Borderline Z-scores in Non-Invasive Prenatal Screening: Does Its Presence Hold Clinical Significance?

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Xiaoli pan, Lixin Wen, Yun Pan, Shuqing Pan, Shanshan Wu, Changshui Chen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6781560/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Sep, 2025 Read the published version in Archives of Gynecology and Obstetrics → Version 1 posted 9 You are reading this latest preprint version Abstract Background This study aimed to investigate the utility of repeated testing for the detection of fetal cell-free DNA (cfDNA) from maternal peripheral blood in cases with borderline Z-scores, and to analyze the associated pregnancy outcomes. Methods A retrospective analysis was conducted on 83,443 pregnant women who voluntarily underwent non-invasive prenatal testing (NIPT) at the Affiliated Women and Children's Hospital of Ningbo University between January 2020 and January 2024. Pregnant women whose initial NIPT results indicated borderline Z-scores were subsequently followed up. Results Among 83,443 pregnant women undergoing NIPT, 700 cases (0.84%) initially showed borderline Z-scores. After retesting, this number decreased to 211 cases (0.25%), and further decreased to 26 cases (0.03%) after re-sampling. Among the initial 700 cases, 34 exhibited abnormal NIPT results, corresponding to a positive rate of 5.29%. Subsequent prenatal diagnosis confirmed a total of 6 fetal abnormalities, including one case of trisomy 13 mosaicism, one case of trisomy 21 mosaicism, one case of 47,XXY, two cases of copy number variations (CNVs), and one case with B-ultrasound findings indicating an abnormality (nuchal translucency [NT] of 3.5 mm and omphalocele). The overall rate of fetal abnormalities was 0.9%. Conclusion Initial NIPT outcomes suggested elevated screening positive rates and a higher incidence of fetal abnormalities among cases with borderline Z-scores compared to the general population. Establishing a defined borderline Z-score threshold in NIPT protocols is crucial to mitigate the risk of missed screenings. Implementing reconstruction and/or re-sampling procedures significantly reduces the failure rate attributed to borderline Z-scores, facilitating the accurate identification of most of pregnancies with normal fetal development and decreasing the need for unnecessary invasive prenatal diagnostic interventions. Pregnant women with detection failures due to borderline Z-scores should be actively counseled and encouraged to pursue prenatal diagnosis. NIPT borderline Z- score prenatal diagnosis positive rate library construction Figures Figure 1 INTRODUCTION Non-invasive prenatal testing (NIPT) is a screening method that analyzes cell-free fetal DNA (cffDNA) in the maternal peripheral blood through high-throughput sequencing. The sequencing data is compared with a model of normal pregnant women's data and risk assessment is performed to determine the risk of fetal chromosomal abnormalities. NIPT has high specificity and sensitivity for fetal chromosomal diseases, especially for trisomy 21, trisomy 18, and trisomy 13, making it an important part of prenatal screening [ 1 – 3 ] . For fetal chromosomal aneuploidies, results are typically expressed as Z-scores, indicating whether there is a statistically significant difference between the tested sample and normal pregnant women. The Z-score is a risk judgment value obtained after processing the sequencing data with GC correction and other treatments [ 4 ] . The normal fetal Z-score range is -3 to + 3. When the Z-score is ≥ 3, it suggests a high risk of chromosomal aneuploidies in the fetus, and the higher the Z-score, the higher the positive predictive value (PPV) [ 5 ] . In practical application, due to the inherent variability in experimental data, different NIPT technology platforms set a borderline Z-score threshold, also known as the "gray zone" for Z-scores, it cannot be definitively classified as high-risk or low-risk, leading to uncertainty results. If a borderline Z-score is not established, some normal fetuses may be misclassified as high-risk, resulting in an increased false-positive rate and unnecessary invasive prenatal diagnostic procedures. Conversely, some abnormal fetuses may be missed, leading to false-negative results. Research indicates that the borderline Z-score is a primary cause of NIPT experimental failure, and repeated testing can effectively reduce the false-positive rate associated with the borderline Z-score [ 6 – 7 ] . This study conducted a retrospective analysis of samples that fell within the borderline Z-score range from January 2020 to January 2024, followed up on their pregnancy outcomes, and discussed