Female patients between 20 to 40 years were enrolled in our study; thirty-eight with pelvic pain and downgraded quality of life, suspected of having an endometriosis and forty young women without pain or inflammation, as a control group.
DNA extraction was performed using QIAamp DNA mini-Blood kit (Qiagen), following manufacturer protocol. Briefly, 1 ml of well homogenised plasma sample was mixed with 100 l of protease and 1 ml of AL buffer. Then the mixture was incubated at 56C during 10 minutes. 1ml of 100% ethanol was added and vortexed for 15 seconds. Samples were then transferred to the QIAamp mini spin column to follow the standard procedure. Each DNA was eluted with 60 l H2O. DNA concentration and purity was checked using spectrophotometer Nanodrop ND-1000 (Thermo Scientific). Bisulphite DNA treatment was performed using EZ DNA Methylation Kit (Zymo Research) following manufacturer recommendations. According to the initial concentrations, 35l of DNA sample was used for the reaction. At the end of the treatment, DNA was eluted with 25l of elution Buffer, then diluted using 10l H2O. Gene specific PCR reactions were performed using TaqOzyme HS Mix (Ozyme), 1l of bisulphite treated DNA and final primers concentration 0.4M each in 20l final reaction volume using C-100 thermocycler (Bio-Rad). The cycling conditions were 95C/1min; (95C/15s  58C/15s  72C/30s) *34; 72C/5s. All amplicon sizes were checked and validated by electrophoresis before sequencing, and sequencing are performed in Paired-End (2*150bp) on NextSeq Illumina Platform following manufacturer protocol.