No difference between the two groups with respect to the cycle
No difference between the two groups with respect to the cycle phase. None of these patients had received any GnRH analogue, antibiotics, radio-, chemo-, or hormone therapy in the last 6 months prior to the surgery. Endometrium samples were gathered within 10?5 min after hysterectomy and immediately frozen in liquid nitrogen and then preserved in -80 refrigerator until further use. Written informed consent was obtained before surgical procedures, including a consent form and protocol approved by the Institutional Review Boards of China Medical University.Methylation specific PCR (MSP)MethodsPatients and specimensEutopic endometrium samples were collected from PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27385778 60 patients with an average age of 43.65 ?3.99 years, who underwent hysterectomy due to endometriosis stages III and IV according to the Revised American Fertility Society Classification for Endometriosis at the Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University. For controls, endometrium samples were obtained from 20 women with an average age of 43.20 ?2.87 years, who underwent total hysterectomyGenomic DNA was extracted from endometrial tissues by using the TIANamp Genomic DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instructions. Then, 1 g genomic DNA was modified by sodium bisulfite with the CpGenomeTM DNA Modification Kit (Chemicon, Billerica, MA, USA). This modification converts unmethylated cytosine to uracil and leaves 5-methyl cytosine unchanged. Briefly, 20 L of genomic DNA was treated with 550 L of a mixed solution of 3.5 M sodium bisulfite/1 mM hydroquinone (pH 5.0). After incubation at 55 for 16 h, the treated DNA was purified and desulfonated with 0.3 M NaOH. The modified DNA obtained was ethanolprecipitated and dissolved in 50 L of Tris-EDTA buffer. The primers used for the methylated COX-2 genepromoter regions were as follows: (a) COX-2 F, 50-GA AGCGTTCGGGTAAAGATTGC-30 and (b) COX-2R, 50-AAATTACGTAAACCCGATAAAA-30. Primers for unmethylated COX-2 were as follows: (a) COX-2 F, 50-TGGAAGTGTTTGGGTAAAG-30 and (b) COX-2R, 50-AAAATTACATAAACCCAATA-30. To exclude falsepositive and false-negative results, universal unmethylated DNA and universal methylated DNA were purchased from Chemicon and served as controls.Table 1 The cases of the endometriosis group and the control group in different phase of menstrual cycle (n)Phase of menstrual cycle EMs group Control PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25447644 group 2 Early secretory Mid secretory Late secretory 20 27 13 7 9 4 P 0.032 0.Wang et al. European Journal of Medical Research 2012, 17:12 http://www.eurjmedres.com/content/17/1/Page 3 ofQuantitative real-time RT-PCRTotal RNA was isolated by using the RNAsimple total RNA kit (Tiangen, China) according to the manufacturer’s instructions. One microgram total RNA was reverse transcribed using the TIANScript RT Kit (Tiangen, China). Quantitative real-time RT-PCR was performed using SYBR green (Tiangen, China) on ExicyclerTM 96 Real-Time Quantitative Thermal Block (Bioneer, Daejeon, Korea). The specificity of the PCR was confirmed by examining the dissociation reaction plot subsequent to real-time RT-PCR. GAPDH served as the constitutive control. Forward and reverse primers were 50-GAATCATTCACCAGGCAAATT G-30 and 50-TCTGTACTGCGGGTGGAACA-30 for COX2, respectively; 50-GCACCGTCAAGGCTGAGAAC-30 and 50-ATGGTG-GTGAAGACGCCAGT-30 for GAPDH, Necrostatin-1 site respectively. PCR reactions of each sample were done in triplicate. We took the average value of the control group as in.