the necessity of setting a Z-score threshold in the detection of cell-free fetal DNA in peripheral blood and the value of repeat testing. METHODS Study Subjects Data were collected from 83,443 pregnant women who underwent NIPT at Ningbo University Affiliated Women and Children's Hospital from January 2020 to January 2024. Inclusion criteria: voluntarily accepted NIPT testing and were at a gestational age of 12 to 26 + 6 weeks. Exclusion criteria: received allogeneic blood transfusion, transplantation surgery, etc., within the past year; received immunoglobulin treatment within 4 weeks; triplets or more; ultrasound indicated fetal structural abnormalities; had a clear history of chromosomal abnormalities or a family history of genetic diseases; coexistence with malignant tumors. For pregnant women at high risk of NIPT, invasive prenatal diagnosis was performed after informed consent was obtained. All pregnant women signed an informed consent form. This study has been reviewed and approved by the hospital's ethics committee (2024KYSL-068). Research Methods NIPT Testing Peripheral blood samples of 10 mL were collected from pregnant women using K- tubes, and plasma was separated within 72 hours. The extraction and construction of cell-free DNA libraries were performed using the Fetal Chromosomal Aneuploidy (T21, T18, T13) Detection Kit (based on the Combinatorial Probe-Anchor Synthesis Sequencing Method) and the MGISP-960 High-Throughput Automated Nucleic Acid Extraction and Library Preparation System. High-throughput sequencing was conducted using the Sequencing Reaction Universal Reagent Kit (based on the Combinatorial Probe-Anchor Synthesis Sequencing Method) and the MGISEQ-2000 sequencing platform. All reagents and instruments were procured from BGI Genomics Co., Ltd., Shenzhen, China. Following sequencing, the generated data were analyzed to determine the Z-scores for chromosomes 21, 18, and 13. A Z-score within the range of -3 to + 3 indicates a low-risk, while a Z-score of ≥ 3 suggests a high-risk. Any other abnormalities were reported in an additional supplementary report format. In this study, the BGI HALOS Informatics Analysis System was employed, which has established a borderline Z-score based on accumulated experimental data. When the NIPT results showed a Z-score within the range of + 1.96 to + 3, the system flagged the sample as falling within the borderline Z-score zone, prompting a re-construction of the DNA library. If the re-constructed library results still indicated a borderline Z-score, the pregnant woman was notified via telephone to re-sampled for further verification. For cases where both blood draws consistently indicated a borderline Z-score, invasive prenatal diagnostic procedures were recommended. Prenatal diagnosis For pregnant women at high risk based on NIPT results, amniocentesis is performed under ultrasound guidance after informed consent, and 35ml of amniotic fluid is extracted for prenatal diagnosis (chromosomal karyotype analysis and/or CMA testing). Chromosomal karyotype analysis: Amniotic fluid cells are cultured, harvested, prepared, and analyzed for karyotype after G-banding, with karyotype descriptions based on the International System for Human Cytogenetic Nomenclature (ISCN2016). CMA testing uses the whole-genome CytoScantm 750k chip (Affymetrix, USA), and the ChAS4.0 (Chromosome Analysis Suite 4.0, Affymetrix) software is used to calculate and analyze the scanning image results, referring to international public databases including DGV, ClinGen, ClinVar, etc., to assess the clinical significance of copy number variations. Statistical Methods Data were statistically analyzed using SPSS 19.0 software. Categorical data are represented as n, and inter-group comparisons were made using the chi-square test, with a P-value of less than 0.05 indicating statistical significance. Quantitative data are expressed as percentages. RESULTS General Situation of NIPT Test Failures Among 83,443 pregnant women, the initial NIPT results indicated a borderline Z-score in 700 cases (0.84%, 700/83,443). Following library re-construction, 211 cases (0.25%, 211/83,443) remained within the borderline Z-score. After repeat blood sampling, 26 cases (0.03%, 26/83,443) still exhibited borderline Z-scores. Among the 700 cases requiring library re-construction, the most frequently observed borderline Z-scores were associated with T18 (37.9%, 265/700), followed by T21 (35.3%, 247/700) and T13 (22.7%, 159/700). Similarly, among the 206 cases requiring re-sampled, the most common borderline Z-scores were for T18 (45.1%, 93/206), followed by T21 (28.1%, 58/206) and T13 (18.4%, 38/206).Out of the 700 pregnant women, 34 cases (5.29%, 34/700) showed abnormal