Association Of GRP 78 Promoter Polymorphisms And Serum GRP 78 Level With Risk Of Asthenozoospermia

Background: Infertility,a common clinical problem,is defined as the inability to conceive naturally without any contraceptive measures within one year,and affects about 13~15% of couples of child-bearing age worldwide.According to the investigation of the World Health Organization(WHO),the incidence of infertility is increasing,affecting human reproductive health.Male infertility,which accounts for about half of all infertility cases,is caused by a variety of factors,ranging from complete absence of sperm in the testicles to significant changes in sperm quality.With the popularity and development of various male infertility tests,some identified causes of male infertility include: anatomical structure defects,sperm abnormalities,molecular genetic disorders,urogenital tract infections,endocrine disorders,genetic variation or environmental toxins.Asthenospermia,also known as low sperm motility,is one of the important factors of male sterility,and forward progressive motility is less than 32%.Asthenospermia is caused by infections,immune abnormalities,long-term abstinence,unhealthy lifestyles and genetic factors.However,the genetic causes of asthenospermia are still relatively unknown.In recent years,studies have found that microRNA expression characteristics,SNP correlation and proteomics are becoming more and more important for the occurrence and development of asthenospermia,and these findings provide clues for the pathophysiological basis of asthenospermia.This study explores the genotype distribution of GRP 78 gene polymorphism in patients with asthenospermia and normal men,and further analyzes the relationship between genotype and sperm motility,which is helpful for understanding of the mechanism of asthenospermia.It is of great significance for the diagnosis and treatment of patients with asthenospermia.Objective:Single Nucleotide Polymorphisms(SNP),the most common human genetic variation,is a DNA sequence Polymorphisms caused by mutations of a Single Nucleotide.SNPs can be used to explain the phenotypic differences in the susceptibility of different groups and individuals to diseases,especially to complex disease or environmental factors.The aim of this study was to investigate the distribution of the rs3216733,rs17840761 and rs17840762 polymorphisms in the promoter region of the glucose-regulated protein 78(GRP 78)gene in males with normal sperm motility(control)and asthenospermia,and to compare the distribution of genotypes and allele frequencies between the two groups.In addition,in order to further clarify the relationship between GRP78 polymorphism and the risk of asthenospermia,and to analyze the relationship between genotypes and sperm motility parameters,and the effect of GRP 78 gene SNPs on serum GRP78 protein expression.Method: The semen,whole blood and serum samples of the subjects were collected.Semen quality analysis was performed using Computer-aided sperm analysis(CASA),and Snapshot SNP genotyping assays was applied to GRP 78 rs3216733,rs17840761 and rs17840762 polymorphisms.The corresponding statistical method was to assess a link between gene polymorphism and asthenospermia.The serum GRP 78 level was measured by enzyme-linked immunosorbent assay(ELISA).Results: The study included 400 asthenospermia infertile men and 400 healthy men with a history of fertility.The percentage of forward progressive motility in the asthenospermia group and the control group was(20.09±8.18)% and(57.16±13.45)%,respectively,the two groups differ in forward progressive motility(P<0.001).There were three genotypes of dd,Gd and GG in the rs3216733.The frequencies in the control group were 46.5%,43.7%,9.8%,respectively,and the asthenospermia group were 38.0%,49.8%,12.2%,respectively;There were CC,CT and TT genotypes at rs17840761.The genotype frequencies in the control group were 27.3%,45.2% and 27.5%,respectively,and those in the asthenospermia group were 28.0%,47.5% and 24.5%,respectively.The CC,CT and TT genotypes were present at rs17840762.The genotype frequencies in the control group were 74.3%,23.0%,and 2.7%,respectively,and the asthenospermia group was 75.0%,23.5%,and 1.5%,respectively.In rs3216733,the frequences of Gd,Gd/GG genotype and G allele were statistically significant in two groups.(Gd vs.dd : OR=1.42,95%CI,1.06-1.93,P=0.020;Gd/GG vs.dd: OR=1.43,95%CI,1.08-1.91,P=0.013;G vs.d: OR=1.26,95%CI,1.03-1.56,P=0.027).However,this study has not confimed the difference in genotype and allele frequencies between the two groups of rs17840761 and rs17840762(P>0.05).The haplotype analyses showed that the G-C-C haplotype was 33.9% and 29.2% in both cases and controls,respectively.The G-C-C haplotype significantly increased the risk of AZS compared to the control group(P=0.026).We also compared the association of sperm motion kinetic parameters(progressive motility,curvilinear velocity,straight linear velocity and linearity)with different rs3216733 genotypes.In rs3216733,asthenospermia patients with Gd,Gd/GG genotypes showed significantly lower progressive motility,curvilinear,straight linear velocity and linearity compared with dd genotypes(P<0.01).In addition,The concentration of GRP 78 in the serum of AZS patients(0.479±0.104 ng/ml)was significantly lower than that in controls(0.661±0.225 ng/ml,P<0.001).Besides,we also found that patients carrying rs3216733 Gd/GG genotypes(0.414±0.069 ng/mL)had lower levels of GRP 78 than carrying dd genotype(0.558±0.082 ng/ml,P<0.001).Conclusion: In summary,our findings suggest that rs3216733 polymorphisms of GRP 78 is correlated with the pathogenesis of asthenospermia.The rs3216733 Gd/GG genotypes may reduce sperm motility by down-regulating GRP 78 protein expression,thus leading to the occurrence of asthenospermia.