Ethical committee of Avicenna Research Institute approved all experiments in this study and samples were collected after getting signed consent forms from participants. Endometrial tissues were obtained from fifteen apparently normal women between 25–45 years of age referring to Avicenna infertility clinic for hysteroscopic tubal ligation who had no evidence of pathology in their hysteroscopic evaluations. Participants had no history of infertility, miscarriage or preeclampsia, thyroid disease, sign of vaginal infection or confounding pathology such as myoma in their endometrium and all had history of at least one successful delivery. All subjects had regular menstrual cycles and did not take vitamin D3 or hormone in the last three months.
Endometrial samples were collected by gynecologists using pipelle pipette in proliferative phase of menstrual cycle and transferred to the laboratory in a cold chain in DMEM containing antibiotics in the sterile condition. Samples were divided into three parts for pathological examination, RNA extraction, and cell isolation. Sample for RNA extraction was immediately immersed in RNAlater and frozen at -20ᵒ C.
Isolation of whole endometrial cells (WECs) and stromal cells (ESCs)
Isolation of WECs and ECSs were preformed according to the protocol published elsewhere . In brief, tissues pieces were cut into small pieces and digested for 1.5 h at 37ᵒ C with collagenase D and DNAse (Roche, USA). Isolated cells were used as WECs. For isolation of ESCs, a part of WECs was cultured for 24 h and adherent cells were collected. Purity of isolated ESCs were determined by immunofluorescent staining and flow cytometry. Vimentin+, nestin+, cytokeratin-, CD10 + and CD45- cells were regarded as ESCs .
Treatment with LPS, LTA and 1,25 (OH)2 D3
We previously showed that WECs expressed TLR2 and TLR4, while ESCs only expressed TLR4  and therefore; in this study WECs were treated with either LPS or LTA, while ESCs were exposed to only LPS. Indeed, using a kinetic study on production of pro-inflammatory cytokines following LPS and LTA treatment of WECs and ESCs, we had previously shown that LTA with the concentration of 1000 ng/ml were optimal doses to drive pro-inflammatory cytokines by those cell types . To this end, WECs and ESCs were treated with pre-optimized concentration of LTA or with 100-10000 ng/ml LPS. Moreover, according to our previous reports[15, 16, 21], physiologic concentration of vitamin D3 was selected for assessing its effects on LPS- and LTA-induced target genes and proteins expression. To evaluate the effects of 1,25 (OH)2 D3 on cytokines production by WECs and ESCs, 1 × 105 cells were cultured in wells of 96 well culture plate and treated with 10− 7M 1,25 (OH)2 D3 for 48 h followed by LPS or LTA stimulation for 24 h. Conditioned media were then harvested and stored at -70ᵒ C cytokine assay. In order to assess potential effect of 1,25 (OH)2 D3 on modulation of LPS- or LTA-stimulated expression of TLR2, TLR4 and MyD88, 106 endometrial cells were cultured in 6 well plates and treated with 10− 7M 1,25 (OH)2 D3 for 48 h. After then pre-optimized concentrations of LPS or LTA was added to the wells for 8 h. Based on the results of preliminary kinetic experiments, this time period was optimal for gene expression following endometrial cell stimulation with either LPS or LTA. After then, cells were harvested in RNA Bee and stored at -70ᵒ C.
The concentrations of IL-6 (ebioscience, USA), IL-8 and TNF-α (BD biosciences, USA) were measured in cell culture supernatants by sandwich ELISA. The minimal detection limit for aforesaid cytokine assay sets were 7.5, 3.1, and 3.1 pg/ml for TNF-α, IL-6 and IL-8, respectively. All measurements were performed in triplicate.
Reverse transcriptase real time quantitative polymerase chain reaction (RT-qPCR):
Total RNA was extracted using commercial RNA extraction kit (Biosite-Sweden) according to manufacturer’s protocol. The cDNA was synthesized for each sample using reverse transcriptase (Fermentas–Lithuania) based on the manufacturer instructions. The following primers were used for RT-qPCR: TLR2 forward: 5’-ATTGTGCCCATTGCTCTTTC-3’ Reverse: 5’-TTCTTCCTTGGAGAGGCTGA-3’ ; TLR4 forward: 5’- GCTTCTTGCTGGCTGCATAA-3’ reverse: GAAATGGAGGCACCCCTTC-3’ ; MyD88 forward: 5’-GAGCGTTTCGATGCCTTCAT-3’ reverse: 5’- CGGATCATCTCCTGCACAAA-3’  and β-actin was used as a housekeeping gene with following primers: Forward: 5’- AGCCTCGCCTTTGCCGA-3’ reverse: 5’- CTGGTGCCTGGGGCG-C3’. To carry out qRT-PCR, SYBR Green (Takara) master mix was used. The amplification efficiency of target and reference genes were determined, then comparative ΔΔCT method was used to analyze qRT-PCR data. Target genes expression were relatively compared and results were analyzed by Rest-rg software.
TLR4 expression at the protein level was also studied using western blot according to the protocol published elsewhere . In brief, cell lysate was prepared in a lysis buffer containing %2 SDS, 10% glycerol, 50 mM Dithiothreitol, and 0.1% Bromophenol blue. Protein content of each cell lysate was measured using BCA assay. Proteins were resolved in a 10% SDS-PAGE gel and bands were electrotransfered onto the PVDF membrane (Millipore, Bedford, USA). Immunobloting was performed using goat anti-human TLR4 (R&D, USA) at 1 µg/ml for 2 h followed by washing and 45 min incubation with rabbit anti-goat Ig-HRP (Sinabiotech, Iran). Bands were visualized using ECL reagent (GE Healthcare, Sweden). Membranes were later stripped using Western Re-Probe (Calbiochem, SanDiego, CA, USA), re-incubated for 2 hr with agitation in polyclonal rabbit anti-beta actin antibody (Sigma, USA) as loading control followed by 45 min incubation in HRP-conjugated sheep anti-rabbit Ig (Sinabiotech) and processed as above. In negative control blots, primary antibody was substituted by equivalent dilutions of species-matched normal sera.
Data were expressed as Scattor plot and mean. For comparison of different groups, either T-test or one way ANOVA analysis was used. The relative expression of target genes was normalized first with reference gene (β-actin) and then with corresponding control. All data were analyzed and plotted by GraphPad Prism 6.07.