Research Of Early Abortion With Copy Number Variations

Background and ObjectiveSpontaneous abortion?SA?is a common clinical abnormal pregnancy disease.Recurrent spontaneous abortion in early pregnancy will bring trouble to women and their families.Clinically,spontaneous abortion in early pregnancy accounts for about 15%of the total number of confirmed pregnancies,and the incidence is increasing year by year.The etiology of early pregnancy spontaneous abortion includes chromosomal factors,uterine factors,endocrine factors,immune factors,infectious factors,uterine artery blood flow factors,environmental factors,sperm factors,unexplained early pregnancy spontaneous abortion,the most important disease because of the embryo chromosome abnormalities.The majority of embryos with chromosomal abnormalities are difficult to survive to birth,and the embryos that survive to birth may die within a short time,or have serious genetic defects,which seriously affect the health and quality of life of the children.Whether the karyotype of the previous pregnancy is normal is a predictor of the risk of subsequent abortion.Array_based comparative genomic hybridization?aCGH?is a high efficient molecular karyotype analysis technology developed in recent years,the advantage is that without the cell culture can be directly on the sample DNA testing,high resolution,high throughput,high accuracy,can be a hybridization experiment of whole genome chromosome non_equilibrium mutation detection,to discover the traditional karyotype analysis is difficult to detect the submicroscopic structure of copy number variation,The next generation sequencing?NGS?is developed in recent years,and the sequencing of samples may be carried out through whole_genome scanning and whole_genome coverage testing,so as to analyze the detailed gene map and detect the duplication and deletion of small fragments of chromosomes,so as to achieve higher accuracy.Reviews genetic cause of miscarriages and its related influencing factors,the early pregnancy embryos for genetic causes of spontaneous abortion,and to lead to analyze the related factors of the chromosome abnormality occurs,for a reasonable guide next pregnancy,reduce the recurrence risk of spontaneous abortion,and genetic birth defects prevention is of great significance.At present,the application of aCGH and NGS technologies on embryos at early abortion genome copy number variation?copy number variations,CNV?for testing is still less,the different types of chromosome abnormality in the statistical analysis of the distribution of each chromosome is not much,and lack for pregnant women age factors and correlation analysis of CNV miscarriage embryo.This study aims to apply aCGH technology and NGS technology for pregnant early abortion embryonic tissue genome copy number variation for testing,to detect the genome copy number variation from the distribution of different types of CNV in each chromosome of age,pregnant women with different types of CNV correlation analysis,to explore the distribution characteristics of early abortion of pregnancy embryo CNV,pregnant women age influence on embryo dyeing CNV happen,providing theoretical basis for guiding the next pregnancy,in order to better avoid the risk of future pregnancy recurrent miscarriage and the risk of birth children with chromosomal abnormalities,It also provides a theoretical basis for the selection of the detection technology for the histogenetic etiology of early pregnancy spontaneous abortion.Materials and methods:1.Sources:Research collected in December 2015_March 2019 in prenatal diagnosis center in henan province people's hospital and diagnosed with early pregnancy of spontaneous abortion embryos,excluding the uterus,endocrine,immune factors,infection factors and uterine artery blood flow factors,environmental factors and sperm factors cause miscarriage cases,554 cases with get pregnant early abortion embryos,which in December 2015_December 2018 application of micro array comparative genomic hybridization analysis of miscarriage embryo 433cases,121 cases of aborted embryos were analyzed by second_generation sequencing technology?including 30 cases of pre_experiment in January 2019 and 91 cases of samples from February to March 2019?.All participants have signed informed consent,and this study was approved by the ethics committee of henan people's hospital.2.Experimental methods:The clinical diagnosis of pregnancy in patients with early abortion in outpatient artificial abortion operation negative pressure aspirator aseptic drain villi tissue 5₁5 mg,soak in a sterile PBS centrifugal tube,organization cut into small