| Objective: The aim of this study was to provide a theoretical and experimental basis for the genetic diagnosis / treatment of non-obstructive azoospermia.Based on the study of high-throughput sequencing,the molecular genetics method was used to verify the association between MTRR,PIWIL1,TAF4 B gene polymorphism and non-obstructive azoospermia in Northeast China Han population.Methods: The object of this study were patients who diagnosed with non-obstructive azoospermia between January 25,2013 and February 26,2016 in First Hospital of Jilin University reproductive center.Inclusion criteria:(1)G-karyotype analysis was normal(2)Y chromosome detection did not report microdeletion(3)No history of high fever recently(4)No long history of exposure to radiation(5)No history of mumps(6)Varicocele No genital trauma,a total of 121 patients were screened by criteria.Collected from October 14,2014 until November 27,2015 to Jilin Province,human sperm bank sperm donation volunteer as the control group,Inclusion criteria same with the NOA test group,a total of 256 cases.The semen routine information of the study group and the control group were analyzed and recorded.The level of various genital hormones in the subjects was measured by commercialization test kit and the results were analyzed by electrochemiluminescence;Routine chromosome karyotype analysis and Y chromosome AZF gene deletion were performed on the subjects;In this study,we used the DNA kit to extract the peripheral blood DNA and LDR-PCR technique to detect and classify the polymorphic loci,Sequencing results were analyzed by 3730 sequencer.the Statistical method was chi-square test and binary logistic regression analysis.The SNPs were analyzed by haplotype software.SPSS19.0 for Statistical analysis.Results are as follows:1.The mean age and BMI of 121 patients with NOA were significantly higher than those of the control group(30.00 ± 5.69 vs25.35 ± 5.51,25.31 ± 5.13 vs 22.89 ±3.83);the levels of FSH,LH and PRL in serum were significantly higher than those in the control group(19.42 ± 11.62 vs 3.35 ± 1.58,9.17 ± 5.20 vs 4.96 ±2.05,409.28 ± 215.19 vs 305.11 ± 168.37).The levels of fructose,α-glucosidase and seminal plasma zinc in seminal plasma were significantly higher than those in control group(66.47 ± 57.96 vs 20.40 ±12.88、41.25±43.95 vs 30.32±18.99、7.45±5.31 vs 2.91±2.88)Sperm density,semen volume,serum T level and serum B level were significantly lower than those in the control group(0 vs 66.88 ±11.11,2.74 ± 1.49 vs 3.69 ± 11.11,1.29,12.46 ± 6.90 vs 17.72 ± 6.43,51.90 ±84.52 vs200.93 ± 71.27).There was no significant difference in PH and serum E2 between the two groups.2.The results of relevance analysis between polymorphism and NOA are as follows.NOA group : the MAF value of MTRR(rs162036),PIWIL1(rs1677016),MTRR(rs161870),TAF4B(rs1106042)is 19.83,86.32,19.83,11.36,PHWE value is 0.314,0.335,0.314,0.691;Control group : MAF value is18.35,82.42,18.35,8.40,PHWE value is 0.568,0.740,0.568,0.142,There was no statistically significant difference between the two groups after Chi-square test and binary logic regression analysis。(p=0.629,0.171,0.629,0.205;0.291,0.264,0.291,0.329).3.Analysis of the relationship between NOA-related SNPs and genetic implication by binary logistic regression analysis。The results showed that the P values of the four loci(rs162036,rs1677016 R,rs161870,rs1106042R)were 0.995,0.520,0.989,0.395(p>0.05).There was no significant difference between the two groups.4.Rs162036,rs161870 form a haploid block with a coverage of about 7 kb on chromosome 5.The phenotype was TA,CG and the P value of haplotypes analysis were 0.629,0.629.There was no significant difference between the two groups(p> 0.005).5.The following results were obtained by analyzing the association between the four SNPs and the reproductive hormone and seminal plasma biochemical levels.Results of the prolactin study group(rs162036,161870):MAF / PHWE = 8.70 /0.448(case);MAF / PHWE = 18.10 / 0.292(control);p-value between groups was0.014(p<0.05).Results of the fructose study group(rs162036,161870):MAF /PHWE = 34.60 / 0.494(case);MAF / PHWE = 12.20 / 0.485(control);p-value between groups was 0.002(p<0.05).The p value of other sites between two groups was greater than 0.05,no statistically significant difference.Conclusions:1.Our results suggest that age and body mass index abnormalities are closely related to non-obstructive azoospermia.2.This study suggested that rs162036,rs161870(MTRR),rs1106042(PIWIL1)and rs1677016(TAF4B)polymorphisms in Han population of Northeast China had no significant correlation with non-obstructive azoospermia.3.Our results suggest that rs162036,rs161870(MTRR)polymorphisms may lead to changes in reproductive hormone PRL levels and then feedback to regulate spermatogenesis..4.The results of this study suggest that rs162036,rs161870(MTRR)polymorphisms are associated with elevated levels of fructose in patients with non-obstructive azoospermia. |
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