A clinical study of CNV-seq techniques in 103 cases of spontaneous abortion

A clinical study of CNV-seq techniques in 103 cases of spontaneous abortion | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Method Article A clinical study of CNV-seq techniques in 103 cases of spontaneous abortion meihua xie, Hongxia Zhou, Jianlong Zhuang, Jingyi Chen, Hening Li, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4330610/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective: To detect the relationship between spontaneous abortion and chromosomal abnormalities by genome copy number variation sequencing (CNV-seq), and explore this relationship’s application to the genetic diagnosis of spontaneous abortion. Methods: From June, 2020 to August, 2022, 103 different embryonic tissue samples from patients who suffered spontaneous abortion in Yueyang Central Hospital were detected by CNV-seq, and the results were analyzed and interpreted after excluding maternal cell contamination. Results: 97 of the 103 cases were detected successfully. A total of 49 cases (50.5%) of abnormal karyotypes were found, including 33 cases of abnormal chromosome number, 12 cases of structural abnormalities (including 14 pathogenic CNVs and 3 variants of uncertain significance,), 3 cases of triploid, and 1 cases of absence of heterozygosity (AOH). The detection rate of chromosomal abnormalities in abortion tissues in the ≥ 35 years old group was higher than that in the <35 years old group (58.8% vs. 48. 7%). Conclusion: Fetal chromosomal abnormalities are an important cause of abortion, CNV-seq technology can efficiently detect chromosomal number abnormalities, large fragment deletions/duplications and pathogenic genome copy number variants, which greatly improves the detection rate of chromosomal abnormalities and provides a valuable genetic risk assessment to couples trying to reproduce again. Spontaneous abortion Genome copy number variation sequencing Genetic diagnosis Figures Figure 1 Introduction Spontaneous abortion (SA) is the most common complication among women of reproductive age. It occurs in approximately 15% of pregnancy cases in the first trimester of pregnancy, and this rate has been growing in recent years(COLLEY et al., 2019; QUENBY et al., 2021). Any non-voluntary termination of pregnancy that occurs within 28 complete weeks of gestation is generally considered to fall under the category of SA. Researchers have made many efforts to study the causes of natural abortion, and although all causes of natural abortion remain unknown, they have found that chromosome abnormalities are the main cause of early pregnancy abortion(YANG et al., 2016). Among these abnormalities, both chromosome copy number variation and chimera account for a significant proportion of abortion cases. Studies have shown that approximately 6–13% of stillbirths are associated with chromosomal abnormalities(DU et al.,2018), and 5–40% of abortion with structural abnormalities are related to chromosomal abnormalities(CHEUNG et al., 2018). Genetic analysis of aborted tissue is of great value in analyzing the causes of miscarriage and stillbirth(SHAFFER et al., 2004), and may help in assessing the risk of recurrence as well as prenatal diagnosis. Traditional chromosome karyotyping is considered the “gold standard” for chromosomal abnormalities, but it has a long detection cycle and cannot detect copy number variations (CNVs) 5-10Mb in the genome. However, with the rapid development of molecular genetics in recent years, the advantages of copy number variation sequencing (CNV-seq) in the detection of aneuploid and copy number have become increasingly evident. CNV-seq can be used for genetic testing of miscarriage, stillbirth, or fetal tissues that require clarification of genetic etiology. In this study CNV-seq technology combined with elimination of maternal contamination detection technology was used to detect CNV with a resolution of more than 100kb in 103 abortion tissue samples in Yueyang Central Hospital(Yueyang, Hunan Province, China), and the genomic regions with detected chromosome abnormalities were analyzed to determine the potential CNVs associated with miscarriage. We aimed to explore the application of this technology to genetic diagnosis of embryonic tissues and to provide genetic risk assessment and guidance for couples seeking to conceive again. Materials and methods Research object This study focused on 103 patients who were treated at Yueyang Central Hospital from June, 2020 to August, 2022. All patients were diagnosed with embryonic arrest, stillbirth, or miscarriage and required further investigation into the cause, and the aborted tissues (fetal tissues or villi) were collected for CNV-seq detection. The age range of the patients was 21–43 years, and none of the pregnant women had a history of infection or exposure to toxic substances. All tests were conducted with informed consent and signed by the patient and family. This study protocol was reviewed and approved by Ethics Committee of Yueyang Central Hospital, approval number [2023-026]. Specimen collection and extraction The villi tissue or fetal tissue was rinsed with sterile normal saline 3–5 times, and the rinsed tissue was placed in a sterile saline sampling box for transport and preservation at low temperature. About 25mg of the villi or fetal tissue was cut into fragments, and the whole-genome DNA was extracted by Guangzhou Darui extraction kit. High-throughput sequencing Library construction of fragmented genomic DNA after fragmentation: ( 1 ) The extracted genomic DNA was first fragmented using a sonicator. ( 2 ) The fragmented DNA was then used for library construction. ( 3 ) The libraries that passed quality control were subjected to fluorescence quantitative PCR analysis and then sequenced using a DA8600 sequencer. The sequencing data was then analyzed using the hg19 reference genome sequence (at a resolution of 100 kb) to obtain copy number values for each chromosome. This allowed determination of chromosome segment duplications or deletions, and a detection result plot was generated to visualize the findings. CNV Databases and Interpretation: The copy number variations (CNVs) detected were analyzed for their clinical significance by searching various databases including the Database of Genomic Variants (DGV) ( http://dgv.tcag.ca/dgv/app/home ), the ClinGen database ( https://dosage.clinicalgenome.org/ ), the Ensembl resources (DECIPHER) ( https://www.deciphergenomics.org/disorders/syndromes/list ) database, the UCSC database ( https://genome.ucsc.edu/ ), and the Online Mendelian Inheritance in Man (OMIM) database ( http://www.omim.org/ ). The clinical significance of the detected CNVs was interpreted based on the guidelines provided by the American College of Medical Genetics (ACMG) for CNV-seq results (KEARNEY et al., 2011) and was categorized into five classes: benign CNV, likely benign CNV, CNV of variant of uncertain significance (VUS), likely pathogenic CNV, and pathogenic CNV. Statistical Methods For intergroup comparison of count data, the χ 2 test was used, with a significance level of P < 0.05 indicating a statistically significant difference. This analysis was performed using SPSS 19.0 software. Results In this study, 6 of the 103 total cases turned out to be maternal tissues without fetal tissues, for a success rate of testing of 94.2%. Among the samples, 49 cases (50.5%) were found to have abnormal karyotypes, including 33 cases (34.0%) with aneuploidy abnormalities, 12 cases (12.4%) with structural abnormalities, 3 cases (3.1%) with triploidy, and 1 case (1.03%) with absence of heterozygosity (AOH), the latter two through combined testing with short tandem repeats (STR). The distribution of positive samples is shown in Fig. 1 . Aneuploidy 30 cases of aneuploid abnormal karyotype were detected, including X Monosomy in 10 cases, Triploidy in 2 cases, 1 repeat mosaicism No. 12, X Deletion in 2 cases, and Trisomy in 20 cases (chromosomes 22, 21, 18, 15, 16, 13, 12, 9, 7, 6) (Table 1 ). Table 1 Detection of aneuploidy chromosomal abnormalities Abnormality types Number Percentage(%)(%) Abnormality types Number Percentage(%)(%) 45,X 12** 36.4 47,XN,+6 1 3.03 47,XN,+21 4 12.1 47,XN,+9 1 3.03 47,XN,+18 18三体 2 6.06 47,XN,+15 1 3.03 47,XN,+13 3 9.09 47,XN,+12 2* 6.06 47,XN,+22 3 9.09 47,XN,+7 1 3.03 47,XN,+16 3 9.09 Total 33 100% * indicates 1 case of chimerism, ** indicates 2 cases of chimerism Structural abnormality (Table 2 ) In the 97 successfully tested samples, 12 cases were found to have chromosomal structural abnormalities, with a total of 17 CNVs detected. Among the 17 CNVs detected, 14 were pathogenic CNVs, and among the detected pathogenic CNVs, a deletion in 4p16.3 was associated with Wolf-Hirschhorn syndrome, and a region in 17p13.2-p13.3 was associated with Miller-Dieker syndrome. In 3 cases with CNVs of unclear clinical significance, one patient had CNV-seq testing on the peripheral blood of the parents that revealed that the 4q34.3 deletion was inherited from the mother, who did not exhibit any phenotypic abnormalities. Among the 5 cases with coexistence of deletion/duplication CNVs, one case underwent parental chromosomal karyotype analysis, and this showed that the CNV was inherited from the mother with a balanced chromosomal translocation. Table 2 Detection of structural chromosomal abnormalities Abnormality types Fragment size(Mb) Pathogenicity assessment 46XN,dup( 3 )(p14.1-p26.3), del( 5 )(p14.3-p15.33) 66.3, 21.05 Pathogenic 46XN,del( 16 )(p11.2) 0.55 Pathogenic 46XN,dup( 2 )(p24.1-p25.3),del( 17 )(p13.2-p13.3) 22.74, 3.45 Pathogenic 46XN,dup( 5 )(q33.3-q35.3),del( 7 )(q32.1-q36.3) 24.8, 31.5 Pathogenic 46XN,dup( 9 )q21.12q34.3, del( 4 )p16.3 66.9, 3.66 Pathogenic 46XN,del( 15 )15q26.2q26.3 7.5 Pathogenic 46XN,dup( 8 )8q23.3-q24.23 21.7 Pathogenic 46XN,del( 12 )(q24.21-q24.22) 0.4 Pathogenic 46XN,dup( 6 )(q16.3q27)del( 7 )( 7q33q36.3) 66.45, 21.64 Pathogenic 46XN,del( 4 )(q34.3) 4.95 VUS 46XN,dup( 7 )p22.1-p22.2 1.4 VUS 46XN,dup( 2 )p25.2 1.25 VUS Total 12 Comparison of chromosomal abnormalities at different ages Among the 97 successfully tested patients, there were 17 cases in the ≥ 35 years age group, with 10 cases (58.8%) exhibiting chromosomal abnormalities, and there were 80 cases in the < 35 years age group, with 39 cases (48.7%) showing chromosomal abnormalities. The comparison of chromosomal abnormality rates between the ≥ 35 group and the 0.05) (Table 3 ). Table 3 Chromosomal abnormalities in different age groups Group Total Normal Abnormal χ 2 P ≥ 35 age group(n=) 80 41(51.3%) 39(48.7%) 0.596 0.451 0.05, indicating no statistically significant difference. Discussion CNVs can occur anywhere in the human genome, and although most CNVS are benign or VOUS, a significant number of CNVs are associated with human diseases(YANG et al., 2018). In addition, with the rapid advancement of genomics, the OMIM database adds an average of 1.5 genes per day, but the design of probes used in Chromosome Microarray Analysis (CMA) cannot keep up with this rate of updates, leading to missed detection of some known pathogenic regions (LAN et al., 2020). The high cost and low throughput of CMA limit its widespread use in prenatal diagnosis. Since the first report in 2009 that CNV-seq can be used for accurate analysis of chromosomal CNVs, more and more studies have confirmed its reliability in detecting them, however. CNV-seq is a low-depth whole-genome sequencing technique based on next-generation sequencing technology that uses bioinformatics analysis to discover the CNVs of the tested samples. This technique has the advantages of high throughput, low sample requirements, and broad detection range. However, it has limitations such as the inability to detect triploidy, polyploidy, uniparental disomy, balanced translocations, or inversions. Therefore, it is necessary to combine STR to solve the problems of triploidy, uniparental disomy, and maternal contamination. In this study, the combined use of STR and CNV-seq analysis revealed six cases of maternal contamination, three cases of triploidy, and one case of absence of heterozygosity (AOH). There are many causes of spontaneous abortion, including embryo chromosomal abnormalities, genetic factors, reproductive tract abnormalities, hormonal imbalances, maternal systemic diseases, immune factors, infectious diseases, toxins, environmental factors, poor lifestyle habits, as well as other unknown factors(QUINTERO-RONDEROS et al., 2020; KARIM et al., 2017; ZWIERZCHOWSKA et al., 2018; TOSIC-PAJIC et al., 2017), and despite advances in medicine, spontaneous abortion remains a significant health issue. Many couples may face the risk of miscarriage when planning to have children. Genetic factors are one of the main causes, and fetal chromosomal abnormalities are an important genetic factor in fetal miscarriage (ZHANG et al., 2018). Among them, abnormal embryo chromosomal numbers and CNVs are the main causes of spontaneous abortion. In this study, chromosomal structural abnormalities were detected in 12 cases out of 97 cases successful fetal tissue samples (13.9%), with a total of 17 CNVs identified. Among the 17 CNVs detected, 14 were pathogenic CNVs. These pathogenic CNVs can lead