pieces,add 1.5 ml without RNas trace centrifuge tube,the whole genome DNA extraction organization,using the NanoDrop 2000 DNA quantitatively,and quality control of the sample,the sample DNA restriction digestion.Comparative genomic hybridization?aCGH?analysis:genomic DNA 400 ng,SurePrint G3 Human CGH Microarray Kit,8x60K G4450A?Agilent,USA?chip for enzyme digestion,cy5_dutp and cy3_dutp labeling,purification,hybridization,film washing and other steps.Then,the SureScan Microarray Scanner system was applied to scan the probe signal of the chip,and the results were analyzed by Agilent CytoGenomics 2.9software.NGS:extraction of abortion group whole genome DNA,with high throughput sequencing library construct DNA purification kit?magnetic beads method??berry and kang?of genomic DNA purification,mining with HS Qubit ds DNA assay kit to quantitative DNA samples,build CNV library,in a Real_Time PCR System?Thermo Fisher Scientific American?fluorescence quantitative PCR,use NextSeq CN500 gene sequencing machine for testing samples.The analysis results were retrieved and interpreted against the currently commonly used international public database of genomes and phenotypes.3.Statistical methods:SPSS 18.0 software was used for statistical analysis.The measurement data were expressed as?x±s?,and t test was used for comparison.Risk factors were assessed by?² test,test level?=0.05.Results1.Chromosome abnormality detectionA total of 337 cases of abortion tissues with chromosomal abnormalities were detected in 554 cases of spontaneous abortion tissues in early pregnancy,with an overall abnormal detection rate of 60.83%,238 cases of tissues with chromosomal number abnormalities?42.96%?,and 99 cases of tissues with chromosomal structure abnormalities?17.87%?.There were 433 cases of abortion tissue detected by aCGH technique,259 cases?59.82%?of 200kb or more genome CNV were detected,180cases?41.57%?of abnormal chromosome number,and 79 cases?18.24%?of abnormal chromosome structure.There were 121 cases of abortion tissue detected by NGS technology,and 78 cases of CNV above 100kb were detected,with an abnormal detection rate of 64.46%.There were 58 cases of samples with abnormal chromosome number,accounting for 74.36%of all chromosome abnormalities.There were 200cases of chromosomal structural abnormalities,accounting for 25.64%of all chromosomal abnormalities.2.Distribution of chromosome abnormalitiesChromosomal abnormalities were found in 554 cases involving all chromosomes.The most common type of chromosomal abnormalities was trisomy,accounting for58.75%of all chromosomal abnormalities.Among trisomy abnormalities,the most common type was 16_trisomy,accounting for 26.77%of all trisomy chromosomal abnormalities.X_monomeraccountedfor19.32%ofallchromosome abnormalities.Among the chromosomal abnormalities,chromosome 15 had the highest incidence,with 16 cases in total,accounting for 6.72%of all chromosomal abnormalities.Chromosome structure abnormalities detected by aCGH mostly occurred on chromosome 15?14 cases,3.23%?and X chromosome?9 cases,2.08%?.Chromosome 11,18 and 22 were the most common chromosomal abnormalities detected by NGS.3.Correlation between maternal age and chromosomal abnormalities of embryos induced by spontaneous abortion in early pregnancyIn the 554 cases of spontaneous abortion in early pregnancy,the incidence of chromosome number abnormalities in aborted embryos at different ages of pregnant women showed an increasing trend with the increase of the age of pregnant women,and the incidence of chromosome structure abnormalities did not show an obvious correlation with the age of pregnant women.We carried out statistical analysis on the chromosomal abnormalities detected by aCH technology and NGS technology and the pregnant women's age,respectively.The data of the two groups indicated that the detection rate of chromosome number abnormalities in the early stage of pregnancy in spontaneous abortion tissue increased gradually,while the incidence of chromosome structure abnormality did not increase significantly with the age of the pregnant women.Conclusion1.Chromosome number abnormality is the most important cause of spontaneous abortion in early pregnancy,and chromosome structure abnormality is an important factor of spontaneous abortion in early pregnancy..2.Chromosomal abnormalities of embryos in early pregnancy induced abortion mainly occur on chromosomes 15,16,22 and X.3.There was a positive correlation between maternal age and the incidence of chromosomal abnormalities in embryos,while there was no significant correlation between maternal age and the incidence of chromosomal abnormalities.