d the ability of hCG to interact with different human endometrial cell types and modulate cell receptivity to IL1. Comparable effects were observed in ESCs during the implantation window with, interestingly, a concomitant up-regulation of IL1R1, a downregulation of IL1RN, an increased angiogenic activity and a higher secretion of monocyte chemotactic protein1 . First identified as a specific factor for macrophage recruitment and activation, MCP1 was later found to be endowed with various immune modulating, proangiogenic and growth-promoting properties. The aim of the present work was to gain a broader understanding 
of the global impact of hCG on the modulation of hCG/IL1 Modulate Endometrial Cell Phenotype human endometrial cell responsiveness to IL1 and the acquisition of growth-promoting phenotype capable of sustaining active embryonic implantation and growth. Using micro-array analysis of hCG, IL1 and hCG/IL1-treated ESCs from the implantation window, our data identified several significantly regulated genes AZD-0530 site targeted by hCG/IL1 synergy and implicated in angiogenesis, proliferation, tissue remodeling, cell signaling and immune modulation, which is relevant to early embryo implantation process, and a wide spectrum of targets encompassing IL1 family members. tion of endometrial tissue biopsies was performed using a Pipelle. Tissue samples were kept at 4uC in sterile Hank’s balanced salt solution containing 100 U/mL penicillin, 100 mg/mL streptomycin and 0.25 mg/mL amphotericin B and immediately transported to the laboratory. Cell culture and treatment ESCs were isolated and characterized according to our previously described procedure. Concisely, tissue was minced into small pieces, dissociated with collagenase 10753475 before ESCs were separated by differential sedimentation and adhesion. The purity of primary ESC cultures was tested morphologically by light microscopy and immunocytochemically on parallel cultures, as previously described. Cultures were free of CD452positive leukocytes and contamination by factor VIII-positive endothelial cells was generally less than 1%. ESCs were cultured at 37uC in DMEM:F12 supplemented with 10% fetal bovine serum, insulin, transferrin, and a mix of antibioticsantimycotics. Preconfluent cells were washed with HBSS, incubated overnight with charcoal-treated FBS-supplemented medium, washed with phenol red-free DMEM:F12 and cultured with phenol red- Materials and Methods Subjects and tissue handling hCG/IL1 Modulate Endometrial Cell Phenotype and FBS-free medium containing hCG for 24 h. Cells were then incubated with a fresh phenol red- and FBS-free medium containing IL1B for additional 24 h. hCG and IL1B concentrations were determined based on our previous studies with human ESCs where different doses were used. hCG and IL1B concentrations are within the range of the molecules’ physiological concentrations. RNA preparation and micro-array analysis Total RNA of ESC cultures issued from 3 different women was extracted with Trizol according to the manufacturer’s directions. 24347635 Then they were washed using the micro RNeasy Kit. Total RNA quantity was measured with Nanodrop ND-1000 spectrophotometer and RNA integrity was assessed by capillary electrophoresis using the Bioanalyzer 2100. DNA micro-array analyses were carried out with Affymetrix Human Gene 1.0 ST at the Gene Expression Platform of the Research Centre of Laval University Hospital Centre, Quebec, Canada. The array interrogates 28,869 well-annotated genes w