to severe developmental delays, malformations, intellectual disabilities, delayed language development, distinctive facial features, hypotonia, and congenital heart disease in embryos or fetuses. One case of 4p16.3 loss was found involving Wolf-Hirschhorn syndrome. Patients with this syndrome typically have low birth weight, failure to thrive after birth, microcephaly, developmental delays, and hypotonia. Facial features include downward-slanting eyelids, a ‘Greek warrior helmet’ appearance, a short nose, and a very short midsection. They may also suffer from conditions such as iris tumors and epilepsy (YIN et al., 2019). In this case, both parents underwent peripheral blood karyotype testing, and the father was identified as a carrier of a balanced translocation [46,XX, (t 4;9)]. It was recommended that they undergo preimplantation genetic diagnosis (PGD) for genetic disease screening when planning to have children again. Additionally, one case involving a 17p13.2-p13.3 deletion associated with Miller-Dieker syndrome was also identified in this study. The main characteristic of this syndrome is lissencephaly (“smooth brain” lacking normal brain gyri and sulci). Clinical manifestations also include a high and prominent forehead, vertical furrows, narrowed temples, widely spaced eyes, upward-slanting eyelids, short and upward-turned nose, inverted red lips, long, wide and thick upper lip, congenital heart disease, umbilical hernia, and joint contractures. MDS patients typically have severe intellectual disabilities, epilepsy, and shortened lifespan. Both parents in this case underwent peripheral blood karyotype testing, and it was found that the mother was a carrier of a balanced translocation [46,XX, t(2;17)(p23;p13), 21ph+]. Given that there is only a 2/18 chance of having a normal fetus in future pregnancies, it was recommended that they undergo preimplantation genetic diagnosis (PGD) to assess for genetic diseases during reproductive planning. CNV-seq technology can efficiently and rapidly detect chromosomal structural abnormalities that may lead to abortion, and analysis of the peripheral blood karyotypes of both spouses can guide assessment for future pregnancies. Under normal circumstances, offspring inherit one homologous chromosome from the mother and one from the father. However, cases where a pair of homologous chromosomes in the offspring is entirely or partly derived from either the father or the mother are referred to as absence of heterozygosity (AOH). Although the incidence of AOH is low, it can increase the risk of recessive disease-causing genes that can lead to complex clinical symptoms. In this study, combined STR and CNV-seq testing detected a homozygous phenomenon in the 2p25.3q37.3 region, spanning 242Mb, which may have led to an increased risk of recessive genetic disorders in that specific region. Currently, there is no conclusive evidence that the occurrence of uniparental diploid on chromosome 2 is directly related to embryo termination. Research have found that after ovulation in vitro, oocyte quality decreases with time, and the expression of spindle assembly checkpoint (SAC) in oocytes is negatively correlated with oocyte senescence. Decreased SAC expression also leads to errors in sister chromatid separation during the second meiotic division (MII) of oocytes, thereby increasing the incidence of early embryonic aneuploidy (SHIMOI et al., 2019). In this study, the rate of chromosomal abnormalities in aborted tissues in the ≥ 35 age group accounted for 58.8%, and the rate of chromosomal abnormalities in the < 35 age group accounted for 48.7%, Although the difference was not statistically significant (P = 0.451), the abnormality rate in the ≥ 35 group was 10.1% higher than that in the < 35 group, indicating a positive correlation between the frequency of abnormal karyotypes and maternal age. Chromosomal abnormalities in fetuses aborted in the first trimester may involve any one or multiple chromosomes, although chromosomal abnormalities in fetuses aborted in the second trimester may be trisomy 13, 18, and 21, sex chromosome aneuploidy, or CNV, which are similar to the types of chromosomal aberrations detected in live birth (ZHANG et al., 2021; LAN et al., 2021). With the development of molecular biology technology, CNV-seq technology has been gradually applied to chromosomal testing of fetuses aborted, mostly used in the detection of chromosome number abnormalities, large fragment deletion/duplication, and copy number variation of pathogenic genomes [19] . Although chromosomal karyotyping is considered the gold standard for detecting chromosomal abnormalities, it cannot detect deletions/duplications smaller than 5-10Mb. However, CNV-seq technology can efficiently detect routine chromosomal number abnormalities and identify pathogenic microdeletions/microduplications that cannot be detected by karyotype analysis. It can also detect unknown variations and discover new genetic information associated with diseases, thus improving the detection rate. CNV-seq technology can provide genetic diagnostic evidence for the etiology of miscarriage, guide future pregnancies with reliable laboratory evidence, and offer valuable reference data for the prevention, diagnosis, and treatment of abortion. Declarations Funding: Hunan Natural Science Foundation of China (2023JJ50306). Statement of Ethics: This study protocol was reviewed and approved by Ethics Committee of Yueyang Central Hospital, approval number [2023-026] Conflict of Interest Statement : The authors have no conflicts of interest to declare. Author Contributions: Meihua Xie and Hongxia Zhou: Drafting the work or revising it critically for important intellectual content. