Miaoling Lin1, Rui Hua2, Jing Ma2, Yao Zhou2, Pei Li3, Xiya Xu2, Zhiqiang Yu4, Song Quan5. 1. Department of Obstetrics and Gynaecology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China; Center for Reproductive Medicine, Department of Obstetrics and Gynaecology, The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen 518107, China. 2. Department of Obstetrics and Gynaecology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. 3. Department of Environmental Science, Zhejiang University, Hangzhou 310058, China. 4. State Key Laboratory of Organic Geochemistry, Guangdong Key Laboratory of Environment and Resources, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China. Electronic address: zhiqiang@gig.ac.cn. 5. Department of Obstetrics and Gynaecology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. Electronic address: quansong@smu.edu.cn.
Abstract
BACKGROUND: Bisphenol A (BPA) is a widespread endocrine-disrupting chemical with estrogen like effects, which could interfere with the human reproductive system by disrupting the normal function of granulosa cells (GCs) leading to abnormal ovarian function. However, the mechanism of its toxicity on human GCs has not been clearly described thus far. METHODS: 106 normogonadotropic infertile women undergoing their first in-vitro fertilization-embryo transfer (IVF-ET) cycle were recruited. Urinary BPA level and the early outcomes of IVF-ET were analysed. Patients were divided to low and high BPA exposure groups using the median urinary BPA concentration as the cut-off value. In-vivo and in-vitro studies were conducted using mice and human granulosa cell line (KGN cells). Female Kunming mice approximately 6-8 weeks of age were poisoned with BPA at different dosages (1, 10 or 100 μg/kg) by oral gavage once daily for 2 weeks, while KGN cells were exposed to BPA at the concentration of 1, 10 or 100 nM for 24 h, 48 h or 72 h. BPA-induced ovarian morphologic changes were analysed by histopathology investigation. Cell viability and apoptosis were evaluated using CCK-8, TUNEL and flowcytometric, respectively. Hormone levels were determined using ELISA and the molecular mechanism studies were conducted using immunofluorescence, RT-PCR and western blots. RESULTS: The oocyte retrieval rate, maturation rate and embryo implantation rate significantly decreased with the higher level of urinary BPA concentration. Peak E2 level was lower in high BPA group, but no statistical significance could be observed. In BPA treated mice, cystic dilation of the follicles with a decreased number of GCs could be observed histopathologically. Decreased E2, P4 and AMH level and GCs autophagy could be detected both in-vivo and in-vitro with the activation of AMPK/mTOR/ULK1 signalling pathway. As being confirmed in KGN cells, phosphorylated AMPK and ULK1 increased while phosphorylated mTOR decreased, and by inhibition autophagy using knockdown of AMPK or 3-MA, adverse effects of BPA exposure in-vitro could be reversed. CONCLUSION: BPA exposure might abnormally influence human ovarian functions leading to abnormal folliculogenesis by activation of autophagy in GCs through AMPK/mTOR/ULK1 pathway.
BACKGROUND: Bisphenol A (BPA) is a widespread endocrine-disrupting chemical with estrogen like effects, which could interfere with the human reproductive system by disrupting the normal function of granulosa cells (GCs) leading to abnormal ovarian function. However, the mechanism of its toxicity on human GCs has not been clearly described thus far. METHODS: 106 normogonadotropic infertile women undergoing their first in-vitro fertilization-embryo transfer (IVF-ET) cycle were recruited. Urinary BPA level and the early outcomes of IVF-ET were analysed. Patients were divided to low and high BPA exposure groups using the median urinary BPA concentration as the cut-off value. In-vivo and in-vitro studies were conducted using mice and human granulosa cell line (KGN cells). Female Kunming mice approximately 6-8 weeks of age were poisoned with BPA at different dosages (1, 10 or 100 μg/kg) by oral gavage once daily for 2 weeks, while KGN cells were exposed to BPA at the concentration of 1, 10 or 100 nM for 24 h, 48 h or 72 h. BPA-induced ovarian morphologic changes were analysed by histopathology investigation. Cell viability and apoptosis were evaluated using CCK-8, TUNEL and flowcytometric, respectively. Hormone levels were determined using ELISA and the molecular mechanism studies were conducted using immunofluorescence, RT-PCR and western blots. RESULTS: The oocyte retrieval rate, maturation rate and embryo implantation rate significantly decreased with the higher level of urinary BPA concentration. Peak E2 level was lower in high BPA group, but no statistical significance could be observed. In BPA treated mice, cystic dilation of the follicles with a decreased number of GCs could be observed histopathologically. Decreased E2, P4 and AMH level and GCs autophagy could be detected both in-vivo and in-vitro with the activation of AMPK/mTOR/ULK1 signalling pathway. As being confirmed in KGN cells, phosphorylated AMPK and ULK1 increased while phosphorylated mTOR decreased, and by inhibition autophagy using knockdown of AMPK or 3-MA, adverse effects of BPA exposure in-vitro could be reversed. CONCLUSION: BPA exposure might abnormally influence human ovarian functions leading to abnormal folliculogenesis by activation of autophagy in GCs through AMPK/mTOR/ULK1 pathway.
Authors: Liang Du; Da Wang; Peter W Nagle; Andries A H Groen; Hao Zhang; Christina T Muijs; John Th M Plukker; Robert P Coppes Journal: Cancers (Basel) Date: 2022-04-01 Impact factor: 6.575