Sperm DNA Damage Caused by the Abnormal Methylation of Rad54 Promoter Region Caused by Folic Acid Deficiency

Background:  Sperm DNA injury is one of the common causes of male infertility. Folic acid deficiency would increase the methylation level of the important genes, including DNA double-strand Break(DSB) repair pathway. This study aims to preliminarily investigate the underlying mechanism on how the abnormal methylation caused by folic acid deficiency aggravates sperm DNA damage. Methods:  Through reduced representation bisulfite sequencing(RRBS), Global DNA methylation of sperm of patients in the low folic acid group and the high folic acid group was analyzed, and differential methylation genes were selected. Animal models and cell line were applied to validate the mechanism and function of the genes of interest. Findings: We analyzed the correlation between Seminal plasma folic acid concentration and semen parameters in 157 infertility patients and 91 sperm donor volunteers, and found that there was a significant negative correlation between seminal folic acid concentration and sperm DFI (r=-1.495, p<0.001).The result of global DNA methylation of sperm findings that the methylation level in Rad54 promoter region increased in the folic acid deficiency group compared with the normal folic acid group. Meanwhile, the results of animal model and spermatocyte line(GC-2) also found that folic acid deficiency can increase the methylation level in Rad54 promoter region, increased sperm DFI in mice, increased the expression of γ-H2AX that is DNA injury marker protein, and increased sensitivity of GC-2 to external damage and stimulation. Interpretation:  The study indicate that Rad54 is downregulated by folic acid deficiency via DNA methylation. Funding Statement: This work was supported by the National Natural Foundation of China(81701539) and by National Health and Family Planning Commission's Male Reproductive and Genetic Key Laboratory（KF201803). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: The study protocol was approved by the Reproductive Medicine Institutional Review Board of Tongji Medical College, Huazhong University of Science and Technology. All participants provided an informed written consent.Clinical investigation was conducted in accordance with the Declaration of Helsinki.All animal procedures were approved by the Animal Care and Use Committee of the Huazhong University of Science and Technology.