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Jianlong Zhuang、Jingyi Chen and Hening Li: acquisition and interpretation of data for the work. Yanfei Gong and Bojie Li: analysis, or interpretation of data for the work. Liyun Xie and Airong Pi: Final approval of the version to be published. Data Availability declaration ： The authors confirm that the data supporting the findings of this study are available within the article. References COLLEY E, HAMILTON S, SMITH P, et al. (2019). Potential genetic causes of miscarriage in euploid pregnancies: a systematic review [J]. Hum Reprod Update, 25(4): 452-72. CHEUNG S W, BI W. (2018). Novel applications of array comparative genomic hybridization in molecular diagnostics [J]. Expert Rev Mol Diagn, 18(6): 531-42. DU Y, CHEN L, LIN J, et al. (2018). Chromosomal karyotype in chorionic villi of recurrent spontaneous abortion patients [J]. Biosci Trends, 12(1): 32-9. KARIM S, JAMAL H S, ROUZI A, et al. (2017). Genomic answers for recurrent spontaneous abortion in Saudi Arabia: An array comparative genomic hybridization approach [J]. Reprod Biol, , 17(2): 133-43. KEARNEY H M, THORLAND E C, BROWN K K, et al. (2011). American College of Medical Genetics standards and guidelines for interpretation and reporting of postnatal constitutional copy number variants [J]. Genet Med, 13(7): 680-5. LAN L, WU H, SHE L, et al. (2020). Analysis of copy number variation by sequencing in fetuses with nuchal translucency thickening [J]. J Clin Lab Anal, , 34(8): e23347. LAN L, SHE L, ZHANG B, et al. (2021). Prenatal diagnosis of 913 fetuses samples using copy number variation sequencing [J]. J Gene Med, 23(5): e3324. QUENBY S, GALLOS I D, DHILLON-SMITH R K, et al. (2021). Miscarriage matters: the epidemiological, physical, psychological, and economic costs of early pregnancy loss [J]. Lancet, 397(10285): 1658-67. QUINTERO-RONDEROS P, LAISSUE P. (2020). Genetic Variants Contributing to Early Recurrent Pregnancy Loss Etiology Identified by Sequencing Approaches [J]. Reprod Sci, 27(8): 1541-52. SHIMOI G, TOMITA M, KATAOKA M, et al. (2019). Destabilization of spindle assembly checkpoint causes aneuploidy during meiosis II in murine post-ovulatory aged oocytes [J]. J Reprod Dev, , 65(1): 57-66. SHAFFER L G, BEJJANI B A. (2004). A cytogeneticist's perspective on genomic microarrays [J]. Hum Reprod Update, 10(3): 221-6. TOSIC-PAJIC J, SEKLIC D, RADENKOVIC J, et al. (2017). Augmented oxidative stress in infertile women with persistent chlamydial infection [J]. Reprod Biol, 17(2): 120-5. XIE W, TAN Y, LI X, et al. (2013). Rapid detection of aneuploidies on a benchtop sequencing platform [J]. Prenat Diagn, 33(3): 232-7. YANG X, SONG Z, WU C, et al. (2018). Constructing a database for the relations between CNV and human genetic diseases via systematic text mining [J]. BMC Bioinformatics, , 19(Suppl 19): 528. YIN⁃ G L H Y L J Z, AIHUA H R C Z L Y. (2019). Prenatal diagnosis and study of Wolf⁃Hirschhorn syndrome [J]. The Journal of Practical Medicine, 35: 10. YANG L, TANG Y, LU M, et al. (2016). Novel rapid molecular diagnosis of fetal chromosomal abnormalities associated with recurrent pregnancy loss [J]. Acta Obstet Gynecol Scand, 95(12): 1433-40. ZWIERZCHOWSKA A, IWAN A, HYC A, et al. (2018). Recurrent miscarriage is associated with increased ghrelin mRNA expression in the endometrium- a case-control study [J]. Reprod Biol, 18(1): 12-7. ZHANG T, SUN Y, CHEN Z, et al. (2018). Traditional and molecular chromosomal abnormality analysis of products of conception in spontaneous and recurrent miscarriage [J]. BJOG, 125(4): 414-20. ZHANG X, HUANG Q, YU Z, et al. (2021). Copy number variation characterization and possible candidate genes in miscarriage and stillbirth by next-generation sequencing analysis [J]. J Gene Med, 23(12): e3383. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4330610","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":299939690,"identity":"c9429e21-4121-4867-a4e5-436cced63252","order_by":0,"name":"meihua 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It occurs in approximately 15% of pregnancy cases in the first trimester of pregnancy, and this rate has been growing in recent years(COLLEY et al., 2019; QUENBY et al., 2021). Any non-voluntary termination of pregnancy that occurs within 28 complete weeks of gestation is generally considered to fall under the category of SA. Researchers have made many efforts to study the causes of natural abortion, and although all causes of natural abortion remain unknown, they have found that chromosome abnormalities are the main cause of early pregnancy abortion(YANG et al., 2016). Among these abnormalities, both chromosome copy number variation and chimera account for a significant proportion of abortion cases. Studies have shown that approximately 6\u0026ndash;13% of stillbirths are associated with chromosomal abnormalities(DU et al.,2018), and 5\u0026ndash;40% of abortion with structural abnormalities are related to chromosomal abnormalities(CHEUNG et al., 2018).\u003c/p\u003e \u003cp\u003eGenetic analysis of aborted tissue is of great value in analyzing the causes of miscarriage and stillbirth(SHAFFER et al., 2004), and may help in assessing the risk of recurrence as well as prenatal diagnosis. Traditional chromosome karyotyping is considered the \u0026ldquo;gold standard\u0026rdquo; for chromosomal abnormalities, but it has a long detection cycle and cannot detect copy number variations (CNVs) 5-10Mb in the genome. However, with the rapid development of molecular genetics in recent years, the advantages of copy number variation sequencing (CNV-seq) in the detection of aneuploid and copy number have become increasingly evident. CNV-seq can be used for genetic testing of miscarriage, stillbirth, or fetal tissues that require clarification of genetic etiology.\u003c/p\u003e \u003cp\u003eIn this study CNV-seq technology combined with elimination of maternal contamination detection technology was used to detect CNV with a resolution of more than 100kb in 103 abortion tissue samples in Yueyang Central Hospital(Yueyang, Hunan Province, China), and the genomic regions with detected chromosome abnormalities were analyzed to determine the potential CNVs associated with miscarriage. We aimed to explore the application of this technology to genetic diagnosis of embryonic tissues and to provide genetic risk assessment and guidance for couples seeking to conceive again.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eResearch object\u003c/h2\u003e \u003cp\u003eThis study focused on 103 patients who were treated at Yueyang Central Hospital from June, 2020 to August, 2022. All patients were diagnosed with embryonic arrest, stillbirth, or miscarriage and required further investigation into the cause, and the aborted tissues (fetal tissues or villi) were collected for CNV-seq detection. The age range of the patients was 21\u0026ndash;43 years, and none of the pregnant women had a history of infection or exposure to toxic substances. All tests were conducted with informed consent and signed by the patient and family. This study protocol was reviewed and approved by Ethics Committee of Yueyang Central Hospital, approval number [2023-026].