Endometrial tissues were obtained from fifteen apparently normal women under 45 years of age referring to a gynecology clinic for hysteroscopic tubal ligation who had no evidence of pathology in their hysteroscopic evaluations. These women had no history of infertility or miscarriage, stillbirth and preeclampsia in their previous pregnancies. Also, participants had no sign of vaginal infection or confounding pathology such as myoma in their endometrium as judged by clinical and ultrasound examinations. All subjects had regular menstrual cycles and had not used hormones for at least 3 months. This study was approved by the institutional review board and ethics committee of Avicenna Research Institute and Tehran University of Medical Sciences and informed consent was taken from all subjects enrolled in the study.

Endometrial biopsies were obtained using endometrial biopsy curette in proliferative phase of menstrual cycle and immediately transferred on ice to the laboratory in tissue culture medium containing antibiotics. Tissue samples were divided into three parts. A small fragment was processed for pathologic examination. The remaining tissue fragment was immersed in RNAlater and frozen at −20°C for RNA extraction. From remaining tissue, single cells were obtained by enzymatic digestion.

Whole endometrial cells (WECs) and endometrial stromal cells (ESCs) were separated by enzymatic digestion according to the established protocol. In brief, endometrial tissues were minced into small pieces and digested at 37°C for 1.5 hr in collagenase D 0.2% and DNAse 0.03%. Viability of single cell suspension was determined by trypan blue exclusion. Digested single cell suspension was used as WECs. For isolation of ESCs, cells were cultivated in 24-well culture plate at 37°C, 5% CO₂ in DMEM-F12 supplemented with 10% fetal calf serum (FCS) and additives. Non-adherent cells were removed by several washes and adherent stromal cells were allowed to propagate. The ESCs were characterized as vimentin+, nestin+, cytokeratin-, CD10+ and CD45- cells (Figure 1) (²³).

RNA extraction, cDNA synthesis and RT-PCR were performed according to the protocols published elsewhere (²⁴, ²⁵). Oligonucleotide primers used in this study were as follows: β-actin: F;5'-GTG-GGG-CGC-CCC-AGG-CAC-CA-3', R; 3'- CTC- CTT- AAT-GTC-ACG- CAC- GAT-TTC-5', TLR2: F; 5'-GTA-CCT-GTG-GGG-CTC-ATT-GT-3', R; 3'-CTG-CCC-TTG-CAG-ATA-CCA-TT-5', TLR4: F; 5'-GAG-CTT-TAA-TCC-CCT-GAG-GCA-3', R; 3'-GAT-TGG-ATA-AGA-TTG-TGA-GCC-5' and MyD88:F;5'-GAC-CCA-GCA-TTG-AGG-A GG-ATT-G-3', R;3'-GCT-TCT-GAT-GGG-CAC-CTG-GAG-AG-5'. After initial heating at 94°C for 3 min, PCR was performed for 30 (for β-actin) or 35 cycles (for TLR2, TLR4 and MyD 88) with annealing temperatures of 62°C, 54.8°C, 60°C and 60°C for TLR2, TLR4, MyD88 and β-actin, respectively. For all PCR programs, denaturation and extension temperatures of 94°C and 72°C, respectively were applied for 30 s. Final extension for 7 min was performed for all amplifications. PCR systems devoid of template cDNA and no amplification controls (no reverse transcriptase added) were included as negative controls. cDNA prepared from PBMCs served as the positive control.

For TLR2 staining, 5×10⁵ living cells were incubated with 5 μg/ml of Alexa fluor 488-conjugated anti-TLR2 antibody (BD) diluted in PBS-BSA for 30 min. All incubations were performed on ice. For TLR4 staining, cells were first incubated with biotin-conjugated anti-TLR4 (BD) for 60 min. After three washes, cells were stained with PE-Cy5.5-conjugated streptavidin (Invitrogen, USA) for 30 min. In negative reagent control tubes, isotype matched irrelevant antibodies (BD) were used instead of primary antibodies. Monocyte gate of PBMC served as the positive control. After three washing steps with PBS-BSA, cells were analyzed by Partec flowcytometer (Partec, Germany).

Western blotting of TLR2 and TLR4 in ESCs was performed according to the protocol published recently with some modifications (²⁵). Blots were incubated for 2 hr with agitation in goat anti-TLR4 antibody (R&D, USA) at 1 μg/ml or 1:100 dilution of goat anti-TLR2 (Santa Cruz, USA) followed by 45 min incubation in HRP-conjugated rabbit anti-goat Ig (Sinabiotech, Iran). Bands were detected using an ECL Western blotting substrate kit and digital images were obtained with a Gel Logic 2200 imaging system (Kodak, Tokyo, Japan). 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) as loading control followed by 45 min incubation in HRP-conjugated sheep anti-rabbit Ig (Sinabiotech, Iran) and processed as above. In negative control blots, primary antibodies were substituted by equivalent dilutions of species-matched normal sera. Band densities of PCR and Western blot experiments were quantified as described elsewhere (²⁵).

