*4.1. Human Subject Participants*

**4. Materials and Methods**  *4.1. Human Subject Participants* Women ages 18 to 60 years, undergoing tubal ligation or having non-endometriosis disorders (controls, *n* = 12) or patients with endometriosis ("endo", laparoscopically diagnosed followed by pathological confirmation and/or patients with symptoms, *n* = 14) were recruited from Obstetrics-Gynecology clinic at Cabell Huntington Hospital, Joan C Edwards School of Medicine, Marshall University, in Huntington, WV, USA. This HIPAA compliant study was approved by the Institutional Review Board of the Marshall University School of Medicine and was carried out per the principles of the Declaration of Helsinki. All patients were consented prior to the study. The inclusion criteria included women ages 18–60 years old, with normal menstrual cycles and otherwise in normal health (except for pain and endometriosis) who have not been on any hormonal medica-Women ages 18 to 60 years, undergoing tubal ligation or having non-endometriosis disorders (controls, *n* = 12) or patients with endometriosis ("endo", laparoscopically diagnosed followed by pathological confirmation and/or patients with symptoms, *n* = 14) were recruited from Obstetrics-Gynecology clinic at Cabell Huntington Hospital, Joan C Edwards School of Medicine, Marshall University, in Huntington, WV, USA. This HIPAA compliant study was approved by the Institutional Review Board of the Marshall University School of Medicine and was carried out per the principles of the Declaration of Helsinki. All patients were consented prior to the study. The inclusion criteria included women ages 18–60 years old, with normal menstrual cycles and otherwise in normal health (except for pain and endometriosis) who have not been on any hormonal medication for at least one month before sample collection. Exclusion criteria included subjects with current medical illnesses such as diabetes, cardiovascular disease, hyperlipidemia, hypertension, systemic lupus erythematosis or rheumatologic disease, positive HIV/AIDS, active infection. Subjects were asked to stop multivitamins that contain high levels of antioxidants and anti-inflammatory medications prior to sample collection.

tion for at least one month before sample collection. Exclusion criteria included subjects with current medical illnesses such as diabetes, cardiovascular disease, hyperlipidemia, hypertension, systemic lupus erythematosis or rheumatologic disease, positive HIV/AIDS, active infection. Subjects were asked to stop multivitamins that contain high levels of antioxidants and anti-inflammatory medications prior to sample collection. In this study, majority of the samples where from endo patients diagnosed with stage I/II and only one at stage IV. Pathological confirmation for endo patients classified the patients as mostly belonging to the peritoneal or uterine serosa pathology. All women completed a gynecologic/infertility history form, a pre-operative quality of life questionnaire and assessment of pain using a visual analog scale for assessment of endometriosis associated pain (dysmenorrhea, non-menstrual pelvic pain, dyspareunia, and dyschesia)

Date of their last menstrual period was used to assess their cycle time.

*4.2. RNA and Protein Isolation in Peritoneal Fluid-Treated Cells*

I/II and only one at stage IV. Pathological confirmation for endo patients classified the patients as mostly belonging to the peritoneal or uterine serosa pathology. All women completed a gynecologic/infertility history form, a pre-operative quality of life questionnaire and assessment of pain using a visual analog scale for assessment of endometriosis associated pain (dysmenorrhea, non-menstrual pelvic pain, dyspareunia, and dyschesia) (adapted from the validated International Pelvic Pain Society's Pelvic Assessment Form).

Peritoneal fluid (PF) (devoid of blood contamination) was collected on ice from all

women during laparoscopic surgery. Peritoneal fluid was spun at 2000× *g* to remove any cellular debris. The supernatant was used immediately for studies or stored in a −80 °C (adapted from the validated International Pelvic Pain Society's Pelvic Assessment Form). Date of their last menstrual period was used to assess their cycle time.
