| Objective: based on the increasing problem of male infertility,the research on gene polymorphism has become a hot spot.High resolution melting analysis(HRM)as a practical convenient and accurate molecular technique for genotyping,has attracted more and more attention.This study intends to use HRM technology to study the gene polymorphism of c.919A>G(rs6165)and c.2039A>G(s6166)on follicle-stimulating hormone receptor(FSHR)in infertile men in Inner Mongolia.To explore the relationship between infertility and FSHR gene polymorphism in infertile men.Materials and Methods.1.Materials.In a case-control study,176 blood samples of male infertility and 80 normal male blood samples were collected from the Reproductive Center of the affiliated Hospital of Inner Mongolia Medical University from November 2017 to November 2019.After routine physical examination,medical history inquiry,semen examination and chromosome examination.Patients with obstructive azoospermia,varicocele,mumps and chromosome abnormalities were excluded.All the subjects in the experiment were unrelated,aged between25 and 35 years old,and denied the history of radiation and exposure to toxic substances.According to semen quality,they were divided into four groups: oligospermia,asthenospermia,azoospermia and normal.Venous blood was collected on an empty stomach and ethylenediamine tetraacetic acid anticoagulant was stored in the refrigerator at-80 ℃ for a long time.2.Method.Whole blood DNA extraction and detection using nucleic acid kit(Shanghai Baiao Science and Technology Co.,Ltd.)to extract whole blood genomic DNA.The purity of DNA was determined by ultraviolet spectrophotometer and the concentration of DNA was determined by OD260.Finally,the concentrations of samples were between 30-100ng/ml and OD260/280:1.60-1.85,which met the requirements of conventional PCR operation.The extracted dna samples were frozen at-80 ℃ without repeated freeze-thaw cycles until testing.High resolution dissolution curve(high resolution melting analysis,HRM)was used to detect FSHR polymorphic genotypes.Primers were designed and synthesized according to references.Primer synthesis was completed by biological companies.The HRM reaction system was established,the pre-experiment was carried out,and the amplified samples were sent to the company for sequencing,the results were completely consistent with the sequencing results,and routine experiments were carried out.The experimental results were analyzed by SPSS statistical software and SHEsis online software.Results.1.The HRM dissolution curves of the pre-experimental samples and 20 infertile patients at sites 919 and 2039 were identified,and the computer analysis software successfully separated them into different curves.According to the results of routine experiment,the Tm values and curves of different genotypes were significantly different,and the effect of distinguishing each genotype was good.All the 176 cases and 80 controls were detected successfully.2.The sequencing results of 20 HRM pre-experimental samples and 1Mel 20 experimental samples showed that there were three genotypes of GG,AA and GA in 919 and 2039,and the coincidence rate was 100% with the HRM test results of the same samples.3.There was no significant difference in the allele frequencies of 3.FSHR c.919A>G(rs6165)and c.2039A>G(rs6166)between the experimental group and the control group.There was no significant difference in the composition of AA,AG and GG genotypes among the experimental groups,and there was no significant difference among the haploids.4.The allele frequencies of 4.FSHR c.919A>G(rs6165)and c.2039A>G(rs6166)loci were in accordance with HW equilibrium,and there was linkage disequilibrium among all genotypes between the experimental group and the control group.Conclusion:1.HRM is a reliable method for genotyping,which can be used to detect nucleotide polymorphism of FSHR.2.No association was found between c.919A>G(rs6165)and c.2039A>G(rs6166)gene polymorphisms and male infertility. |
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