Abstracts / Placenta 35 (2014) A1eA112 A47

Bioanalyzer. DNA and total RNA samples were analyzed by a variety ofmethods including genotyping, qPCR, microRNA analysis, RNA sequencing,and Next Generation Sequencing.Results: Median times to first and second collections were 0.68 hours and65.5 hours, respectively. RIN was significantly higher in specimens fromfirst collections vs. second collections (median+SD: 5.1+2.4 vs. 2.4+1.2;Wilcoxon: Z¼-6.3, p<0.00). RIN > 5 has been a typical standard to definespecimen quality for gene expression analysis. First collection samples hadan equal number of samples above or below RIN¼5. Second collections hadsignificantly more samples with RIN < 5 (c2¼65.7, p<0.00). qPCR wasperformed on all samples regardless of RIN to determine expression ofknown placenta-expressed genes (e.g., NFKB1A, PLA2G4A, PLA2G4B,PTGER, OXTR, PGR) and normalized to ACTB, and 18S. Genotyping ofknown variants for these genes was reliable in the DNA extracted fromthese samples.Discussion: Knowledge of collection times is important to help charac-terize sources of preanalytical variability in research tissue samples.Placental gene expression values showed only small changes with ten-foldvariation in median time between first and second collections. Neithertime to collection nor RIN were significant factors influencing placentalgene expression levels, suggesting that the placenta is a robust source ofgenetic material well suited to collection in clinical settings for perinatalgenomic research.

P1.117-N.RNA-SEQ ANALYSIS OF PLACENTAL TRANSCRIPTIONAL LANDSCAPE INNORMAL AND COMPLICATED PREGNANCIES

Siim S~ober a, Mario Reiman a, Kristiina Rull a,b, Pille Vaas b,Jaana M€annik a,c, Maris Laan a aHuman Molecular Genetics Group, Instituteof molecular and cellular biology, University of Tartu, Tartu, Estonia;bDepartment of Obstetrics and Gynecology, Tartu University Hospital,Tartu, Estonia; cDepartment of Biochemistry, Cellular and MolecularBiology, University of Tennessee, Knoxville, Tennessee, USA

In mammals, placenta is a temporary organ of the fetus that exists andfunctions only during the pre-birth developmental stage and is requiredfor fetal oxygen and nutrient supply. Aberrant gene expression in theplacenta may adversely affect placental function and result in pregnancycomplications.

To characterize the transcriptional landscape over a wide range of normaland complicted pregnancies we performed total RNA and small RNAsequencing in 40 samples from term placentas. Samples were selectedfrom the REPROMETA sample collection and represent normal pregnancies(n¼8) along with the most clearly defined (extreme) cases of preeclampsia(n¼8) and gestational diabetes (n¼8). In addition, samples were selectedfrom small (n¼8) and large (n¼8) for gestational age births. Sequencingyielded ~3.4 Billion 50 +50 bp paired end reads (Mean: 84.4 M reads persample; range: 48.4M e 145.2M). Of the total 164 billion sequenced bases121 billion (73.5%) were successfully mapped to the human genome afterquality control, filtering and alignment steps (49.2% mapped to knownmRNAs). These data, to our knowledge, represent one of the deepestsequencing efforts of the placental transcriptome to date with 600-800fold mean coverage of exonic regions.This dataset establishes the range and variation in placental geneexpression and allows detailed exploration of overlaps and differences inplacental transcription over diverse pregnancy courses. In addition, thesedata enable identification and investigation of key molecular changes inplacentas from complicated pregnancies.

P1.118.EVOLUTION OF PLACENTAL FUNCTIONS THROUGH GENE CO-OPTION

Brian Cox, Jordana Lowe University of Toronto, Toronto, Ontario, Canada

Objectives: How do new biological systems as complex as an organevolve? Approximately 150 million years ago the placenta, the uniqueand essential organ to mammalian pregnancy, evolved. The placenta

has diversified into a number of different morphological structuresadapted to the particular gestational and physiological needs of eachspecies. In humans and mice the placental trophoblast has several corefunctions, including: angiogenesis, immune-modulation, vascularmimicry, transport and endocrine. We hypothesize that the placentafunctions arose by co-option of existent gene networks that regulatethese core functions in other tissues. Additionally, both humansand mice have order specific expansion of placental trophoblastexpressed microRNAs. We hypothesis the evolution of placenta specificmicroRNAs may serve to prune co-opted networks for optimalplacental function without affecting the genetic network, which wouldbe disruptive to other organ systems that still require the intactnetwork

Methods: To address our hypothesis on a genome wide scale, analysis ofdevelopmental microarray datasets of trophoblast cells and other em-bryonic cell types has identified sets of co-expressed developmentallyregulated genes.Results: A subset of genes involved in cardiomyocyte and endothelialdevelopment are expressed in trophoblast. We identified knowngenes, Myoferlin and Dysferlin involved in syncytialization of tropho-blast and cardiac cells. Novelly, we identified transcriptional regula-tors, GATA3, EGR1, EPAS1 and HAND2 as co-expressed. Interestingly,these transcription factors are also essential to immune system pro-cesses. We are currently assembling and testing predicted mRNA tar-gets of trophoblast specific microRNAs that are co-expressed with co-opted genes.Conclusions: Sets of co-expressed genes related to functions shared be-tween the placenta and different organ systems have been identified.These suggest common and unique genetic aspects of placenta andtrophoblast function.

P1.119-N.GLOBAL REWIRING OF MOLECULAR NETWORKS IN PLACENTA PREVIAAND ACCRETE

Roberta Hannibal, Janet Song, Ann Folkins, Deirdre Lyell, Amy Heerema-McKenney, Julie Baker Stanford University School of Medicine, Stanford,CA, USA

Objectives: The incidence of the serious pregnancy complicationsplacenta previa and accreta is rapidly increasing, yet the molecularmechanisms behind these disorders are unknown. Research has foundthat scaring from previous uterine surgery and trauma increase the riskof developing previa/accreta. While this suggests that local factors maybe involved, the identity of these factors is unknown. Our objective is toidentify and understand the molecular mechanisms behind placentaprevia/accreta.

Methods: We used the Stanford Translational Integrated Database Envi-ronment (STRIDE) to identify formaldehyde fixed paraffin embedded(FFPE) placentas banked in the Stanford Pathology archive. We preparedRNA from these samples for high-throughput RNA sequencing, alignedsequence data against the human genome, and determined relative tran-script levels.Result: We sequenced RNA from pregnancies classified with previa,accreta, previa and accreta, pre-eclampsia, spontaneous preterm birth,and term. For each placenta, we sequenced RNA from both the mem-branes and the decidua basalis. Surprisingly, we found 20 genes that areupregulated in both the membranes and the decidua basalis in previa/accreta compared to pre-eclampsia and spontaneous preterm birth,suggesting a global signature. We are currently identifying the specificcell type(s) that express these genes using in situ hybridization. We arealso examining levels of these proteins in maternal blood for use asbiomarkers.Conclusions: There are 20 genes upregulated in both the membranes andthe decidua basails of previa/accreta pregnancies compared to pre-eclampsia and spontaneous pre-term birth. While previous data suggestthat local scar tissue may cause previa/accreta, our data indicate a globalrewiring of molecular networks.