| Objective:The genetic cause of non-obstructive azoospermia(NOA)has not been fully elucidated.The study explored the possible genetic causes of NOA patients,analyze the incidence and the genetic pathogenesis of clinical genetic causes.The study further screened genetic variation in NOA by using target gene capture high-throughput sequencing technology,and to provide theoretical and experimental basis for the future clinical diagnosis of non-obstructive azoospermia.Methods:This study was a retrospective study.Male patients,who were diagnosis with infertile in center for reproductive medicine and center for prenatal diagnosis of the First Hospital of Jilin University,were recruited from September 2013 to April 2017.NOA was diagnosed for each patient after routine clinical semen analysis,seminal plasma biochemical analysis or testicular fine needle puncture cytology.The case group andcontrol group were included in the study of high-throughput sequencing studies for target gene capture.The case group was the patients with NOA,who excluded chromosomal abnormalities and AZF microdeletions.The participants of control group were collected from January 2014 to December 2016 to human sperm bank of Jilin province,and were confirmed to be fertility with normal semen parametersm,and meet the following criteria:(1)no recent history of high fever,long-term exposure to radiation;(2)no history of mumps,major systemic history of disease,history of genital trauma,history of radiotherapy and chemotherapy,etc.;(3)testicular examination normal;(4)no severe(3 degrees)varicocele;(5)routine analysis of semen were in the normal range;(6)karyotype analysis was normal;(7)The AZFa,AZFb,and AZFc regions of the AZF geneexists in chromosome Y.The questionnaire collected the related information of the study subjects,and recorded clinical information such as varicocele.The subjects were abstinent for 3 to 7 days,and the semen was taken by masturbation.After complete liquefaction,it was directly used for routine semen analysis.If the sperm density is 0,a microscopic examination is performed after centrifugation.It is usually clinically excluded from patients who have not seen sperm after three times of centrifugation and no semen after retrograde ejaculation.The seminal plasma biochemical is detected by the quantitative detection kit for the berry sugar,the neutral ?-glucosidase quantitative detection kit,and the seminal plasma zinc quantitative detection kit.Serum follicle stimulating hormone(FSH),luteinizing hormone(LH),estradiol(E2),pituitary prolactin(PRL),testosterone(T)were detected by using electrochemiluminescence detection.Inhibitor B(INHB)was detected by using enzyme-linked immunosorbent assayt.Karyotype analysis was detected acorreding to routine peripheral blood inoculation culture G banding.AZF microdeletion detection was used for 9 sites of s Y84,s Y86,s Y127,s Y134,s Y143,s Y152,s Y254,s Y255,s Y157 by multiplex PCR.The SRY gene was detected by PCR and fluorescence in situ hybridization,respectively.The SOX9,DAX1,and WNT4 gene variants were detected using a first-generation sequencing method.The marker chromosome was detected by applying to the chromosome microarray chip.Target gene capture sequencing high-throughput sequencing technology was used to detect related genes with NOA,and statistical analysis was used to study the relationship between single nucleotide variation and NOA.Results:One thousand and seventy-five cases of NOA were collected in this study.A total of 175 chromosomal abnormalities were detected,accounting for 16.3% of all NOA patients.A total of 74 AZF microdeletions were detected,accounting for 6.9% of all NOA patients.12 patients with AZF microdeletions accounted for 1.1% of all NOA patients;838 patients(77.9%)were not diagnosed clinically.Among 135 cases of sex chromosome abnormalities,Klinefelter syndrome was the most common,95 cases(70.3%,95/135);followed by sexual reversal(46,XX male)9 cases(6.7%,9/135),sex chromosome chimera 5 cases(3.7%,5/135),4 cases of XYY syndrome(3.0%,4/135),3 cases of abnormality of Y chromosome structure(2.2%,3/135),1 case of male Turner syndrome(0.7%,1/135),1 case of sex chromosome-autosomal balance translocation(0.7%,1/135).In 9 cases of 46,XX male patients,8 cases had SRY gene,and all translocated to the short arm end of X chromosome;1 case of SRY negative patient detected a mutation in exon 1 of DAX1 c.557G>A.For patients with 