NIPT results. Further details are summarized in Table 1 and Figure 1. Table 1. The distribution of borderline Z-score types among the 700 pregnant women after re-construction or re-sampling. borderline Z-score types negative after re-construction positive after re-construction other chromosomal abnormalities after re-construction borderline after retest total negative after re-sampled positive re-sampled other chromosomal abnormalities after re-sampled borderline after re-sampled no re-sampled total T13 117 3 0 29 1 1 7 1 39 159 T21 181 2 4 48 1 3 6 2 60 247 T18 163 4 3 72 8 2 11 2 95 265 T13、T18 2 0 0 5 0 0 1 0 6 8 T13、T21 6 0 0 1 0 0 0 0 1 7 T21、T18 2 0 0 5 1 0 1 0 7 9 T13、T21、T18 1 0 1 3 0 0 0 0 3 5 Total 472 9 8 163 11 6 26 5 211 700 Prenatal Diagnosis Situation Among the 700 pregnant women, 65 cases were identified by NIPT as high-risk or other chromosomal abnormalities, no re-sampled, and experienced NIPT detection failure due to borderline Z-scores.. They were advised to seek prenatal diagnostic consultation. Among the 65 pregnant women, 31 underwent invasive prenatal diagnosis, 29 refused invasive prenatal diagnosis, and 5 were lost to follow-up. The prenatal diagnosis rates for cases with high-risk results, other chromosomal abnormalities, no re-sampled, and borderline Z-scores were 80%(16/20), 50%(7/14), 0%(0/5), and 23.1%(6/26), respectively（Table 2）. Table 2. Prenatal Diagnosis Situation of 65 Pregnant Women NIPT results n underwent prenatal diagnosis did not underwent prenatal diagnosis loss to follow-up true positive other chromosomal abnormalities False positive total induced abortion birth total high-risk 20 2 1 13 16 1 3 4 0 other chromosomal abnormalities 14 0 1 7 8 0 6 6 0 no re-sampled 5 0 0 0 0 0 4 a 4 1 borderline Z-scores 26 0 1 6 7 0 15 15 4 合计 65 2 3 26 31 1 28 29 5 Note: a: In one case of a twin pregnancy, selective reduction was performed due to prenatal diagnosis ultrasound indicating abnormalities in one fetus, while the other fetus was born. Prenatal Diagnosis Results Among the pregnant women who underwent prenatal diagnostic consultation, 5 cases of chromosomal abnormalities were detected, including 1 case of trisomy 13 mosaicism,1case of trisomy 21 mosaicism, 1 case of Klinefelter syndrome, and 2 cases of Copy Number Variation (CNV) abnormalities. Among these, 1 of the CNV abnormalities was classified as a variant of uncertain clinical significance (VUS), the pregnant woman chose to continue the pregnancy, and no abnormalities were observed postnatally. The remaining 4 pregnant women opted for induced abortion. Among the pregnant women who did not undergo prenatal diagnosis, 1 case with a borderline trisomy 18 at 16 weeks of gestation experienced an unexplained miscarriage, and 1 case with borderline trisomy 18 in a twin pregnancy chose fetal reduction due to abnormal findings on prenatal diagnostic ultrasound(NT 5.3 mm, omphalocele)in one of the fetuses. In summary, the fetal abnormality rate was 0.9%(6/700).Follow-up did not identify any false-negative cases. For details, see Table 3. Table 3. Prenatal Diagnosis Results of 6 Pregnant Women case Age (years) Gestational age(weeks) clinical indicators borderline Z-score types First Z-score Z-score after re-construction NIPT results prenatal diagnosis results follow-up 1 27 18 +4 abnormal MOM T13 2.727 3.000 T13 arr[hg19]15q21.1(44,927,065-45,904,982)x3 birth 2 34 13 +3 voluntary request T21 2.912 3.570 T21 46，XN，1qh+,rob(21:21)[17]/46,XN,1qh+[40] induced abortion 3 39 17 maternal age>35 T13 2.146 3.29 T13 47,X?,+13[34]/46,X?[4] induced abortion 4 36 14 +2 maternal age>35 T21 2.997 0.558 XXY 47，XXY induced abortion 5 23 21 +4 serological screening borderline risk T21 2.877 2.048 detection failure arr[hg19]3q29(195,703,616-197,356,334)x1 induced abortion 6 34 15 IVF twins T18 2.014 2.541 detection failure NT 5.3 mm, omphalocele fetal reduction True Positive Detection Rates for Different Borderline Z-score Types Using invasive prenatal diagnosis and/or prenatal diagnostic ultrasound as the gold standard, the true positive detection rates for different types of borderline Z-score were analyzed. The results showed that the true positive rates for T13,T21,and T18 borderline Z-score were 1.3%,1.2%,and 0.4%,respectively,with positive predictive values(PPVs)of 40%,30%,and 6.7%,respectively.No true positives were detected in the other cases(Table 4). Table 4. True Positive Detection Rates for Different Borderline Z-score Types borderline Z-score types number of individuals in the borderline zone number of individuals in the borderline zone after retest underwent prenatal diagnosis true positive PPV（%） n % a T13 159 38 5 2 1.3 40 T21 247 58 10 3 1.2 30 T18 265 93 15 1 0.4 6.7 other b 29 17 1 0 