental proteome differed between lean and obese women with a stronger separation between those with low vs high insulin sensitivity (Principal Component Analysis). Proteins (n=89) enriched (p<0.05, FDR<0.05) in the low insulin sensitive mothers were selected for pathway analysis (KEEG). The top enriched pathways were related to DNA replication and cell cycle (FDR<0.001). Six members of the Minichromosome Maintenance Complex (MCM), involved in DNA damage response and cell cycle arrest, were associated with low maternal insulin sensitivity. There was a significant correlation (R2=0.45 p<0.001) between MCM6 and γH2AX (DNA damage marker) concentration in total tissue. Furthermore, MCM6 co-localized with γH2AX in the nuclei of cytotrophoblast cells, suggesting increased DNA-damage in this specific cell type, which was indeed found by semiquantitative γH2AX immmunohistochemistry. Challenging ACH-3P cells with palmitoleoyl ethanolamide resulted in increased (p<0.05; n=4) MCM6 gene expression after 24h treatment. CONCLUSION: Low insulin sensitivity of pregnant women in in the early first trimester of pregnancy associated with obesity induced changes in the placental proteome related to DNA replication and cell cycle. This may reflect trophoblast response to increased DNA damage in these placentas.