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSpecimen collection and extraction\u003c/h2\u003e \u003cp\u003eThe villi tissue or fetal tissue was rinsed with sterile normal saline 3\u0026ndash;5 times, and the rinsed tissue was placed in a sterile saline sampling box for transport and preservation at low temperature. About 25mg of the villi or fetal tissue was cut into fragments, and the whole-genome DNA was extracted by Guangzhou Darui extraction kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHigh-throughput sequencing\u003c/h2\u003e \u003cp\u003eLibrary construction of fragmented genomic DNA after fragmentation: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) The extracted genomic DNA was first fragmented using a sonicator. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) The fragmented DNA was then used for library construction. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) The libraries that passed quality control were subjected to fluorescence quantitative PCR analysis and then sequenced using a DA8600 sequencer. The sequencing data was then analyzed using the hg19 reference genome sequence (at a resolution of 100 kb) to obtain copy number values for each chromosome. This allowed determination of chromosome segment duplications or deletions, and a detection result plot was generated to visualize the findings.\u003c/p\u003e \u003cp\u003eCNV Databases and Interpretation: The copy number variations (CNVs) detected were analyzed for their clinical significance by searching various databases including the Database of Genomic Variants (DGV) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dgv.tcag.ca/dgv/app/home\u003c/span\u003e\u003cspan address=\"http://dgv.tcag.ca/dgv/app/home\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the ClinGen database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://dosage.clinicalgenome.org/\u003c/span\u003e\u003cspan address=\"https://dosage.clinicalgenome.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the Ensembl resources (DECIPHER) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.deciphergenomics.org/disorders/syndromes/list\u003c/span\u003e\u003cspan address=\"https://www.deciphergenomics.org/disorders/syndromes/list\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database, the UCSC database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://genome.ucsc.edu/\u003c/span\u003e\u003cspan address=\"https://genome.ucsc.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the Online Mendelian Inheritance in Man (OMIM) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.omim.org/\u003c/span\u003e\u003cspan address=\"http://www.omim.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The clinical significance of the detected CNVs was interpreted based on the guidelines provided by the American College of Medical Genetics (ACMG) for CNV-seq results (KEARNEY et al., 2011) and was categorized into five classes: benign CNV, likely benign CNV, CNV of variant of uncertain significance (VUS), likely pathogenic CNV, and pathogenic CNV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Methods\u003c/h2\u003e \u003cp\u003eFor intergroup comparison of count data, the χ\u003csup\u003e2\u003c/sup\u003e test was used, with a significance level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicating a statistically significant difference. This analysis was performed using SPSS 19.0 software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eIn this study, 6 of the 103 total cases turned out to be maternal tissues without fetal tissues, for a success rate of testing of 94.2%. Among the samples, 49 cases (50.5%) were found to have abnormal karyotypes, including 33 cases (34.0%) with aneuploidy abnormalities, 12 cases (12.4%) with structural abnormalities, 3 cases (3.1%) with triploidy, and 1 case (1.03%) with absence of heterozygosity (AOH), the latter two through combined testing with short tandem repeats (STR). The distribution of positive samples is shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eAneuploidy\u003c/h2\u003e\n \u003cp\u003e30 cases of aneuploid abnormal karyotype were detected, including X Monosomy in 10 cases, Triploidy in 2 cases, 1 repeat mosaicism No. 12, X Deletion in 2 cases, and Trisomy in 20 cases (chromosomes 22, 21, 18, 15, 16, 13, 12, 9, 7, 6) (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDetection of aneuploidy chromosomal abnormalities\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAbnormality types\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePercentage(%)(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAbnormality types\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePercentage(%)(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45,X\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47,XN,+6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47,XN,+21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47,XN,+9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47,XN,+18\u003c/p\u003e\n \u003cp\u003e18三体\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47,XN,+15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47,XN,+13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47,XN,+12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47,XN,+22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47,XN,+7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47,XN,+16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003e* indicates 1 case of chimerism, ** indicates 2 cases of chimerism\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003eStructural abnormality\u003c/strong\u003e (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\n \u003cp\u003eIn the 97 successfully tested samples, 12 cases were found to have chromosomal structural abnormalities, with a total of 17 CNVs detected. Among the 17 CNVs detected, 14 were pathogenic CNVs, and among the detected pathogenic CNVs, a deletion in 4p16.3 was associated with Wolf-Hirschhorn syndrome, and a region in 17p13.2-p13.3 was associated with Miller-Dieker syndrome. In 3 cases with CNVs of unclear clinical significance, one patient had CNV-seq testing on the peripheral blood of the parents that revealed that the 4q34.3 deletion was inherited from the mother, who did not exhibit any phenotypic abnormalities. Among the 5 cases with coexistence of deletion/duplication CNVs, one case underwent parental chromosomal karyotype analysis, and this showed that the CNV was inherited from the mother with a balanced chromosomal translocation.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDetection of structural chromosomal abnormalities\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAbnormality types\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFragment size(Mb)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePathogenicity assessment\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46XN,dup(\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e)(p14.1-p26.3), del(\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e)(p14.3-p15.33)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.3, 21.