0 0 total 700 206 31 6 0.9 19.4 Note: PPV = True Positives / Number of Prenatal Diagnostic Procedures; a: True Positive Rate = True Positives / Number of Critical Zone Individuals (initial); b: Individuals with two or more types of critical zone categories. DISCUSSION The National Health and Family Planning Commission issued the "Technical Specifications for Prenatal Screening and Diagnosis of Cell-Free Fetal DNA in Maternal Peripheral Blood," which states that the failure rate of NIPT should not exceed 5% [ 8 ] . An increasing number of domestic and international scholars have conducted analyses and discussions on the reasons of NIPT failure [ 9 – 10 ] . The NIPT testing process is lengthy and complex, susceptible to various influencing factors, including sample-related issues such as hemolysis and coagulation, as well as experimental procedural issues such as DNA extraction and library construction failures. Therefore, in practical clinical practice, NIPT testing often requires library reconstruction and/or re-sampled to reduce the failure rate. The borderline Z-score is a common reason for library re-construction and/or re-sampled in NIPT. The Technical Specifications for Prenatal Screening and Diagnosis of Cell-Free Fetal DNA in Maternal Peripheral Blood defines the serum screening risk value between the high-risk cutoff and 1/1000 as the borderline risk [ 8 ] . The chromosomal abnormality rate of the serum screening borderline risk group is significantly higher than that of the low-risk group [ 11 ] . Therefore, the author believes that a borderline Z-score may be established in NIPT. In this study, among the 700 pregnant women whose initial NIPT results indicated a borderline Z-score, 34 cases with abnormal results were detected, with a screening positive rate of 5.29% (34/700). Following prenatal diagnosis, a total of 6 fetal abnormalities were detected (including 1 case with prenatal diagnosis ultrasound indicating abnormalities and 5 cases with invasive prenatal diagnosis indicating chromosomal abnormalities), with a fetal abnormality rate of 0.9% (6/700). Without the establishment of a borderline Z-score, these abnormal fetuses might have been missed. This underscores the necessity of setting a borderline Z-score zone in NIPT to reduce the rate of missed screenings. Pan Shuqing et al. [ 12 ] analyzed over 50,000 NIPT screening cases from this laboratory, reporting screening positive and fetal abnormal rates of 0.6% (326/54957) and 0.2% (135/54957), respectively, which are significantly lower than those observed in this study. Similarly, Xiang L et al. [ 1 ] and Liu Jing et al. [ 3 ] analyzed NIPT screening data from the general population, and their screening positive and fetal abnormal rates were significantly lower than those in this study. These findings indicate that the screening positive rate and fetal abnormality rate in cases with initial NIPT results indicating a borderline Z-score are higher than those in the general population, which is consistent with the studies by Luo Yanmei et al. [ 6 ] , Chan N et al. [ 13 ] , and Lu Y et al. [ 14 ] . This study analyzed NIPT-related data from over 80,000 pregnant women and identified 700 cases (0.84%, 700/83,443) with initial borderline Z-scores (Z-scores between 1.96 and 3), which is lower than the 2.62% (160/6110) reported by Qi Hong et al. [ 7 ] and the 1.64% (663/40311) reported by Luo Yanmei et al [ 6 ] . This may be due to different sequencing platforms and differences in the definition range of the borderline Z-score, resulting in a significant difference in the detection failure rate (borderline Z-score). After reconstruction, 211 cases were indicated as the borderline Z-score, indicating a repeat sampling rate (borderline Z-score) of 0.25% (211/83,443), which is consistent with the aforementioned studies [ 6 – 7 ] . After re-sampled, NIPT results indicated that 26 cases failed due to the borderline Z-score, accounting for 0.03% (26/83443). This demonstrates that library reconstruction and/or re-sampled can greatly reduce the NIPT detection failure rate (borderline Z-score), decreasing it from 0.84–0.03%, possibly because as the gestational age increases, the fetal concentration increases, enhancing the accuracy of NIPT detection. Ultimately, among the 700 pregnant women, 635 cases (90.71%, 635/700) received low-risk results, 34 cases (4.86%, 34/700) indicated abnormal NIPT results, 26 cases indicated NIPT detection failure, and 5 pregnant women did not cooperate with re-drawing blood. Follow-up did not find any false negatives. It can be seen that reconstruction and/or re-drawing can screen out most normal pregnant women, effectively reducing unnecessary invasive prenatal diagnoses and alleviating