Deviations of endometrial immune cells during pregnancy in the cow

Abstract

s / Placenta 35 (2014) A1eA112 A61 expressed on the fourthmonth of pregnancy and in cloned animals as early as day 34-36 of pregnancy. These discrepancies of MHC-I expression between cloned and normal placenta observed during the first trimester of pregnancy may play a role on high rates of fetal losses in clones. Objective: Determine if there is an increased expression of MHC-I in the placenta throughout pregnancy and to determine if NC isoforms are differentially expressed in normal and cloned placenta in the first and third trimester of pregnancy. Methods: Bovine control and cloned placentomes, from first (n1⁄45) and third (n1⁄46) trimester of pregnancy were used to assess the NC isoforms by IHC and qPCR. Results: No expression of MHC-I in first trimester from control pregnancies, whereas the cloned pregnancies showed strong staining of MHC-I. Furthermore, in the third trimester of pregnancy the expression of MHC-I was not different between normal and cloned placenta. In regards the NC expression, all four NC isoforms were expressed in all groups. The isoform NC1 do not differ between normal and cloned pregnancies in first trimester, but increase in third (p 0.001) and downregulated in third (p>0.01) while NC4 showed no difference in expression normal and cloned pregnancy in both stages. Conclusion: The increased expression of MHC class I proteins associated to the increased expression of NC1 and NC2 in cloned pregnancy suggests that the deregulation of expression MHC proteins can compromise pregnancy establishment and maintenance. P1.162-N. DEVIATIONS OF ENDOMETRIAL IMMUNE CELLS DURING PREGNANCY IN THE COW Ana Carolina Furlanetto Mancanares , Rodrigo da Silva Nunes Barreto , Naira Caroline Godoy Pieri , Aline Fernanda de Souza , Carlos Eduardo Ambrosio , Flavio Vieira Meirelles , Daniele dos Santos Martins , Lilian de Jesus Olivera a a Faculdade de Medicina Veterin aria e Zootecnia Universidade de S~ ao Paulo, S~ ao Paulo, SP, Brazil; b Faculdade de Zootecnia e Engenharia de Alimentos Universidade de S~ ao Paulo, Pirassununga, SP, Brazil Modulation of maternal immune response to fetal antigens is one key involved in pregnancy success, and the mechanisms involved are not completely understood. Recent studies showed that successful embryo implantation requires perfect synchronization between maternal and fetal factors to promote endometrial receptivity and fetal development. Objective:Quantify the subsets of T lymphocyte in bovine endometrium in early and late pregnancy.We hypothesized that there is an accumulation of T cells as pregnancy progresses in the cow. Methods: Pregnant endometrium from first (30-40 days, N1⁄43) and third trimester (230-250 days, N1⁄43) were obtained from a slaughterhouse. Samples were frozen and processed for single-color immunofluorescence analysis to identify the presence and abundance of T lymphocyte subsets. We used anti-CD3 as pan-T cells, anti-CD4 as T helper, anti-CD8 as T cytotoxic and anti-WC1 as gdT marker. The samples were examined under epifluorescence microscope and five random fields from each sample were documented for further analysis. The number of positive cells was counted and total number was normalized for cell per mm2. The statistical analysis was performed using ANOVA with Tukey’s post hoc test. Results: There was an increased number of CD3+, CD4+ and WC1+ cells in third trimester when comparedwith first (p value1⁄4 0.00029; 0.00009 and 0.0305, respectively) in the shallow stroma of the pregnant endometrium. While, there was no change in the number of CD8+ cells in both stages (p value 1⁄4 0.33174). Conclusion: Our results suggest that there is a constant recruitment to the pregnant endometrium along the pregnancy in the cow. Perhaps, the increased number of endometrial CD3+, CD4+ and WC1+ cells occurs in response of an increased exposure of local maternal immune system to fetal antigens due to more intimate connection between maternal and fetal cells in later stages of bovine pregnancy. Financial support: FAPESP (2012/11212-6) P1.163. TARGETING PLACENTAL LEUKEMIA INHIBITORY FACTOR WITH A UNIQUE INHIBITOR: A NOVEL TREATMENT STRATEGY FOR ECTOPIC PREGNANCY Amy Winship , Tara Trishnan , Ellen Menkhorst , Andrew Horne , Jeremy Brown , Stephen Tong , Jian-Guo Zhang , Nick Nicola , Eva Dimitriadis a,e MIMR-PHI Institute of Medical Research, Melbourne, Australia; b The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia; c The University of Edinburgh, Edinburgh, UK; d The University of Melbourne, Melbourne, Australia; Monash University, Melbourne, Australia Objective: Ectopic pregnancy is unique to humans and a leading cause of maternal morbidity/mortality however the etiology remains unknown. Leukemia inhibitory factor (LIF) has roles in extravillous-trophoblast adhesion/ invasion and is expressed in ectopic pregnancy. We hypothesised that LIF facilitatesblastocyst adhesion/invasion in theFallopian tube (FT), contributing to ectopic pregnancy. LIF blockade could serve as a treatment strategy. Methods: We used an oviduct epithelial cell line-OE-E6/E7 and HTR-8/ SVneo cell-line (trophoblast-derived) spheroid co-culture to model blastocyst attachment to the FT. LIF signaling was determined by Western blot. The effect of LIF/LIF inhibition (using a unique PEGylated-LIFantagonist (PEGLA)) on first-trimester placental outgrowth was determined. To demonstrate the effect of LIF blockade to reverse trophoblast invasion in vivo, pregnant mice were administered with PEGLA (500mgx2/ day PEGLA/PEG) on gestation days (D)8-10 or 10-13. Implantation sites at D10/13 were stained with cytokeratin (trophoblast), isolectin-B4 and aSMA (vascular). Results: LIF receptor (R) was immunolocalised to villous and extravillous trophoblast and FT epithelium in ectopic pregnancy. LIF activated STAT3 but not ERK in OE-E6/E7 and stimulated HTR-8/SVneo-spheroid adhesion to OE-E6/E7 which was blocked by PEGLA. LIF promoted placental-explant outgrowth, which was blocked by PEGLA. In mice, PEGLA blocked LIF action, reduced decidua (D10), and reduced placental spongiotrophoblast/ labyrinth and vascular cells (D13). Our data suggests LIF facilitates the development of ectopic pregnancy by stimulating blastocyst adhesion and trophoblast outgrowth in the FT. Conclusion: Ectopic pregnancy is usually diagnosed after 6 weeks gestation; pharmacologically targeting LIF-mediated trophoblast-outgrowth may be useful as a novel treatment for ectopic pregnancy. P2.1. CONGENITAL INFECTION OF PROTOZOA: HISTOPATHOLOGICAL AND HISTOCHEMICAL ANALYSIS OF HUMAN CHORIONIC VILLI EXPLANTS INFECTED EX VIVO WITH TRYPANOSOMA CRUZI AND TOXOPLASMA