| Background and ObjectiveSpontaneous Abortion(SA)is a common disease,which generally occurs within28 weeks of pregnancy.In the etiology of abortion investigation,embryonic chromosome abnormalities account for more than 50%.As the etiology of SA is complex and heterogeneous,the etiological diagnosis is typically combined with a series of auxiliary tests in the laboratory.Among chromosome detecting methods,chromosome karyotype analysis is widely used in diagnosis.Even though karyotype analysis can keep abortion material,it still suffers from a high failure rate of spontaneous abortion villus.With the development of chromosome detecting methods,chromosome microarray analysis(CMA)and low depth genome sequencing(CNVSeq)received much attention.Limited by the high cost and micro degradation of abortion samples,CMA is not popularly applied in chromosome detection.By contrast,CNV-seq takes advantage of the cost and resolution of sample analysis,which can potentially be used in large-scale applications.However,CNV-seq also has its limitation in detecting polyploid and uniparental diploids in SA.Therefore,the combination of CNV-seq and short tandem repeat(STR)is proposed to address the issues of CNV-seq limitation in abortion sample detection.In this study,the joint analysis strategy,the combination of CNV-seq and short tandem repeat(STR)is used in the abortion tissue samples analysis.Meanwhile,chromosome karyotype analysis was also used for comparison to evaluate the feasibility and application value of the proposed strategy.This study gives a unique insight into chromosome detection and provides a reliable solution for SA clinical diagnosis.Methods(1)572 patients at the Maternal and Child Health Hospital of Henan Province were selected,who were diagnosed with early SA from June 2019 to June 2021.With the permission of the patients,aseptic uterine debridement,villous tissue samples,and the peripheral blood of both couples were conducted.(2)572 SA tissue samples were detected by CNV-seq and STR typing at the same time and the different results were verified by Chromosomal microarray analysis(CMA).Results comparison between CNV-seq scheme and STR scheme was investigated or was analyzed.(3)236 of 572 SA tissue samples were randomly selected for G-banding chromosome karyotype analysis.Results comparison between CNV-seq+STR scheme and karyotype analysis scheme was investigated or was analyzed.(4)The pedigrees of 10 embryonic tissues with abnormal chromosome structures were verified.According to different variation types,the peripheral blood of parents was uptaken for chromosome karyotype analysis or CNV-seq detection.Special cases were analyzed by using the MLPA method.Results(1)572 SA tissue samples were detected by CNV-seq and STR typing at the same time.275 cases of chromosome abnormalities were detected by CNV-seq scheme,accounting for 48.1%.313 cases of chromosome abnormalities were detected by CNV-seq+STR combined scheme,accounting for 54.7%.Among the negative samples detected by CNV-seq scheme,38 cases of abnormal samples were detected by CNV-seq+STR combined scheme,accounting for 6.6%,The detection rate of the two schemes was statistically significant(P < 0.05).(2)CNV-seq + STR typing and G-banding karyotype analysis: 236 SA tissue samples were detected at the same time.236 cases were successfully detected by CNV-seq + STR,with a success rate of 100%.210 cases were successfully detected by G-banding karyotype,and 26 cases failed,with a success rate of 89.0%.The success rate of the two methods was statistically significant(P < 0.005).(3)236 SA tissue samples were detected by CNV-seq+STR typing and G-banding chromosome karyotype analysis.The results of CNV-seq+STR typing showed that 99 cases of the chromosomes were normal,accounting for 41.9%.109cases(46.2%)were found to be normal by karyotype.There was no significant difference between the two methods(P > 0.005).137 cases(58.1%)were found to be abnormal by CNV-seq+STR typing.The results of chromosome abnormalities detected by G-banding karyotype were 101 cases,accounting for 42.8%;There was a significant difference in the detection rate of chromosome abnormality between the two methods(P < 0.005).102 cases of chromosome number abnormality were detected by CNV-seq+STR typing accounting for 43.2%,and likewise 91 cases of chromosome number abnormality were detected by chromosome karyotype accounting for 38.6%.There was no significant difference in the detection rate of chromosome number abnormality between the two methods(P > 0.005).24 cases(10.2%)of chromosome structural abnormalities(CNVs)were detected by CNV-seq+STR and 8 cases(3.4%)were detected by karyotype analysis.There was a significant difference in the detection rate of chromosome structural abnormalities(CNVs)between the two methods(P < 0.05).9 cases of chimera were detected by CNV-seq+STR typing accounting for 3.8%,and 2 cases of chimera were detected by chromosome karyotype analysis accounting for 0.8%.There was a significant difference in the detection rate of a chimera between the two methods(P < 0.05).The difference between CNV-seq+STR typing and karyotype analysis was statistically significant(P < 0.05).(4)233 cases of chromosomal aneuploidy were detected by CNV-seq,including chromosomes 2～11,13～18,20～22,and sex chromosomes.The concentrated distribution of 16,22,and sex chromosome aneuploidy: 50 cases of trisomy 16(21.5%),41 cases of trisomy 22(17.6%),and 39 cases of abnormal sex chromosome number(including compound trisomy and chimera),accounting for 16.7%.(5)Family verification results of 10 SA samples: CNV-seq was used to verify the family of 6 cases of SA embryos: 2 cases VOUS were inherited from the mother,2cases VOUS were inherited from the father,1 case VOUS was inherited from mother,and 1 case VOUS was a new variant;Chromosome karyotype analysis was used to verify the pedigree of 4 cases of SA embryos: 2 cases p CNVs were from mother of balanced translocation,1 case was p CNVs inherited from father that was balanced translocation,and 1 case were p CNVs that was a denovo mutation.(6)572 Patients were divided into two groups,i.e.,the non-elder group(< 35 years old)and the elder group(≥ 35 years old),according to their age.The number abnormality rate and genomic structure variation rate in the two groups were compared.The number abnormality rate of the non-elderly group was 43.3%,which was lower than that of the elder group(61.6%).The comparison between the two groups was statistically significant(P< 0.005).The genomic structure variation rate of the non-elder group was 8.9%,which was slightly higher than that of the elderly group(8.0%).Thus,there was no significant difference between the two groups(P >0.005).According to the number of abortions,they were divided into a single group(<2 times)and multiple group(≥ 2 times).The number abnormality rate and genomic structure variation rate of the two groups were compared.The chromosome number abnormality rate of the single group was 50.8%,which was slightly higher than that of the multiple group(44.2%).There was no statistical significance between the two groups(P > 0.005);The variation rate of genomic structure in the multiple disease group was 7.5%,which was slightly lower than that of the multiple disease group(9.5%).There was no statistical significance between the two groups(P > 0.005).Conclusion(1)CNV-Seq can quickly and accurately detect chromosomal aneuploidy and unbalanced genome copy number variation,and the combined detection with short STR typing can effectively complete the detection of polyploidy and uniparental diploidy abnormalities.CNV-Seq and STR typing can be widely used in the detection and analysis to find the cause of abortion,and it has a high clinical application value.(2)The chromosome aneuploidy of 233 early aborted embryos in Zhengzhou,Henan Province showed the distribution characteristics of high concentration of chromosome 16,22,and sex chromosome. |
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