vere endometriosis were studied. Complete excision of the ovarian endometrioma was performed. The diagnosis of endometriosis was confirmed by 
anatomopathological examination of all specimens obtained. GSK-126 web Endometrial biopsies from women with moderate or severe endometriosis were performed using a cornier device without curettage, which takes the functional layer. Controls. Normal endometrial tissues were obtained from fertile women without endometriosis who underwent surgery for tubal sterilization and pelvic pain. The absence of endometriosis was confirmed by meticulous examination of the pelvic and extrapelvic peritoneum, ovaries, intestine, and diaphragm to detect typical or atypical endometriotic lesions. Biopsies of potential areas of endometriosis were confirmed to be negative in these women. Other gynaecologic pathologies such as adhesions or ovarian or uterine masses were also confirmed to be negative in this control group by preoperative gynaecologic ultrasound and systematic laparoscopic examination of the abdominal cavity during the intervention. Both peritoneal fluids, from control and patients, were collected immediately after the establishment of the pneumoperitoneum and before laparoscopic manipulation. The peritoneal fluids were centrifuged at 1,5006g for 30 min at 4uC, filtered through a 0.2mm pore size membrane, and stored at 280uC. Women affected by menorrhagia or hypermenorrhea or women who had been pregnant or breast feeding during the previous 6 months were excluded from the study. None of the women had received any form of hormone therapy for at least 3 months before the study. Tissue Samples Endometrial tissue from 11 women with moderate or severe endometriosis , ovarian endometrioma from 11 women with moderate or severe endometriosis and control endometrial 2876749 tissue from 8 women without the disease were obtained for stromal cell isolation. No statistical significant differences in the age of the groups were observed. miRNAs in Endometrial Cultures from Endometriosis Peritoneal Fluid Pools 10 peritoneal fluids from women with endometriosis and 10 peritoneal fluids from fertile women without endometriosis in the proliferative phase of the menstrual cycle were thawed and pooled. and proteolytic factors to determine the relationship among these parameters. RNA Extraction Total RNA from primary cell cultures were extracted using the mirVana miRNA isolation kit, according to the manufacturer’s protocol. Yield and purity of RNA were measured using a NanoDrop ND-1000 spectrophotometer. Primary Cell Culture of Stromal Cells from Endometrial and Endometriotic Tissues Primary cultures of endometrial cells were prepared as previously described with minor modifications. Endometrial biopsies were collected in PBS containing 50 U penicillin/mL and 50 mg streptomycin/mL and rinsed to remove blood cells, stored at 4uC and processed within 218 h. No significant correlations between the sample processing times and the studied parameters were observed in the different groups. Tissues were cut into 1 mm3 pieces and incubated at 37uC for 60 minutes in the presence of collagenase. Dissociated tissues were filtered through a nylon sieve to remove undigested material. 22884612 Purity of the endometrial stromal cells was higher than 95%, as evaluated by positive cellular staining for vimentin and negative cellular staining for cytokeratin and CD68, as previously described. Cell suspension was centrifuged at 5506g for 5 minutes and the pellet resuspende
