Briefly, rat tail collagen was extracted from tendon bundles of sterilised rat tails in sterile diluted inhibitor CHIR99021 acetic acid at 4 C, and the acid soluble collagen was col lected aseptically and used as stock as previously described. Collagen biomatrix was generated by neutralising acid collagen with 0. 34 N NaOH and diluted with 10X medium to a concentration of 1. 2 0. 1 mgml at pH 7. 4 on ice and then gelled at 37 C with 5% carbon dioxide. Cells were allowed to attach with a daily change of medium at 72 hours when cells had grown to 80% confluence, serum free DMEM F12 medium supplemented with insulin, transferrin, selen ium, hydrocortisone, gentamicin, penicillin, streptomycin and fungizone was added. Serum free cell cultures in all three groups were exposed to Inhibitors,Modulators,Libraries different concentrations of rhCG in triplicate.

After 24 hours, cultures were terminated, and gels containing cells were used for RNA extraction and quantitative gene expression using RT PCR. The remaining conditioned media were collected and centrifuged at 10,000 g for 5 minutes, Inhibitors,Modulators,Libraries and the supernatant was stored at 80 C for immunoassay of multiple cytokines and immuno blotting experiments. Five replicates in duplicate were con ducted for each set of experiments. hCG binding to isolated endometrial cells Different sets of experiments were performed to examine the binding of biotinylated rhCG Inhibitors,Modulators,Libraries to the cells grown in culture. Biotin labelling of hCG was performed using a commercially available biotin labelling kit from Thermo Scientific according to the manufacturers protocol.

Isolated endometrial cells were grown separately on collagen coated cover slips in DMEM Inhibitors,Modulators,Libraries F12 complete medium as described above. The cells were then washed twice in serum free DMEM F12 and exposed to biotinylated rhCG at 37 C in a humidified incubator with 5% CO2 for 40 minutes these parameters were determined based on initial op timisation with three concentrations of biotinylated rhCG Inhibitors,Modulators,Libraries and two time points. To assess the specificity of hCG binding, parallel cultures of attached cells after 72 hours of culture were incubated with biotinylated scrambled sham rhCG, unconjugated biotin, unconju gated rhCG in place of biotinylated rhCG or without any of these reagents. The incubation was stopped with ice cold PBS, and the cells were fixed in 4% para formaldehyde to assess the bound hCG by immuno fluorescence using an Alexa488 conjugated anti biotin monoclonal antibody and a Confocal Laser Scanning Microscope.

Multiplex assays of cytokines in conditioned media The concentrations of 48 cytokines, chemokines and growth factors that have been reported to be synthesised and secreted by the human endometrium were analysed. they Cell culture supernatants were assessed by quantitative cytokine assays using a BioplexTM Pro human cytokine 27 plex panel and a cytokine 21 plex panel based on xMAP technology according to the pre optimised protocol.