Preliminary Research On The Relationship Between Sperm MtDNA Mutation And Male Infertility

Purpose:By detecting the mitochondrial gene sequences of sperm and blood in men with primary or secondary infertility,and then analyzing the mutation and differences of mtDNA in sperm and blood,the relationship between sperm mtDNA mutations and male infertility was preliminarily explored.Materials and methods:(1)By comparing the quality of NGS sequencing data based on Long PCR and probe capture,a more suitable detection method for detecting full-length mtDNA mutations was determined.(2)Then,the semen and blood samples of 19 male infertile patients from the sample bank of Reproductive and Genetics Hospital of CITIC-Xiang Ya were collected,and their clinical data were collected at the same time.We extracted g DNA from semen and blood samples respectively,and then used the obtained g DNA to construct an NGS library.Finally,the NGS library was hybridized and captured with mtDNA probes,and then the mtDNA NGS library was obtained,which was sequenced using a next-generation sequencer(Illumina-Mi Seq).Results:(1)Compared with full-length mtDNA amplified by Long PCR,the sequencing results of mtDNA captured by CHRMT probes have better uniformity,coverage and accuracy.(2)There was a statistically significant difference between the number of mtDNA mutations in sperm of infertile men and the number of mtDNA mutations in blood(P<0.001).(3)The mtDNA mutation in sperm of infertile men was statistically significantly different at different ages(P<0.01),and the mtDNA mutation in the blood of infertile men was statistically different under different BMI.difference(P<0.05).(4)A total of 792 variants were detected in the non-coding control regions,r RNA genes,t RNA genes and polypeptide coding regions of mtDNA of study participants,and most of them were transition mutations.38 of these variants were assessed as pathogenic.According to literature reports,there were 10 variants screened in ND3,ND5,ND6 and CYTB genes that may be associated with male infertility.Conclusions:CHRMT probe capture is a more optimized strategy for the detection of mtDNA mutations.The mutation sites of sperm mtDNA were statistically significantly more than those of blood mtDNA.The mutation of sperm mtDNA in infertile men may be related to age,and the mutation of sperm mtDNA is more serious in infertile men older than 30 years.There were 792 variants that detected in sperm and blood of infertile men.Compared with blood,more pathogenic variants and loci that may be associated with male infertility were found in sperm mtDNA.The differences in mtDNA mutations in cells from different tissues suggest that mtDNA mutations in sperm may be more direct and more important in the effect on male infertility than blood.