Application Of Chromosomal Microarray Analysis In Early Spontaneous Abortion

Objective: 1.To explore the value of applying the Chromosomal Microarray Analysis(CMA)technique in early spontaneous abortion and the causes of early spontaneous abortion.2.To analyze the influence of factors of both maternal age and embryo gender on early spontaneous abortion.As well as to explore the relationship between the age of women who had a spontaneous abortion,the sex of spontaneous abortion embryos and various chromosomal abnormalities.Methods: 1.A total of 335 patients(age from 22 to 44)who were diagnosed as stoping the development of the embryo by ultrasound at 6~12 weeks' gestation from Guangzhou Women and Children's Hospital from January 2014 to January 2017 were elected.Both chorionic villi and peripheral blood were collected for the test from these patients 2.As per the manufacturers' protocol,genomic DNA(g DNA)was extracted from the villous tissue and peripheral blood of patients with abortion,and the concentration and purity of the extracted g DNA were measured.3.Fluorescence quantitative polymerase chain reaction(QF-PCR)was used to preliminarily detect whether or not the aborted villous samples were 13-trisomy,18-trisomy,21-trisomy or sex chromosome X monomers.QF-PCR is to select 2-4 short tandem repeats(STR)on sex chromosomes 21,18,13 and sex chromosomes so as to use this as a target sequence to design and synthesize primers and then label different fluorescent dyes.The human g DNA in the sample was used as a template for PCR amplification.Capillary electrophoresis was perfromed after PCR amplification.Different gene peaks of STR loci were obtained after the electrophoresis results were analyzed.The number of each gene peak or the ratio of the area of the allele peaks was examined to determine whether there are abnormalities in 21,18,13 and the sex chromosomes.Meanwhile,the peripheral blood samples of pregnant women were detected and the STR peak comparison was used to determine whether the fetal samples were contaminated by the maternal tissue cells.4.According to the Cyto ScanTM 750 K Array manufacturer's protocol,the g DNA extracted from chorionic villi were tested by the CMA technique.The intensity(CEL)file data was created after scanning the Array chip and then ran the Csh AS software to analyze the probe level on CEL file data.5.In order to determine the nature of CNVs,the result of CNVs detected by using the CMA technique was compared with online databases,such as the database of DECIPHER,OMIM,DGV,UCSC,CAGdb,and ISCA.6.Peripheral blood samples from both husband and wife would be required for karyotype analysis when CMA results of aborted villi indicated cases of chromosome deletion or duplication of more than 5M.Results: 1.Results tested with QF-PCR: 33 samples were unqualified in the 335 cases.7 samples were found maternal tissue contamination after being tested by the QF-PCR technique.In the 295 cases which underwent QF-PCR test,chromosomal abnormalities were found in 76 of 295 cases,and the rate is 25.8%.There were 5 cases of abnormal 13-trisomy,5 cases of abnormal 18-trisomy,16 cases of abnormal 21-trisomy,28 cases of numeral abnormalities of abnormal sex chromosome and 22 cases of polyploidy.2.Results tested with CMA: All the 295 qualified samples were successfully tested by the CMA.96 cases were found chromosomal normals or chromosomal polymorphisms,a rate of 32.5%;199 cases were defined chromosomal anomalies,rate 67.5%.In these anomalies,165 cases were numeral abnormalities,rate 82.9%,most of which were aneuploid(134/165),such as 16-trisomy,22-trisomy,45 X,etc.16 of the 165 cases were detected involving pathologic CNVs,and 18 of the 165 cases were detected involving VOUS.3.Pathologic CNVs:24 pathologic CNVs sized form 301 kb to 46870 kb,were detected in 18 cases(2 of 18 cases were numeral abnormalities),mainly of which distributed on 3,5,20,22 and X chromosomes.58.3%(14/24)of these pathological CNVs were below 5Mb size large.4 cases were diagnosed with microdeletion syndrome or microduplication syndrome with the comparison of databases of OMIM,ISCA,DECIPHER and Pub Med.10 of these pathological CNVs' sizes were over 5 Mb.2 cases were diagnosed with cri du chat syndrome.4.Variants Of Unknown Significance(VOUS):62 VOUS were mainly distributed on 4,5,6,10 and 22 chromosomes.There were 51 cases with size from 204 kb to 60640 kb,including 4 cases of 45 X,19 cases with autosome trisomy and 1 case of both numeral abnormality and 45 X.Only 18 cases were diagnosed with involved VOUS alone.5.Karyotype of these couples: According to the results of the CMA,exclusively to the polyploids and aneuploids,8 cases were detected with bigger pathological CNVs.We analyze 4 couples' karyotypes.Only 1 couple's karyotypes were translocational,whereas others were completely normal.6.The relationship between the CMA results and the maternal age: in this study,pregnant women with spontaneous abortion were aged between 22 and 44 years with a median age of 31 years.CMA results were used to classify pregnant women with spontaneous abortion into groups with the chromosomal numeral abnormality,pathogenic microdeletion or micro-duplication,VOUS or normal.Among them,the difference of age in the group of pregnant women with either chromosomal numeral abnormality or normality was statistically significant(P=0.008).Furthermore,the incidence of chromosomal numeral abnormality of aborted villi increased with the increase of age after the age of 25,whereas the incidence of VOUS decreased.Surprisingly,the incidence of pathogenic CNVs did not change significantly.7.The relationship between CMA results and embryonic gender: in this study of 295 pregnant women with spontaneous abortion,149 male embryos and 146 female embryos were detected by CMA.The ratio of males to female embryos is 1.02:1.CMA results were used to classify pregnant women with spontaneous abortion into groups with a chromosomal numeral abnormality,a pathogenic microdeletion/micro-duplication,VOUS and normal karyotypes.Comparing the sex differences of embryos in each group,the difference in embryo genders between the normal group and the chromosomal numeral abnormality group was statistically significant(P=0.034).The difference of sex between the chromosomal numeral abnormality group and the VOUS group was statistically significant(P=0.006).Conclusion: 1.Chromosomal aneuploidy abnormality is still the main cause of spontaneous abortion.2.The aging in pregnant women is one of the causes of spontaneous abortion.Women over the age of 25 are more likely to miscarry as their age growth.The risk of chromosomal microdeletion or microduplication in the fetus is irrelevant to the age of the pregnant woman.3.There is no obvious gender difference in spontaneously aborted embryos.However,female embryos are more prone to chromosomal numeral abnormalities,whereas male embryos are more likely to have chromosomal abnormalities other than numeral abnormalities or to be affected by other factors.4.CMA technology is applied to the analysis of spontaneous abortion etiology,which can significantly increase the success rate of the detection of spontaneous abortion chorionic villus chromosomes and the detection rate of abnormality.