Transcriptome analysis of human spermatozoa with different DNA fragmentation index using RNA sequencing

The study is aimed to screen the differential expressed genes (DEGs) related to sperm DNA fragmentation in men and provide reference basis of the sperm selection in assisted reproduction based on DNA fragmentation. We evaluated 60 semen samples from patients with high, medium or low sperm DNA fragmentation index (DFI). Using multicolor flow cytometry, we measured the content of reactive oxygen species (ROS), phosphatidylserine (PS) externalization and mitochondrial membrane potential (MMP) in these sperm samples. The results revealed that the more ROS content and PS externalization were detected in the sperm with higher DFI, but there was lower MMP level in the high DFI sperm. Next, we conducted RNA sequencing (RNA-seq) on 3 groups of sperm samples with high, medium and low DFI. Then, Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were performed on DEGs. Furthermore, we utilized qRT-PCR to validated the significantly DEGs from the RNA-seq assay. The transcriptome results showed a total of 5334 DEGs were found in the sperm sample with high, medium and low DFI. According to GO and KEGG analysis 421 downregulated genes in the high DFI group were related to oxidative stress and spermatogenesis. Thirteen novel genes were also identified that most likely were involved in sperm DNA fragmentation, which were further validated by qRT-PCR. In conclusion, our study suggested that the sperms with highly fragmented DNA were accompanied by down-regulation of a series of genes related to antioxidant and spermatogenesis.