| Objective Infertility,defined as the absence of pregnancy after 12 months of regular unprotected intercourse,affects approximately 8%-12% of couples of reproductive age worldwide.In recent years,with the continuous development of assisted reproductive technology(ART),many infertile families have achieved pregnancy.Successful pregnancy requires normal meiosis to produce normal gametes,in which genetic factors play a crucial role.Any abnormal expression of genes involved in meiosis will lead to defective gametes and subsequent abnormal embryo development.Therefore,patients with idiopathic ovarian function decreased and non-obstructive azoospermia(NOA)were included in this study.Using whole exome sequencing(WES)and strict bioinformatics technology to find the pathogenic genes of related diseases and track the outcome of ART in these patients,in order to provide new targets for gene diagnosis,genetic counseling and individualized treatment for these patients.Methods The subjects included total 60 male and female infertility patients with ovarian function decreased and NOA phenotypes from the Reproductive Center of the First Affiliated Hospital of Anhui Medical University.Through strict medical history inquiry,physical examination,laboratory examination,imaging examination,and testicular biopsy,non-genetic causes that may lead to decreased ovarian function and nonobstructive azoospermia were excluded,and then the peripheral blood of these patients was subjected to WES.First,we screened the original sequencing data,removed the highfrequency variants,and evaluated the pathogenicity of the remaining variants to retain pathogenic and possibly pathogenic variants.Secondly,we performed a comprehensive analysis of various databases to verify whether the known or candidate genes of highly suspected pathogenic genes were consistent with family co-segregation by Sanger sequencing to clarify the family origin of gene variations.In addition,minigene assay was used to verify the pathogenicity of splicing site variations in vitro,and SWISSMODLE software was used to predict the impact of frameshift-deletion variations on protein structure.In order to verify whether the gene variation caused abnormal embryo development,we detected the copy number of the patient’s embryo and the products of abortion after embryo arrest for the presence of abnormal chromosomes,and verified their origin by base contrast.In terms of clinical treatment,we tracked the outcome of ART in these patients to determine whether patients with gene variations could achieve successful pregnancy through ART.Results WES results of 60 male and female infertility patients with the main phenotypes of ovarian function decreased and NOA showed that one patient with Poor ovarian response(POR)and one patient with NOA proband carried homozygous variation of HFM1(F1 II-1 and F1 II-2 nm＿001017970.6: c.1730-1G>T).Two probands with DOR carried homozygous variations of MSH4(F2 II-1 NM＿002440.4: c.2220＿2223del,p.K741Rfs*2;F3 II-1 NM＿002440.4: c.2712 del G,p.D905Ifs*9).In addition,female patients with splicing variation of the HFM1 can obtain high-quality and poor-quality blastocysts during the ART cycles,and exhibit the phenotype of RIF after multiple transplantation.For the splicing variation of the HFM1,in vitro minigene results showed that this variation would lead to abnormal alternative splicing.Through copy number variant sequencing,we found that the embryos of female patient diagnosed with POR of HFM1 biallelic variation had euploidy and aneuploidy.However,these embryos have chromosomal microduplication from the mother.The male patient diagnosed with NOA with HFM1 biallelic variation had testicular puncture indicating spermatogenesis arrest,and his partner finally had a successful pregnancy with the help of artificial insemination by donor semen.For MSH4 frameshift deletion variations,SWISS-MODLE software was used to assess the protein structure changes caused by this variant,and two women were unable to get pregnant by ART.Conclusion(1)Biallelic variation of HFM1 may have different pathogenicity due to different variation sites,resulting in different ovarian reserve,which can lead not only to POI but also to POR in women,and manifest as recurrent implantation failure(RIF)in ART cycles.Our study revealed the potential risk of chromosomal abnormalities underlying the RIF phenotype.Clinically,patients with biallelic variation of HFM1 who can obtain embryos during the ART cycle are advised to perform preimplantation genetic diagnosis before transplantation to reduce the risk of RIF.(2)Novel biallelic variation of MSH4 lead to DOR and poor ART outcomes in female patients.The pathogenicity of biallelic variation of MSH4 should be fully evaluated before initiating ART in female patients. |