Immunofluorescent staining of TLR2 and TLR4 was performed as described elsewhere (²⁶). Briefly, cells were cyto-spinned and fixed with either ice cold acetone (for TLR2) or natural buffered formalin (for TLR4). Primary antibodies (goat anti-TLR2, 1:100 for overnight or goat anti-TLR4, 10 µg/ml for 3 hr) were added followed by 1:50 dilution of FITC-labeled rabbit anti-goat Ig for 60 min. Cells were then washed and counterstained with 4'-6-diamidino-2-phenylindole (DAPI) for nucleus staining. Fluorescent signal was visualized and photographed by a BX51 Olympus microscope equipped with a DP71 CCD camera.

Based on the results of gene and protein expression analysis, WECs were stimulated separately with both LPS and LTA, while ESCs were treated only with LPS. To study the effect of TLR engagement on cytokine production, cells (1×10⁵/well in 96-well plate) were treated with different concentrations of LPS (0.1 ng/ml-10000 ng/ml) or LTA (0.1 ng-1000 ng/ml) for 24 hr and after that cell culture supernatants were collected, centrifuged and stored at −70°C for future analysis. Indeed, to evaluate the effect of LPS and LTA stimulation on TLR4, TLR2 and MyD88 gene expression, WECs were stimulated with LPS (100 ng/ml) or LTA (10000 ng/ml) for 8 hr followed by cell harvesting and gene expression analysis as described above. Control cells received PBS only. ESCs, however, were treated with LPS for extended time periods of 2 hr, 4 hr, 8 hr, 12 hr and 24 hr.

Concentrations of TNF-α, IL-6 and IL-8 in supernatant of cultured cells were measured using ELISA according to the protocol provided by the manufacturer. The sensitivity of the assays for TNF-α, IL-6 and IL-8 were 7.5, 3.1 and 3.1 pg/ml, respectively.

All data are presented as median±range. Pairwise comparisons of gene expression and cytokine production after TLR stimulation was performed using non-parametric Wilcoxon rank test and statistically significant differences were accepted at p < 0.05.

TLR4, TLR2 and MyD88 transcripts were detected in samples derived from human endometrium. Likewise, it was demonstrated that ESCs expressed TLR4 and MyD88 at mRNA level but failed to express TLR2 transcript (Figure 2A). To confirm the results of gene expression analysis of TLR4 and TLR2, three readout systems, flow cytometry, Western blotting and immunofluorescent staining were used. The results of flow cytometric analysis showed that ESCs expressed neither TLR4 nor TLR2 at their surface (Figure 2B). Western blotting clearly demonstrated TLR4, but not TLR2 protein expression by ESCs (Figure 2C). Consistent with flow cytometry results, no surface expression of TLR4 was observed; instead TLR4 was localized in perinuclear compartment. In line with Western blot results, ESCs failed to express TLR2 protein in immunofluorescent staining (Figure 2D).

The results showed that different concentrations of LPS at different time intervals had only marginal effect on TLR4 gene up-regulation (Figures 3A, B). At the protein level, however, expression of TLR4 was significantly increased by LPS activation (p < 0.05) (Figure 3E). Treatment of this cell type with 100 ng/ml of LPS significantly increased MyD88 expression up to 8 hr after LPS stimulation (p < 0.05) (Figures 3C, D). More importantly, effect of LPS stimulation on MyD88 expression was gradually subsided thereafter reaching to the control level after 24 hr.

Akin to ESCs, expression of TLR4 and MyD88 in WECs was investigated after LPS stimulation. Whole endometrial cells were treated with 100 ng/ml LPS for 8 hr and the expression of TLR4 and MyD88 transcripts were then investigated. In parallel with the results from ESCs, it was demonstrated that LPS treatment marginally increased TLR4 gene expression but significantly intensified the expression of MyD88 transcript (p < 0.05) (Figures 4A, B).

Lower female reproductive tract is more frequently populated with Gram positive bacteria. With this end in view, WECs were also stimulated with LTA to see the effect of such treatment on TLR2 and MyD88 expression. Although such treatment increased the levels of TLR2 and MyD88 gene expression, but this did not reach statistically significant level (Figures 5A, B).

Endometrial stromal cell cultures were established and analyzed for the potential effect of LPS at different concentrations on pro-inflammatory cytokine production by this cell population. The results showed that ESCs produce significantly higher amounts of IL-6, IL-8 and TNF-α in response to LPS stimulation in a dose-dependent manner (p < 0.05) (Figures 6A–C).

Stimulation with 100 ng/ml LPS and all concentration range of LTA significantly increased production of IL-6 in WECs (p < 0.05) (Figures 7A, D). Similar results for IL-8 production were also achieved when WECs were stimulated with the same concentration of LPS or 1000 ng/ml LTA (p < 0.05) (Figures 7B, E). Although LPS and LTA stimulation increased TNF-α production by WECs, the results were not statistically significant (Figures 7C, F).