47,X,i(Y)(q10),+mar,it was further found that the marker chromosome is actually i(Yp).Among 40 cases of autosomal abnormalities,chromosomal abnormality included Yqh±17(29.8%),1,9,16qh±15(37.5%,15/40),D/G group with 13 cases(32.5%,13/40),inv(9)7 cases(17.5%,7/40),4 cases of balanced translocation(10%,4/40),and 1 case of pericentric inversion(2.5%,1/40).Further studies and comments on the clinical characteristics of male balanced translocation carriers involving chromosome 6 found that the 6q15 breakpoint is closely related to pre-gestational infertility,and other breakpoints are associated with gestational infertility.In 74 patients with AZF microdeletions,5 cases of AZFa deletion,8 cases of AZFb deletion,23 cases of AZFc deletion,21 cases of AZFb+c deletion,1 case of AZF part b deletion,1 case of AZF part c deletion,AZFc+ part b deletion 2 For example,13 cases of AZFa+b+c are missing.In addition,a total of 12 chromosomal abnormalities were accompanied by AZF microdeletions.Many gene data were provided after target gene capture high-throughput detection technology.A total of 36,929 mutations were detected in the NOA group in the exon region,and 43135 mutations in the control group.70,328 mutations were detected in the NOA group in the intron region.There were 107,041 mutations in the group.Among the known variants,frameshift(NOA group 0.3% vs.control group 0.23%),nonframeshift mutation(nonframeshift)(NOA group 0.39% vs.control group 0.53%),none the sense of stopgain(0.12% vs.0.09% in the NOA group)and splicing(1.57% vs.0.88% in the NOA group)were statistically significant(p<0.05).The unknown variation detected was significantly lower in the NOA group than in the control group(71.87% vs.73.55%,p< 0.001).The 282713 variants distributed in the exon region included 80064 mutations,of which 72272 were SNV mutations,covering 2189 sites in 441 male infertility-related genes.In this study,the screening sites with a frequency of more than 60%(n>120)were statistically calculated,and a total of 131 loci of 88 genes were obtained.In the equilibrium analysis of Hardy Weinberg between the two groups,the distribution of 131 genotypes was consistent with Hardy Weinberg equilibrium,and the minimum allele frequencies of 131 spots in the two groups were statistically different(p<0.05).There were statistical differences between 116 SNV loci in the two groups,which could further explore the correlation between SNV locus and NOA,.In the genotypic dominant model,there were statistically significant differences in 64 SNV loci(p< 0.05).Meanwhile,in the genotypic recessive model,65 SNV loci were statistically different in the NOA group and the control group(p< 0.05).Further analysis of the correlation between SNV and NOA,haplotype correlation analysis was performed on SNV sites located on the same chromosome,and 61 SNV loci were screened and distributed on 19 chromosomes.In summary,21 SNV loci were closely related to NOA,which were located in 12 genes,CHD5,APOB,,PMS2,GTF2A1 L,TXNDC2,LHCGR,SIRPG,ANKS1 A,CLCA1,NOTCH1,,PTGDS,SLC9C1 genes.Conclusions:(1)Chromosomal abnormalities were detected in 16.3% cases with NOA,and AZF microdeletions of Y chromosomes were 6.9%.77.9% of patients were unable to be diagnosed in the clinic.Klinefelter syndrome is the most common chromosomal abnormalities in patients with NOA,AZF microdeletionswere in the carriers of chromosomal abnormalities with NOA.(2)The positive of SRY gene was in 88.9% of the male patients with 46,XX,and translocated to the chromosome Xp.DAX1 gene mutation was found in a case of SRY gene negative patients.The breakpoint 6q15 on chromosome 6 translocation is closely related to pre-gestational infertility.(3)116SNV loci were screened for NOA.Among them,8 genes include CHD5,APOB,PMS2,GTF2A1 L,TXNDC2,LHCGR and SIRPG are closely related to NOA.(4)The study also found that the 13 sites of ANKS1 A,CLCA1,NOTCH1,PTGDS and SLC9C1 genes were associated with NOA,but further research is needed.(5)61 SNV loci were screened through allele and genotype dominance and recessiveness analysis from 131 loci with statistical differences in minimal allele frequencies in the inter-group control study.46 SNV loci formed monosomic blocks,which were respectively distributed on 14 chromosomes of 1,2,3,6,7,9,18 and 20.The genetic effects of these haplotype blocks on NOA phenotype need to be further verified. |
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