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46XN,del(\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e)(p11.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46XN,dup(\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e)(p24.1-p25.3),del(\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e)(p13.2-p13.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.74, 3.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46XN,dup(\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e)(q33.3-q35.3),del(\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e)(q32.1-q36.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.8, 31.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46XN,dup(\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e)q21.12q34.3, del(\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e)p16.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.9, 3.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46XN,del(\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e)15q26.2q26.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46XN,dup(\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e)8q23.3-q24.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46XN,del(\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e)(q24.21-q24.22)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46XN,dup(\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e)(q16.3q27)del(\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e)( 7q33q36.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.45, 21.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46XN,del(\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e)(q34.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVUS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46XN,dup(\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e)p22.1-p22.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVUS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46XN,dup(\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e)p25.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVUS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eComparison of chromosomal abnormalities at different ages\u003c/h2\u003e\n \u003cp\u003eAmong the 97 successfully tested patients, there were 17 cases in the \u0026ge;\u0026thinsp;35 years age group, with 10 cases (58.8%) exhibiting chromosomal abnormalities, and there were 80 cases in the \u0026lt;\u0026thinsp;35 years age group, with 39 cases (48.7%) showing chromosomal abnormalities. The comparison of chromosomal abnormality rates between the \u0026ge;\u0026thinsp;35 group and the \u0026lt;\u0026thinsp;35 group did not show any statistically significant difference, however (\u0026chi;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.596, P\u0026thinsp;=\u0026thinsp;0.451, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eChromosomal abnormalities in different age groups\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAbnormal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ge;\u0026thinsp;35 age group(n=)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41(51.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39(48.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e0.596\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e0.451\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;35 age group (n=)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7 (41.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10(58.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003eNote: The \u0026chi;\u003csup\u003e2\u003c/sup\u003etest with a 2x2 contingency table was used, and the result showed that P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, indicating no statistically significant difference.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCNVs can occur anywhere in the human genome, and although most CNVS are benign or VOUS, a significant number of CNVs are associated with human diseases(YANG et al., 2018). In addition, with the rapid advancement of genomics, the OMIM database adds an average of 1.5 genes per day, but the design of probes used in Chromosome Microarray Analysis (CMA) cannot keep up with this rate of updates, leading to missed detection of some known pathogenic regions (LAN et al., 2020). The high cost and low throughput of CMA limit its widespread use in prenatal diagnosis. Since the first report in 2009 that CNV-seq can be used for accurate analysis of chromosomal CNVs, more and more studies have confirmed its reliability in detecting them, however.\u003c/p\u003e \u003cp\u003eCNV-seq is a low-depth whole-genome sequencing technique based on next-generation sequencing technology that uses bioinformatics analysis to discover the CNVs of the tested samples. This technique has the advantages of high throughput, low sample requirements, and broad detection range. However, it has limitations such as the inability to detect triploidy, polyploidy, uniparental disomy, balanced translocations, or inversions. Therefore, it is necessary to combine STR to solve the problems of triploidy, uniparental disomy, and maternal contamination. In this study, the combined use of STR and CNV-seq analysis revealed six cases of maternal contamination, three cases of triploidy, and one case of absence of heterozygosity (AOH).\u003c/p\u003e \u003cp\u003eThere are many causes of spontaneous abortion, including embryo chromosomal abnormalities, genetic factors, reproductive tract abnormalities, hormonal imbalances, maternal systemic diseases, immune factors, infectious diseases, toxins, environmental factors, poor lifestyle habits, as well as other unknown factors(QUINTERO-RONDEROS et al., 2020; KARIM et al., 2017; ZWIERZCHOWSKA et al., 2018; TOSIC-PAJIC et al., 2017), and despite advances in medicine, spontaneous abortion remains a significant health issue. Many couples may face the risk of miscarriage when planning to have children. Genetic factors are one of the main causes, and fetal chromosomal abnormalities are an important genetic factor in fetal miscarriage (ZHANG et al., 2018). Among them, abnormal embryo chromosomal numbers and CNVs are the main causes of spontaneous abortion. In this study, chromosomal structural abnormalities were detected in 12 cases out of 97 cases successful fetal tissue samples (13.9%), with a total of 17 CNVs identified. Among the 17 CNVs detected, 14 were pathogenic CNVs. These pathogenic CNVs can lead to severe developmental delays, malformations, intellectual disabilities, delayed language development, distinctive facial features, hypotonia, and congenital heart disease in embryos or fetuses. One case of 4p16.3 loss was found involving Wolf-Hirschhorn syndrome. Patients with this syndrome typically have low birth weight, failure to thrive after birth, microcephaly, developmental delays, and hypotonia. Facial features include downward-slanting eyelids, a \u0026lsquo;Greek warrior helmet\u0026rsquo; appearance, a short nose, and a very short midsection. They may also suffer from conditions such as iris tumors and epilepsy (YIN et al., 2019). In this case, both parents underwent peripheral blood karyotype testing, and the father was identified as a carrier of a balanced translocation [46,XX, (t 4;9)]. It was recommended that they undergo preimplantation genetic diagnosis (PGD) for genetic disease screening when planning to have children again.