The Study Of Sperm DNA Fragmentation On The Outcome Of Assisted Reproduction And Its Mechanism

Infertility has become a major reproductive health issue in recent years,afflicting about 15%of couples of reproductive age worldwide.Sperm quality reflects male fertility,and abnormal sperm can lead to male infertility.Conventional semen analysis still has some limitations in assessing male infertility,and routine semen analysis cannot detect cytospermia dysfunction.Germ cells undergo mitotic and meiotic processes to produce haploid sperm.Sperm are highly differentiated male germ cells consisting of a head,midsection and tail.The head contains the haploid genome and sperm DNA and contributes 50%of the embryonic genome.Its integrity is essential for sperm fusion with the maternal genome and accurate transmission of paternal genetic information.Chromatin is highly concentrated in the process of sperm production and maturation.Factors such as in vivo and in vitro may lead to sperm DNA fragmentation and damage,showing increased sperm DNA fragmentation index(DFI).The influence of other aspects has always been a research hotspot of male infertility.With the development of science and technology,the application of transcriptome sequencing technology and protein high-resolution mass spectrometry technology has greatly promoted our understanding of sperm cells.The study of male sterility etiology can provide a reference for male sterility(or male breeding efficiency)of animal genetics and breeding.In this research,the results of routine semen examination,the clinical outcomes of artificial insemination and in vitro fertilization-embryo transfer assisted reproduction,sperm transcriptome and proteome,sperm DNA fragmentation and the impact of sperm DNA fragmentation were analyzed from multiple perspectives.The main results showed as follows:1.In the studies on the clinical outcome of sperm DFI and artificial insemination cycles,the increased sperm DFI has no significant effect on clinical outcomes of IUI,such as biochemical pregnancy rate(P=0.386),clinical pregnancy rate(P=0.433),delivery rate(P=0.456),live birth rate(P=0.484),and pregnancy loss rate(P=1.000).2.Sperm DNA fragmentation index(DFI)was significantly positively correlated with male age(r=0.140,P<0.001),percentage of sperm hyperstaining(HDS)(r=0.171,P<0.001),percentage of immotile sperm(IM)(r=0.171,P<0.001),semen volume(r=0.089,P<0.001)and abstinence days(r=0.07,P<0.001),and significantly negatively correlated with sperm concentration(r=-0.330,P<0.001),percentage of sperm anterior movement(PR)(r=-0.465,P<0.001),percentage of normal sperm morphology(r=-0.324,P<0.001),the rate of sperm non-forward movement(NP)(r=0.08,P<0.001)and the specific sperm movement parameters(including curvilinear velocity(VCL),straight-line(rectilinear)velocity(VSL),average path velocity(VAP),beat-cross frequency(BCF),beat-cross frequency(ALH),etc).3.In the studies on the clinical outcome of sperm DFI and in vitro fertilization embryo transfer cycles,the increased sperm DFI could significantly reduce the delivery rate of fresh embryo transfer cycles(P=0.014),but has no significant effect on the biochemical pregnancy rate(P=0.232),clinical pregnancy rate(P=0.072)and pregnancy loss rate(P=0.098),suggesting that sperm DFI may affect late embryo development.The increased sperm DFI could significantly reduce the biochemical pregnancy rate(P=0.006)and clinical pregnancy rate(P=0.027)of frozen embryo transfer cycles,and had no significant effect on the delivery rate(P=0.074)and the pregnancy loss rate(P=0.919),suggesting that sperm DFI may affect the early embryonic development in frozen embryo transfer cycles.The sperm DFI in the rescue ICSI group was significantly higher than that in the conventional IVF group(P=0.003),suggesting that sperm DFI elevated may affect sperm and egg fusion(normal fertilization).4.The whole transcriptome results showed that compared with the normal DFI group,352 differentially expressed genes(DEG)were identified in the high and normal DFI enrichment analysis found the main differential including endocytosis,p53 signaling pathway,PI3K-Akt signaling pathway,etc.5.The difference in protein expression between spermatozoa of high DFI group and normal group was compared by SWATH technology.A total of 10761 peptides were obtained,of which 2186 are spliced proteins and 4088 are quantifiable peptides,and 1591 are quantifiable proteins.Among 252 differential proteins,124 were significantly increased and 128 were significantly decreased.DNA damage and repair gene DFFA is highly expressed in the experimental group,suggesting that DFFA may play an important role in sperm DNA damage and repair.Preliminary results of protein modification found that post-translational modifications such as ubiquitination,acetylation and lactation may play important roles in sperm DNA fragmentation.6.The combined analysis of transcriptome and proteome data showed that there are 19,970 genes from the transcriptome data,1,591 quantitative expression genes from the proteome data.And the matched genes are 1,548 with matchedrates of 7.75%and 97.30%,respectively.One gene(LRRC47)was increased and 5 genes(CFAP53,UBTD2,DNAH3,CFAP107,CBLN1)were decreased at both transcription and translation levels.The main biological processes of the GO enrichment analysis(after adjustment)of the correlation between the two groups are GTPase activator activity,GTPase regulator activity,ATPase activity,enzyme activator activity,DNA recombination,response to reactive oxygen species,regulation of leukocyte activation.The significantly enriched pathways of the two group correlation KEGG analysis were the p53 signaling pathway,endocytosis,and glycolysis/gluconeogenesis,suggesting that these biological processes and pathways may play important roles in the process of sperm DNA damage and repair.