Screening Of Genetic Variation In Non-obstructive Azoospermia

Infertility has been a major health problem in the world.Infertility affects approximately 10%-15% of couples wordwide,and the incidence of infertility also exceeds 10% in China.Among these couples,male factor accounts for approximately 30%.Spermatogenic impairment has become the major cause of male infertility.The common genetic factors that cause Spermatogenic impairment include: Y chromosome microdeletions,autosomal structure abnormalities,X chromosome linkage abnormalities,and gene mutations.Y chromosome microdeletions are responsible for account 6%-18% of Spermatogenic impairment caused by genetic factors.Similarly,the abnormal number of sex chromosomes such as Klinefelter's syndrome also is a common cause of Spermatogenic impairment.Single nucleotide variations(SNVs)were found also the factors of Spermatogenic impairment in recent years.Previous study,we also found that some single nucleotide polymorphisms(SNPs)were significant associated with non-obstructive azoospermia(NOA)by genome-wide association studies(GWAS).Nevertheless,known genetic factors are responsible for approximately 20% of cases of NOA,and there are still nearly 80% of factors for NOA is unknown that is referred to as “idiopathic” NOA.Part ? Screening and mechanism of genetic variation in the whole exon of non-obstructive azoospermiaNon-obstructive azoospermia is the most severe manifestation of spermatogenic impairment,accounting for about 1% of adult men.Spermatogenic impairment are closely related to environmental chemical contaminants,genetic factors,and abnormal epigenetic modifications.Genetic factors explain 30% of spermatogenic impairment.In the past,we screened genetically-related disease-causing genes mainly dependent on family linkage analysis,but it could not accurately locate pathogenic sites,and result some pathogenic mutation being ignored.But,we combined pedigree with highthroughput sequencing can make up for the inferior of linkage analysis.Therefore,we are based on the pedigree of NOA,and screening the rare genetic mutations and de novo mutations associated with NOA by performing whole exome sequencing.Combining genetic models,we constructed a point mutation mouse model by CRISPR/Cas9 system.First,we performed whole exome sequencing(WES)to the pedigree of NOA and the pathogenicity of mutation was predicted by SIFT,Mutation Taster,Polyphen-2,Ex AC,1000 Genomes,HGMD,and other normal population/disease databases.Combinat with the genetic model,there are two rare genetic variation COG5 chr7:106924058 G>A and NLRP14 chr11:7091668 T>G may be related to NOA.Furthermore,we have tested the function of Cog5 through mouse germ cell and found that knockdown of Cog5 could affect the cycle and proliferation of germ cells.NLRP14 has been reported associated with NOA and is highly expressed in the testis.Thence,we not performed functional verification in vitro.Subsequently,we constructed a two point mutation mouse model by CRISPR/Cas9 technology to further explore its possible mechanism in sperm production.Part ? Screening and Mechanism of X-chromosome Genetic Variation in Non-obstructive AzoospermiaSpermatogenesis is a continuous process of cell division and differentiation.Traditionally,Y chromosome play a decisive role in this complex process.However,recent studies have found that many X chromosome-linked genes do not play a role in women,but they are important for maintaining normal male reproductive function.To some extent,its can influence male gender formation.In males,the X-linked gene is usually subjected to special evolutionary pressure because of the hemizygous state of the X chromosome.Therefore,X-linked genetic diseases are significantly higher in men.In the previous study,We have completed the exomes sequencing of the X chromosome in 96 cases of NOA and 96 controls.Through bioinformatics analysis and SSC functional experimentation,we have included two genes in the follow-up study.CRISPR/Cas9 technology was used to construct a model of gene knockout mouse,and analysis the function of candidate genes from animal levels.The results showed that Bcorl1 knockout mice were infertile,CASA analysis revealed a significant decrease in sperm count and sperm motility.The results of sperm transmission electron microscopy revealed abnormal sperm mitochondria structure.Therefore,we speculate that Bcorl1 affect male fertility may though spermatogenesis and sperm motility.The implementation of the study will provide new ideas and strategies for the pathogenesis,clinical diagnosis and treatment of NOA patients.