An Study On The Expression Of SIRT And FSHR Polymorphism In ART Women Of Mongols And Han Nationality

Objective Female ovarian function is directly related to reproductive function.In recent years,the proportion of assisted reproduction mainly due to abnormal ovarian function has shown an increasing trend.The study of genetic polymorphisms affecting ovarian function has become a hot topic of research at home and abroad in recent years.In this study,we propose to use Mass ARRAY technology to investigate the rs2236319 and Follicle Stimulating Hormone Receptor(FSHR)polymorphisms in the SIRT1(Sirtuinl)gene in Mongolian and Han Chinese women undergoing Assisted Reproductive Technology(ART)treatment in Inner Mongolia.We investigated the relationship between the above genetic polymorphisms and ovarian function in women treated with ART,and provided guidelines and protocols for individualized infertility treatment.Materials and Methods1.Materials.A total of 204 blood samples were collected from 53 Mongolian and 151 Han Chinese female patients who underwent Assisted Reproductive Technology assisted pregnancy at the Reproductive Center of the Affiliated Hospital of Inner Mongolia Medical University from September 2019 to November 2021.Routine physical examination,medical history inquiry,sex hormone examination chromosome examination were performed.Patients with primary and secondary amenorrhea,history of pelvic and abdominal surgery,suffering from remaining endocrine diseases and chromosomal abnormalities were excluded.All subjects in the experiment are unrelated,and have lived stably in Inner Mongolia for more than 20 years.The three generations of direct blood relatives are all Han and Mongolian,aged between 20 and 45 years old,and deny the history of radiation and exposure to toxic substances.Detailed information about the patient’s prolotherapy cycle was recorded.5 ml of venous blood was collected on empty stomach after the patient enteredthe stimulation cycle and stored in a refrigerator at-80℃ with EDTA anticoagulant.2.Method.DNA extraction,agarose gel electrophoresis and PCR amplification:Whole blood genomic DNA was extracted using a nucleic acid kit(Tiangen BiochemicalCo.,Ltd.)For all DNA samples,the NanoDrop2000 instrument was used to detect the OD value.The final sample concentrations were obtained between 20-100 ng/μl,OD260/280:2.2～1.6,and OD260/230 values>0.5.The OD260/230 values were>0.6,with no absorption peak at OD230nm.And 1.25%agarose gel electrophoresis was used to detect the integrity of gDNA without serious degradation,and the quality-checked samples were stored at-20 ℃ for backup.MassARRAY SNP typing technology to detect SIRT1 and FSHR polymorphism genotypes:Agena’s Assay Designer 4.0 software was used to evaluate the primer design of multiple SNP loci and design primer synthesis,which were all done by the biological company.DNA samples were extracted and sent to the company for testing.The results of the experiments were analyzed using SPSS statistical software.Haploview software was applied for chain imbalance analysis;t-test,ANOVA,chi-square test,Fisher’s exact probability method,Mann-WhitneyU test,Kruskal-Wallis test and multiple logistic regression analysis were applied for statistical analysis.Results.1.rs2236319 on the SIRTl gene and rs6166 on the FSHR of 204 Mongolian and Han Chinese ART females were genotyped for SNPs using MassARRAY technique,and the raw data and genotyping were obtained using computer analysis software pieces,and good results were obtained for the differentiation of each genotype,and all 204 samples were successfully tested.2.The results of MassARRAY SNP genotyping technique for 204 experimental specimens showed that there were three genotypes of AA,AG and GG for SIRT1 rs2236319,AA,AT and TT for FSHR rs6165,and NN,NS and SS for rs6166.3.The allele frequencies of SIRT1 rs2236319,FSHR rs6165 and rs6166 loci were in Hardy-Weinberg equilibrium,and there was linkage disequilibrium between FSHR rs6165 and rs6166.4.The allele frequencies and genotype frequencies of SIRT1 rs2236319,FSHR rs6165,and rs6166 loci were not statistically different in composition between Mongolian and Han ART female patient groups.There were no significant differences between Mongolian and Han ART women in terms of general clinical data and basal sex hormones and ovarian reserve function.5.The number of eggs retrieved in GG group with rs2236319 site mutation in SIRT1 gene was significantly higher than that in wild-type AA and mutant heterozygous AG groups.The rs2236319 locus of SIRT1 gene may be related to female function.6.The basal FSH level of AA type at Thr307Ala site of FSHR gene was significantly higher than that of TT type and TA type,and the basal FSH level of SS type at Asn680Ser site was significantly higher than that of NN type and NS type.7.The percentage of ovarian hyporesponsiveness occurred significantly higher in FSHR gene Thr307Ala locus follower type than TT type and TA genotype,and the percentage of ovarian hyporesponsiveness occurred significantly higher in Asn680Ser locus SS type than NN type and NS genotype.8.SIRT1 rs2236319,FSHR rs6165,rs6166 gene polymorphisms were not associated with cumulative pregnancy rate.Conlusion.1.There were no significant differences in general clinical information,basal sex hormones,ovarian reserve function,and gene polymorphisms at the SIRT1 rs2236319,FSHR rs6165,and rs6166 loci between Mongolian and Han Chinese women treated with ART.2.Gene polymorphism at the rs2236319 locus of the SIRT1 gene is associated with ovarian responsiveness in women.3.The FSHR rs6165 and rs6166 loci affect the ovarian response in infertility patients and there is a chain imbalance between them.