Ying Li1, Ruiqi Li1, Nengyong Ouyang1, Kailing Dai1, Ping Yuan1, Lingyan Zheng1, Wenjun Wang2. 1. Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China; Reproductive Medicine Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China. 2. Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China; Reproductive Medicine Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China. Electronic address: wenjungzcn@163.com.
Abstract
OBJECTIVE: To investigate the possible impact of local inflammation on granulosa cells (GCs) and follicular development in endometriosis patients. DESIGN: Prospective study with related paired design. SETTING: Reproductive medicine center. PATIENT(S): A total of 80 endometriosis patients and 104 controls, with cultured GCs collected from control participants younger than 35 years. INTERVENTION(S): Tumor necrosis factor-α (TNF-α) and nuclear factor κB (NF-κB) inhibitor. MAIN OUTCOME MEASURE(S): Intrafollicular concentrations of cytokines measured with ELISA, NF-κB binding levels with electrophoretic mobility shift assay (EMSA), and telomerase activity (TA) with quantitative-telomeric repeat amplification protocol (Q-TRAP) assay, and protein and mRNA expression with Western blot and polymerase chain reaction analyses, respectively. RESULT(S): Patients with endometriosis exhibited a statistically significantly lower antral follicle count (11.48 ± 8.11 vs. 15.68 ± 8.56), lower number of retrieved oocytes (8.28 ± 6.69 vs. 10.87 ± 6.26), and lower number of mature oocytes (6.67 ± 6.09 vs. 8.53 ± 5.69). The GCs from endometriosis patients showed higher NF-κB binding activity and increased expression of inhibitor of NF-κB kinase subunit β (IKKβ, 2.743-fold) and NF-κB inhibitor α (IκBα, 5.017-fold). Their NF-κB p65 expression was negatively associated with mature oocytes (bNF-κB' = -0.304, R2 = 0.195, R = 0.442) but positively associated with intrafollicular TNF-α (r = 0.37); TA showed a negative relationship with NF-κB binding levels (r = -0.667). Tumor necrosis factor-α induced expression of IκBα (5.408-fold) and NF-κB p65 (1.400-fold) but lowered human telomerase reverse transcriptase (hTERT) and TA levels (0.0009 vs. 0.5619) in cultured GCs. However, inhibiting NF-κB obviously increased hTERT expression (1.988-fold). CONCLUSION(S): Endometriosis showed activated NF-κB pathways in GCs, which might negatively affect TA and oocyte quality. Intrafollicular TNF-α might down-regulate TA and hTERT via NF-κB pathway, but further studies are required.
OBJECTIVE: To investigate the possible impact of local inflammation on granulosa cells (GCs) and follicular development in endometriosispatients. DESIGN: Prospective study with related paired design. SETTING: Reproductive medicine center. PATIENT(S): A total of 80 endometriosispatients and 104 controls, with cultured GCs collected from control participants younger than 35 years. INTERVENTION(S): Tumor necrosis factor-α (TNF-α) and nuclear factor κB (NF-κB) inhibitor. MAIN OUTCOME MEASURE(S): Intrafollicular concentrations of cytokines measured with ELISA, NF-κB binding levels with electrophoretic mobility shift assay (EMSA), and telomerase activity (TA) with quantitative-telomeric repeat amplification protocol (Q-TRAP) assay, and protein and mRNA expression with Western blot and polymerase chain reaction analyses, respectively. RESULT(S): Patients with endometriosis exhibited a statistically significantly lower antral follicle count (11.48 ± 8.11 vs. 15.68 ± 8.56), lower number of retrieved oocytes (8.28 ± 6.69 vs. 10.87 ± 6.26), and lower number of mature oocytes (6.67 ± 6.09 vs. 8.53 ± 5.69). The GCs from endometriosispatients showed higher NF-κB binding activity and increased expression of inhibitor of NF-κB kinase subunit β (IKKβ, 2.743-fold) and NF-κB inhibitor α (IκBα, 5.017-fold). Their NF-κB p65 expression was negatively associated with mature oocytes (bNF-κB' = -0.304, R2 = 0.195, R = 0.442) but positively associated with intrafollicular TNF-α (r = 0.37); TA showed a negative relationship with NF-κB binding levels (r = -0.667). Tumor necrosis factor-α induced expression of IκBα (5.408-fold) and NF-κB p65 (1.400-fold) but lowered humantelomerase reverse transcriptase (hTERT) and TA levels (0.0009 vs. 0.5619) in cultured GCs. However, inhibiting NF-κB obviously increased hTERT expression (1.988-fold). CONCLUSION(S): Endometriosis showed activated NF-κB pathways in GCs, which might negatively affect TA and oocyte quality. Intrafollicular TNF-α might down-regulate TA and hTERT via NF-κB pathway, but further studies are required.