the psychological burden on pregnant women. In addition, this study found that the true positive detection rate varies for different borderline Z-score types. The true positive rate for the T21 borderline is the highest (1.3%), followed by the T13 borderline (1.2%) and the T18 borderline (0.4%), with the positive predictive value (PPV) also decreasing accordingly, indicating that the T18 borderline has the highest "false positive rate." When two or more borderline types coexist, no true positives were detected, mostly caused by data fluctuations. This may guide clinical genetic counseling for such populations. Additionally, this study found that when NIPT results indicate abnormal results, pregnant women have good compliance with prenatal diagnosis, but they do not pay enough attention to the borderline Z-score, with 80.6% (25/31) of pregnant women not choosing prenatal diagnosis (see Table 2 ). Therefore, for pregnant women who fail detection due to the borderline Z-score, prenatal genetic counseling should be strengthened, actively guiding pregnant women to undergo further examinations to prevent missed screenings. Mitotic or meiotic non-disjunction errors can lead to the formation of mosaicism, resulting in the existence of two or more cell lines in the fetus and placenta [ 15 ] . Placental mosaicism is the main cause of false negatives and false positives in NIPT [ 16 ] . Among the 5 cases of fetal chromosomal abnormalities, 2 were mosaic types, including 1 case of translocation-type trisomy 21 mosaicism with a mosaic ratio of 40%, and another case of trisomy 13 mosaicism with a mosaic ratio of 43%. Both cases had two cell lines in the fetus, possibly due to meiotic errors forming a triploid zygote, and then through mitotic in subsequent mitosis, losing the extra chromosomes, ultimately leading to the coexistence of normal diploid and abnormal triploid [ 17 ] . Both cases initially showed borderline Z-score, and after library reconstruction, the Z-score exceeded 3, showing that multiple NIPT results with Z-scores near 3 may indicate the possibility of fetal and placental mosaicism. The other 3 cases were other chromosomal abnormalities, possibly due to abnormal chromosome fragments affecting the calculation of Z-scores. In addition, 1 case of twins with one abnormality leading to a borderline Z-scores was found. A 34-year-old pregnant woman transplanted two fresh embryos, and at 13 weeks of pregnancy, ultrasound indicated one twin with NT 5.3mm, dotted nasal bone, and omphalocele. It is speculated that the abnormal fetus may be trisomy 18, and the abnormal fragments released by the abnormal fetus neutralize with the normal fragments of the normal fetus, reducing the concentration of abnormal fragments and resulting in a borderline Z-score for trisomy 18 in NIPT. In summary, among pregnant women with initial NIPT results indicating borderline Z-scores, both the screening positive rate and fetal abnormality rate are higher than those in the general population. Therefore, it is necessary to establish a borderline Z-score in NIPT to reduce the rate of missed screenings. Additionally, library reconstruction and/or re-drawing can significantly decrease the NIPT detection failure rate caused by the borderline Z-score, effectively identifying the majority of normal pregnancies and reducing unnecessary invasive prenatal diagnoses. For pregnant women who fail detection due to the borderline Z-score, active guidance should be provided to encourage further diagnostic testing to prevent missed screenings Declarations Author Contribution Xiaoli Pan wrote the main manuscript text .Wen prepared all figures.Yun pan and Shuqing Pan collected data.Wu、Chen and Li reviewed the manuscript. References Xiang L, Zhu J, Deng K, et al. Non-invasive prenatal testing for the detection of trisomies 21, 18, and 13 in pregnant women with various clinical indications: A multicenter observational study of 1,854,148 women in China [J]. 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Expert Rev Mol Diagn, 2022, 22(3):387-394. doi: 10.1080/14737159.2022.2049245. Zhang HY, Fu ML, Wang W. Analysis of biological causes of false positives and false negatives in non-invasive prenatal genetic testing for chromosomal aneuploidies[J].Chinese Journal of Prenatal Diagnosis(Electronic Edition),2017,9(3):48-58.DOI:10.13470/j.cnki.cjpd.2017.03.010. Fu ML. Research on the Causes of False Negatives and False Positives in Non-Invasive Prenatal Genetic Testing and the Establishment of a Laboratory Investigation Protocol[D].South China University of Technology,2020. Sirchia SM, Garagiola I, Colucci G, et al. Trisomic zygote rescue revealed by DNA polymorphism analysis in confined placental mosaicism [J]. