O-083 Endometriosis induces DNA double-strand breaks and triggers apoptosis in oocytes of primordial follicles and its inhibition by melatonin administration

Abstract

Study question What is the involvement of DNA double-strand breaks (DDSBs) signaling pathway in primordial follicle depletion in endometriosis and the role of melatonin in this pathway? Summary answer Endometriosis increases percentages of γH2AX-positive oocytes and triggers apoptosis in primordial follicles, while melatonin prevents ovarian reserve reduction by rescuing oocytes from DDSBs and apoptosis. What is known already Endometriosis causes ovarian reserve reduction; however, its mechanism remains unclear. The percentage of γH2AX-positive primordial follicles is significantly elevated, and the apoptosis of primordial follicles is evident in cyclophosphamide-treated mice. In addition, the percentage of phosphorylated-Ataxia Telangiectasia Mutated (pATM)-positive oocytes and RAD51-positive oocytes is significantly higher in primordial follicles of gamma irradiated mice. Melatonin may prevent ovarian reserve reduction by protecting oocytes from DDSBs during prophase arrest and enhancing DNA repair. There is still insufficient studies on the DDSBs signaling pathway in endometriosis-induced ovarian reserve depletion and the role of melatonin in this pathway. Study design, size, duration Ovarian cortex tissues were obtained from 16 endometriosis patients (E), aged 29-43, who had undergone oophorectomy and 12 age-matched control women (C) without ovarian pathology. Endometriosis model mice (mE; n = 16) and control mice (mC; n = 4) were established from 5 weeks old BALB/c mice on Day 0. Melatonin (30 mg/kg) was administered to mice (n = 13) daily from Day 3 to Day 14. Mice were sacrificed on Day 14 and the ovarian tissues were extracted. Participants/materials, setting, methods Endometriosis model mice were established by intraperitoneal injection of the minced uterus from homologous mice, while control mice were given phosphate buffered saline. Immunohistochemical study using human and mouse ovarian cortex tissue were performed for H2A histone X (γH2AX), pATM, and RAD51 to detect DDSBs pathway and DDSBs repair response. TUNEL assay was performed to detect apoptosis. The positive-stained oocytes of primordial and growing follicles were counted and analyzed using an unpaired student’s T-test. Main results and the role of chance DDSBs consistently occurs in the oocytes of primordial follicles of endometriosis model mice and human ovaries with endometriosis. The percentages of γH2AX-positive oocytes were significantly higher in E (62.9%) than in C (4.7%, p < 0.05) and mE (87.5%) than in mC (25.0%, p < 0.05). pATM as a global regulator in DDSBs repair response and RAD51 as a marker for homologues recombination pathway were analyzed. The percentage of pATM-positive oocytes was significantly lower in E (11.4%) than in C (25.2%, p < 0.05) and mE (18.25%) than in mC (24.75%, p < 0.05). The percentage of RAD51-positive oocytes was significantly higher in mE (87.0%) than in mC (14.5%, p < 0.05). Apoptosis of primordial follicles oocytes is evident in endometriosis model mice and human ovaries with endometriosis. The percentages of TUNEL-positive oocytes were significantly higher in E (85.2%) than in C (24.7%, p < 0.05) and mE (81.75%) than in mC (20.75%, p < 0.05). Melatonin significantly reduces percentage of γH2AX-positive oocytes (72.1% vs 50.5%, p < 0.05) and TUNEL-positive oocytes (83.0% vs 22.0%, p < 0.05). Therefore, it’s a novel discovery that melatonin rescues oocytes from DDSBs, indicating its pharmacological potential to prevent ovarian reserve reduction in endometriosis. Limitations, reasons for caution The main lesion in our endometriosis model mice is peritoneal endometriosis, meanwhile ovarian and deep infiltrating lesions are rare. Even though the endometriosis lesion in our mouse model does not perfectly mimic the human endometriosis, it represents the evidence of inflammation caused by peritoneal endometriosis resulting in ovarian reserve reduction. Wider implications of the findings Clarifying the signaling pathway in endometriosis-induced ovarian reserve depletion is important for drug discovery that is able to protect ovarian reserve in endometriosis. Further studies of melatonin supplementation in endometriosis patients is warranted to evaluate its effect on ovarian reserve reduction. Trial registration number No