Min Li,* Hedong Lu,* Chunya Ye,* Xiaolei Wang, Dongmei Ji, Zhiguo Zhang, Yunxia Cao, Weiwei Zou

Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Yunxia Cao, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, No. 218 Jixi Road, Hefei, 230022, People’s Republic of China, Email caoyunxia@ahmu.edu.cn Weiwei Zou, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, No. 218 Jixi Road, Hefei, 230022, People’s Republic of China, Email tottizou@126.com

Purpose: Graphene oxide (GO) has promising biomedical applications, but its potential toxicity to the female reproductive system is underexplored. This study investigates the short-term effects of a single dose of GO nanosheets on human ovarian granulosa cells, focusing on mitochondrial damage.
Materials and Methods: First, cell viability was detected by CCK-8 and apoptosis was detected by flow cytometry to assess the cytotoxicity of GO on KGN. Second, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and mitochondrial morphology were observed by confocal microscopy, mitochondrial and sub-mitochondrial structure by transmission electron microscopy (TEM), quantitative analysis of ATP and mitochondrial complex I enzyme activity by luminosity value and autophagy by flow cytometry to assess the mitochondrial toxicity of GO on KGN cells.
Results: The 72h half-maximum effective concentration (EC50) value of GO was determined to be 29.73 μg/mL. GO induced cell death in a dose-dependent manner, with significant effects on cell viability even at low doses (1 μg/mL). Exposure to low GO concentrations resulted in abnormal mitochondrial morphology and function, including mitochondrial breakage, membrane damage, reduced mitochondrial cristae, enhanced autophagy, decreased ATP production, decreased MMP, and decreased enzymatic activity of mitochondrial complex I. Mitochondrial function returned to normal levels on day 7 after KGN cells left the GO-exposed environment.
Conclusion: This study demonstrates that short-term exposure to low-dose GO causes mitochondrial damage in human ovarian granulosa cells, highlighting the need for further research on the safety of GO, particularly regarding its potential effects on reproductive health. However, GO-induced transient mitochondrial damage is highly likely to negatively affect ovarian reserve function, which needs to be further verified in animal models.

Keywords: graphene oxide, human ovarian granulosa cells, mitochondrial damage, cytotoxicity, reproductive toxicity