Altered N6‐methyladenosine methylation level in spermatozoa messenger RNA of the male partners is related to unexplained recurrent pregnancy loss

Abstract Background Understanding the pathogenesis of unexplained recurrent pregnancy loss is paramount for advancing effective treatments. Various biological processes, including spermatogenesis and embryo development, are tightly regulated by N6‐methyladenosine modifications. However, few studies have focused on the impact of sperm N6‐methyladenosine modifications on embryonic development. Therefore, we aimed to study altered N6‐methyladenosine‐mediated messenger RNA methylation modifications in the spermatozoa of male partners from couples experiencing unexplained recurrent pregnancy loss, to identify potential diagnostic markers and explore their potential molecular mechanisms in pregnancy loss and embryogenesis. Methods Methylated RNA immunoprecipitation (MeRIP) sequencing and RNA sequencing were conducted on the spermatozoa of men from couples in the ‘unexplained recurrent pregnancy loss’ group ( n = 6), and the fertility control group ( n = 6). To identify the role of the detected key genes, zebrafish model embryos were studied, and multi‐omics (transcriptomics, proteomics, and metabolomics) analyses helped to explore the molecular mechanism of abnormal embryogenesis. Findings Comparing unexplained recurrent pregnancy loss with the fertility control group, 217 N6‐methyladenosine peaks were significantly upregulated, and 40 were downregulated in the spermatozoa. The combined analyses of spermatozoa‐methylated RNA immunoprecipitation sequencing and RNA sequencing indicated that N6‐methyladenosine methylation and the expression of SEMA5A , MT‐ATP6 , ZNF662 , and KDM4C were significantly different. In zebrafish embryos, the altered expression of the four genes increased embryonic mortality and malformations by disturbing several key signaling pathways and zygotic genome activation. Interpretation This study highlights the paternal epigenome, which could be one of the reasons for faulty embryogenesis leading to pregnancy loss. The N6‐methyladenosine modification, the most prevalent RNA modification, contributes to the exploration and understanding of the paternal epigenome in the maintenance of pregnancy and fetal growth and development. The four genes identified in this study may serve as potential diagnostic markers and elucidate novel molecular mechanisms of embryogenesis.