\u003c/p\u003e \u003cp\u003eAdditionally, one case involving a 17p13.2-p13.3 deletion associated with Miller-Dieker syndrome was also identified in this study. The main characteristic of this syndrome is lissencephaly (\u0026ldquo;smooth brain\u0026rdquo; lacking normal brain gyri and sulci). Clinical manifestations also include a high and prominent forehead, vertical furrows, narrowed temples, widely spaced eyes, upward-slanting eyelids, short and upward-turned nose, inverted red lips, long, wide and thick upper lip, congenital heart disease, umbilical hernia, and joint contractures. MDS patients typically have severe intellectual disabilities, epilepsy, and shortened lifespan. Both parents in this case underwent peripheral blood karyotype testing, and it was found that the mother was a carrier of a balanced translocation [46,XX, t(2;17)(p23;p13), 21ph+]. Given that there is only a 2/18 chance of having a normal fetus in future pregnancies, it was recommended that they undergo preimplantation genetic diagnosis (PGD) to assess for genetic diseases during reproductive planning.\u003c/p\u003e \u003cp\u003eCNV-seq technology can efficiently and rapidly detect chromosomal structural abnormalities that may lead to abortion, and analysis of the peripheral blood karyotypes of both spouses can guide assessment for future pregnancies. Under normal circumstances, offspring inherit one homologous chromosome from the mother and one from the father. However, cases where a pair of homologous chromosomes in the offspring is entirely or partly derived from either the father or the mother are referred to as absence of heterozygosity (AOH). Although the incidence of AOH is low, it can increase the risk of recessive disease-causing genes that can lead to complex clinical symptoms. In this study, combined STR and CNV-seq testing detected a homozygous phenomenon in the 2p25.3q37.3 region, spanning 242Mb, which may have led to an increased risk of recessive genetic disorders in that specific region. Currently, there is no conclusive evidence that the occurrence of uniparental diploid on chromosome 2 is directly related to embryo termination. Research have found that after ovulation in vitro, oocyte quality decreases with time, and the expression of spindle assembly checkpoint (SAC) in oocytes is negatively correlated with oocyte senescence. Decreased SAC expression also leads to errors in sister chromatid separation during the second meiotic division (MII) of oocytes, thereby increasing the incidence of early embryonic aneuploidy (SHIMOI et al., 2019). In this study, the rate of chromosomal abnormalities in aborted tissues in the \u0026ge;\u0026thinsp;35 age group accounted for 58.8%, and the rate of chromosomal abnormalities in the \u0026lt;\u0026thinsp;35 age group accounted for 48.7%, Although the difference was not statistically significant (P\u0026thinsp;=\u0026thinsp;0.451), the abnormality rate in the \u0026ge;\u0026thinsp;35 group was 10.1% higher than that in the \u0026lt;\u0026thinsp;35 group, indicating a positive correlation between the frequency of abnormal karyotypes and maternal age.\u003c/p\u003e \u003cp\u003eChromosomal abnormalities in fetuses aborted in the first trimester may involve any one or multiple chromosomes, although chromosomal abnormalities in fetuses aborted in the second trimester may be trisomy 13, 18, and 21, sex chromosome aneuploidy, or CNV, which are similar to the types of chromosomal aberrations detected in live birth (ZHANG et al., 2021; LAN et al., 2021). With the development of molecular biology technology, CNV-seq technology has been gradually applied to chromosomal testing of fetuses aborted, mostly used in the detection of chromosome number abnormalities, large fragment deletion/duplication, and copy number variation of pathogenic genomes\u003csup\u003e[19]\u003c/sup\u003e. Although chromosomal karyotyping is considered the gold standard for detecting chromosomal abnormalities, it cannot detect deletions/duplications smaller than 5-10Mb. However, CNV-seq technology can efficiently detect routine chromosomal number abnormalities and identify pathogenic microdeletions/microduplications that cannot be detected by karyotype analysis. It can also detect unknown variations and discover new genetic information associated with diseases, thus improving the detection rate. CNV-seq technology can provide genetic diagnostic evidence for the etiology of miscarriage, guide future pregnancies with reliable laboratory evidence, and offer valuable reference data for the prevention, diagnosis, and treatment of abortion.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e Hunan Natural Science Foundation of China (2023JJ50306).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement of Ethics:\u003c/strong\u003e This study protocol was reviewed and approved by Ethics Committee of Yueyang Central Hospital, approval number [2023-026]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e:\u0026nbsp;The authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003eAuthor Contributions:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMeihua Xie and Hongxia Zhou: Drafting the work or revising it critically for important intellectual content. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.\u003c/p\u003e\n\u003cp\u003eJianlong Zhuang、Jingyi Chen and Hening Li: acquisition and interpretation of data for the work.\u003c/p\u003e\n\u003cp\u003eYanfei Gong and Bojie Li: analysis, or interpretation of data for the work.\u003c/p\u003e\n\u003cp\u003eLiyun Xie and Airong Pi: Final approval of the version to be published.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability declaration\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eThe authors confirm that the data supporting the findings of this study are available within the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCOLLEY E, HAMILTON S, SMITH P, et al. (2019). Potential genetic causes of miscarriage in euploid pregnancies: a systematic review [J]. Hum Reprod Update, 25(4): 452-72.\u003c/li\u003e\n\u003cli\u003eCHEUNG S W, BI W. (2018). Novel applications of array comparative genomic hybridization in molecular diagnostics [J]. Expert Rev Mol Diagn, 18(6): 531-42.\u003c/li\u003e\n\u003cli\u003eDU Y, CHEN L, LIN J, et al. (2018). Chromosomal karyotype in chorionic villi of recurrent spontaneous abortion patients [J]. Biosci Trends, 12(1): 32-9.\u003c/li\u003e\n\u003cli\u003eKARIM S, JAMAL H S, ROUZI A, et al. (2017). Genomic answers for recurrent spontaneous abortion in Saudi Arabia: An array comparative genomic hybridization approach [J]. Reprod Biol, , 17(2): 133-43.\u003c/li\u003e\n\u003cli\u003eKEARNEY H M, THORLAND E C, BROWN K K, et al. (2011). American College of Medical Genetics standards and guidelines for interpretation and reporting of postnatal constitutional copy number variants [J]. Genet Med, 13(7): 680-5.\u003c/li\u003e\n\u003cli\u003eLAN L, WU H, SHE L, et al. (2020). Analysis of copy number variation by sequencing in fetuses with nuchal translucency thickening [J]. J Clin Lab Anal, , 34(8): e23347.\u003c/li\u003e\n\u003cli\u003eLAN L, SHE L, ZHANG B, et al. (2021). Prenatal diagnosis of 913 fetuses samples using copy number variation sequencing [J]. J Gene Med, 23(5): e3324.\u003c/li\u003e\n\u003cli\u003eQUENBY S, GALLOS I D, DHILLON-SMITH R K, et al. (2021). Miscarriage matters: the epidemiological, physical, psychological, and economic costs of early pregnancy loss [J]. Lancet, 397(10285): 1658-67.\u003c/li\u003e\n\u003cli\u003eQUINTERO-RONDEROS P, LAISSUE P. (2020). Genetic Variants Contributing to Early Recurrent Pregnancy Loss Etiology Identified by Sequencing Approaches [J]. Reprod Sci, 27(8): 1541-52.\u003c/li\u003e\n\u003cli\u003eSHIMOI G, TOMITA M, KATAOKA M, et al. (2019). Destabilization of spindle assembly checkpoint causes aneuploidy during meiosis II in murine post-ovulatory aged oocytes [J]. J Reprod Dev, , 65(1): 57-66.\u003c/li\u003e\n\u003cli\u003eSHAFFER L G, BEJJANI B A. (2004). A cytogeneticist\u0026apos;s perspective on genomic microarrays [J]. Hum Reprod Update, 10(3): 221-6.\u003c/li\u003e\n\u003cli\u003eTOSIC-PAJIC J, SEKLIC D, RADENKOVIC J, et al. (2017). Augmented oxidative stress in infertile women with persistent chlamydial infection [J]. Reprod Biol, 17(2): 120-5.\u003c/li\u003e\n\u003cli\u003eXIE W, TAN Y, LI X, et al. (2013). Rapid detection of aneuploidies on a benchtop sequencing platform [J]. Prenat Diagn, 33(3): 232-7.\u003c/li\u003e\n\u003cli\u003eYANG X, SONG Z, WU C, et al. (2018). Constructing a database for the relations between CNV and human genetic diseases via systematic text mining [J]. BMC Bioinformatics, , 19(Suppl 19): 528.\u003c/li\u003e\n\u003cli\u003eYIN⁃ G L H Y L J Z, AIHUA H R C Z L Y. (2019). Prenatal diagnosis and study of Wolf⁃Hirschhorn syndrome [J]. The Journal of Practical Medicine, 35: 10.\u003c/li\u003e\n\u003cli\u003eYANG L, TANG Y, LU M, et al. (2016). Novel rapid molecular diagnosis of fetal chromosomal abnormalities associated with recurrent pregnancy loss [J]. Acta Obstet Gynecol Scand, 95(12): 1433-40.\u003c/li\u003e\n\u003cli\u003eZWIERZCHOWSKA A, IWAN A, HYC A, et al. (2018). Recurrent miscarriage is associated with increased ghrelin mRNA expression in the endometrium- a case-control study [J]. Reprod Biol, 18(1): 12-7.\u003c/li\u003e\n\u003cli\u003eZHANG T, SUN Y, CHEN Z, et al. (2018). Traditional and molecular chromosomal abnormality analysis of products of conception in spontaneous and recurrent miscarriage [J]. BJOG, 125(4): 414-20.\u003c/li\u003e\n\u003cli\u003eZHANG X, HUANG Q, YU Z, et al. (2021). Copy number variation characterization and possible candidate genes in miscarriage and stillbirth by next-generation sequencing analysis [J]. J Gene Med, 23(12): e3383.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Spontaneous abortion, Genome copy number variation sequencing, Genetic diagnosis","lastPublishedDoi":"10.21203/rs.3.rs-4330610/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4330610/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e To detect the relationship between spontaneous abortion and chromosomal abnormalities by genome copy number variation sequencing (CNV-seq), and explore this relationship’s application to the genetic diagnosis of spontaneous abortion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e From June, 2020 to August, 2022, 103 different embryonic tissue samples from patients who suffered spontaneous abortion in Yueyang Central Hospital were detected by CNV-seq, and the results were analyzed and interpreted after excluding maternal cell contamination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e 97 of the 103 cases were detected successfully. A total of 49 cases (50.5%) of abnormal karyotypes were found, including 33 cases of abnormal chromosome number, 12 cases of structural abnormalities (including 14 pathogenic CNVs and 3 variants of uncertain significance,), 3 cases of triploid, and 1 cases of absence of heterozygosity (AOH). The detection rate of chromosomal abnormalities in abortion tissues in the ≥ 35 years old group was higher than that in the \u0026lt;35 years old group (58.8% vs. 48. 7%).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Fetal chromosomal abnormalities are an important cause of abortion, CNV-seq technology can efficiently detect chromosomal number abnormalities, large fragment deletions/duplications and pathogenic genome copy number variants, which greatly improves the detection rate of chromosomal abnormalities and provides a valuable genetic risk assessment to couples trying to reproduce again.\u003c/p\u003e","manuscriptTitle":"A clinical study of CNV-seq techniques in 103 cases of spontaneous abortion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-10 20:21:26","doi":"10.21203/rs.3.rs-4330610/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"22c4b065-acc3-4f9e-9d82-1c5106d45db7","owner":[],"postedDate":"May 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-08T13:54:01+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-10 20:21:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4330610","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4330610","identity":"rs-4330610","version":["v1"]},"buildId":"cTy_lsJlmDsVRNrSptgXS","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}