Gene expression profile of Syncytin-1 and methyl-binding proteins in isolated trophoblasts.

<p>A) Histograph showing fold-change differences to gene expression profile (2^−ΔΔCT) of control trophoblasts of IUGR, PE, PE/IUGR and HELLP/IUGR (each n = 7). Red line marks gene expression profile of control trophoblasts set to 1. (*P≤0.05). B) Venn diagram showing for each placental syndrome the statistical significant different expressed genes compared to control VCTs. The intersection of all four placental syndromes represents Syncytin-1.</p

Reduced Syncytin-1 Expression Levels in Placental Syndromes Correlates with Epigenetic Hypermethylation of the ERVW-1 Promoter Region

<div><p>Terminal differentiation of villous cytotrophoblasts (CT) ends in formation of the multinucleated syncytiotrophoblast representing the fetal-maternal interface. Aberrations during this cell-fusion process are associated with Intrauterine Growth Restriction (IUGR), Preeclampsia (PE) and High Elevated Liver and Low Platelets (HELLP) Syndrome. Syncytin-1, the envelope gene of the human Endogenous Retrovirus ERVW-1, is one of the most important genes involved in cell-fusion and showed decreased gene expression during these pathological pregnancies. The aim of this study was to determine the methylation pattern of the entire promoter of ERVW-1 and to correlate these findings with the expression profile of Syncytin-1 in the placental syndromes. 14 isolated villous cytotrophoblasts from control (n = 3), IUGR (n = 3), PE (n = 3), PE/IUGR (n = 3) and HELLP/IUGR (n = 2) placentae were used to determine the mean methylation level (ML) for the ERVW-1 promoter region. ML rose significantly from 29% in control CTs to 49% in IUGR, 53% in PE, 47% in PE/IUGR and 64% in HELLP/IUGR indicating an epigenetic down-regulation of Syncytin-1 by promoter hypermethylation. DNA demethylation of the trophoblast like cell lines BeWo, JEG-3 and JAR with 5-AZA-2′desoxycytidine (AZA) showed an increased Syncytin-1 expression and fusion ability in all cell lines. Promoter activity of the 5′LTR could be inhibited by hypermethylation 42-fold using a luciferase based reporter-gene assay. Finally overexpression of the methyltransferases DNMT3a and LSH could be responsible for a decreased Syncytin-1 expression by promoter hypermethylation of ERVW-1. Our study linked decreased Syncytin-1 expression to an epigenetic hypermethylation of the entire promoter of ERVW-1. Based on our findings we are predicting a broad aberrant epigenetic DNA-methylation pattern in pathological placentae affecting placentogenesis, but also the development of the fetus and the mother during pregnancy.</p> </div

Clinical data.

<p>GOT: Glutamate-Oxaloacetate-Transaminase; GPT: Glutamate-Pyruvate-Transaminase; LDH: Lactate dehydrogenase;</p><p>Significant changes marked in bold.</p

Luciferase Assay.

<p>The 5′LTR of ERVW-1 was cloned in the pGL3-basic vector and transfected in BeWo cells. 48 hr post transfection cells were analysed. Luciferase activity of the pGL3-basic vector was set to 1. All experiments were repeated at least five times. Histographs show fold-induction to pGL3-basic. (**P<0.005).</p

SYBR-Green primers for real time PCR.

<p>SYBR-Green primers for real time PCR.</p

Methylation Profile of the entire promoter of ERVW-1 in isolated trophoblasts.

<p>A) Locus of Syncytin-1 on chromosome 7q21.2 within the ERVW-1 gene. Black needles represent the single CpG sides in the promoter. Arrows mark binding sites for transcription factors ore methyl binding proteins B) All sequenced clones of control (n = 11), IUGR (n = 12), PE (n = 14), PE/IUGR (n = 13) and HELLP/IUGR (n = 17) from one placental trophoblast isolation of each group. White circles represent unmethylated CpGs and black circles methylated CpGs. C) MethylationHeatMap of the promoter region. Mean of three different placentae of each group. Mean of single CpGs are color-coded. From green unmethylated (0%) over red (50% methylated) to black (100% methylated). meanML → mean methylation level of the entire promoter; clones → total analysed clones of each group; Syn1 → mean Syncytin-1 gene expression in mol/ng_cDNA.</p

Influence of factor XIII activity on post-operative transfusion in congenital cardiac surgery—A retrospective analysis

<div><p>Objectives</p><p>Coagulation factor XIII (FXIII) plays a key role in fibrin clot stabilization—an essential process for wound healing following cardiothoracic surgery. However, FXIII deficiency as a risk for post-operative bleeding in pediatric cardiac surgery involving cardiopulmonary bypass (CPB) for congenital heart disease (CHD) is controversially discussed. Thus, as primary outcome measures, we analyzed the association of pre-operative FXIII activity and post-operative chest tube drainage (CTD) loss with transfusion requirements post-operatively. Secondary outcomes included the influence of cyanosis and sex on transfusion.</p><p>Methods</p><p>Our retrospective analysis (2009–2010) encompassed a single center series of 76 cardio-surgical cases with CPB (0–17 years, mean age 5.61 years) that were post-operatively admitted to our pediatric intensive care unit (PICU). The observational period was 48 hours after cardiac surgery. Blood cell counts and coagulation status, including FXIII activity were routinely performed pre- and post-operatively. The administered amount of blood products and volume expanders was recorded electronically, along with the amount of CTD loss. Uni- and multivariate logistic regression analysis was performed to calculate the associations (odds ratios) of variables with post-operative transfusion needs.</p><p>Results</p><p>FXIII activities remained stable following CPB surgery. There was no association of pre- and post-operative FXIII activities and transfusion of blood products or volume expanders in the first 48 hours after surgery. Similarly, FXIII showed no association with CTD loss. Cyanosis and female sex were associated with transfusion rates.</p><p>Conclusions</p><p>Although essentially involved in wound healing and clotting after surgery, FXIII activity does not serve as a valid predictor of post-operative transfusion need.</p></div

Descriptive analysis of post-operative data.

<p>Descriptive analysis of post-operative data.</p

Analysis of the associations between intra-operative volume management and post-operative blood tests / transfusion requirements.

<p>Analysis of the associations between intra-operative volume management and post-operative blood tests / transfusion requirements.</p

Multivariate logistic regression analysis of post-operative transfusion requirement.

<p>Multivariate logistic regression analysis of post-operative transfusion requirement.</p