Assaying the enhancer landscape during early placental development to identify a trophoblast invasion transcriptional code and gene-enhancer network

s / Placenta 36 (2015) A1eA60 A8 by a common set of factors. Of particular interest, we have also devised a novel way of utilizing these data to develop non-invasive methods of screening for real-time biomarkers of placental genome regulation. Together, our findings reveal previously unappreciated aspects of the placental transcriptional landscape. O1.1. PLACENTA AND OBESITY: DIFFERENCES IN REPRODUCTIVE IMMUNOLOGY AND UTEROPLACENTAL PATHOLOGY Terry Morgan. OHSU, Portland, OR, USA Background: Obesity is a growing problem, affecting 30-40% of reproductive age women in our population. This is significant because obesity is associated with an increased risk of gestational diabetes, preterm birth, preeclampsia, and stillbirth. How obesity affects key immune modulators in the placental bed are only beginning to be understood. We hypothesize obesity leads to reduced uterine natural killer cell (uNK) activation, perhaps via differences in uterine T-cell regulation. The consequence is abnormal uterine spiral artery remodeling, abnormal blood flow to the placenta, and placental damage. Methods: Our group studies the effects of obesity on uteroplacental blood flow and placental pathology in women. Contrast-enhanced imaging techniques combined with flow cytometry andmolecular analysis allow us to correlate blood flow with immunologic regulators of uterine spiral artery remodeling in the placental bed. Placental histology and ultrastructure are related to clinical outcomes. Results: Obesity leads to placental insufficiency, which is evident in abnormal uteroplacental blood flow and placental pathology. Uterine natural killer cell activation appears to be decreased in obese women and there may be a relationship with abnormal T-cell polarization in the late first trimester. At delivery there is increased placental pathology associated with pregnancy complications. Conclusions: We are only beginning to understand why and how obesity affects reproductive immunology. Our studies suggest obesity may affect T-cells and uNK cells, which play a key role in spiral artery remodeling and the regulation of uteroplacental blood flow. O1.2. ASSAYING THE ENHANCER LANDSCAPE DURING EARLY PLACENTAL DEVELOPMENT TO IDENTIFY A TROPHOBLAST INVASION TRANSCRIPTIONAL CODE AND GENE-ENHANCER NETWORK Geetu Tuteja , Gill Bejerano . 1 Iowa State University, Ames, IA, USA; 2 Stanford University, Stanford, CA, USA Objectives: In early placental development, trophoblast cells line the implantation site and invade the maternal decidua, establishing adequate blood flow between mother and fetus. Defects in trophoblast invasion can lead to serious pregnancy complications, yet the mechanisms underlying this process are poorly understood. Our goals were to identify co-regulated enhancers that contribute to trophoblast invasion, and then to build a trophoblast invasion gene-enhancer network. Methods: We carried out ChIP-Seq for an enhancer-associated mark on mouse placenta at e7.5 and e9.5. We used 5mg of chromatin and 1.2mg antibody per ChIP, and prepared libraries with 2ng of DNA. We used GLITR software to call peaksonaligned reads.We then identifiedpeakswithhigher activity at e7.5 (e7.5-specific enhancers) by comparing read profiles at both time points. We computationally discovered transcription factor binding motifs that occurred significantly more in the e7.5-specific enhancers. Results: We identified ~8,000 peaks at each time point, and both sets of peaks were associated with placental development genes. Of these peaks, 1,977 were determined to be e7.5-specific enhancers. Our transcription factor motif analysis led to the discovery of a transcriptional code within these enhancers, consisting of motifs for AP1, Ets, and Tcfap2. Interestingly, enhancers containing this code are significantly associated with cell migration genes. We validated differential expression of transcription factors we predicted to bind these enhancers by performing RNA-Seq in the same context. Finally, we constructed a trophoblast invasion geneenhancer network by integrating all of these data with publicly available protein-protein interaction data. Conclusions: By combining experimental genomics with computational analysis, we discovered enhancers sharing a specific transcription factor pattern that is associated with cell migration, and constructed a trophoblast invasion gene-enhancer network. This network opens up multiple avenues of research that will lead to a more detailed understanding of the mechanisms regulating trophoblast invasion. O1.3. PREPREGNANCY MATERNAL VITAMIN D DEFICIENCY AND PLACENTAL DEVELOPMENT IN MICE Alison Gernand, Margherita Cantorna, Francisco Diaz, Lindsay Snyder, James Hester, Sreelakshmi Vasudevan, Manasi Kamat, Troy Ott. The Pennsylvania State University, University Park, PA, USA Objective: Our objective was to examine the effect of prepregnancy and early pregnancy vitamin D deficiency on placental vascular development in mice. Methods: We modeled vitamin D deficiency using a Cyp27B1 genetic knockout (KO) mouse with no dietary vitamin D (n1⁄46); wildtype (WT) mice with a vitamin D sufficient diet were the control (n1⁄46). All mice were fed their respective diet from 3 weeks of age onward. At 8 weeks of age, mice were mated to genetically dissimilar males to produce allogeneic embryos. Mice were euthanized at 12.5 days gestation and uterine horn, placental, and fetal morphometry was measured. Placental mRNA was estimated by qPCR for angiogenic proteins (angiopoeitin1 (Ang1), Ang2, vascular endothelial growth factor (VEGF), and placental growth factor (PlGF)) to assess vascular development. Results: KO mice had fewer viable fetuses at 12.5 days compared to WT (7.0 vs. 8.8, p1⁄40.052). There were no statistically significant differences for KO vs. WT in uterine horn, placental or fetal weights or placental Ang1, Ang2, or PIGF mRNA (all p>0.05). However, KO placentas exhibited a 27% reduction in VEGF expression compared toWT placentas (p1⁄40.047). Fewer KO mice established pregnancy after mating (45% vs. 69%), yet the study design did provide sufficient sample size to adequately test this effect. Conclusion: Prepregnancy maternal vitamin D deficiency in mice caused lower litter size among mice that became pregnant. Further, decreased placental VEGF mRNA abundance indicated impaired angiogenesis (or vascular development) of the placenta in deficient mothers. Ongoing work is examining the effect of vitamin D deficiency on additional aspects of placental implantation and development. O1.4. MITOCHONDRIAL FUNCTION AND GLUCOSE METABOLISM IN THE PLACENTA WITH GESTATIONAL DIABETES MELLITUS Sribalasubashini Muralimanoharan, Alina Maloyan, Leslie Myatt. UTHSCSA, San Antonio, Texas, USA Gestational diabetes mellitus (GDM) affects ~ 7% of all pregnancies. Increasing maternal BMI is a well-recognized risk factor for the development of GDM and the two conditions share a similar metabolic milieu characterized by insulin resistance, hyperglycemia and mild chronic inflammation. First trimester placentae have high rates of aerobic glycolysis but there is a switch to mitochondrial oxidative phosphorylation with advancing gestation to support fetal growth. We have previously shown impaired mitochondrial function in placenta with increasing maternal obesity. Recently it was shown in a rat model that maternal obesity detrimentally alters placental mitochondrial function and hence affects hepatic mitochondrial function in the offspring. Inhibition of anti-oncomiR, miR143 has been shown to upregulate aerobic glycolysis in cancer tissues. We hypothesized that mitochondrial function would be reduced and linked to up-regulation of aerobic glycolysis via miR-143 in the placenta of women with GDM. Villous tissue was collected at term by C section from women with type A1GDM (diet control), A2GDM (treated with glyburide or insulin) and prepregnancy BMI matched controls (n1⁄418). The placentae from GDM mothers were bigger and less efficient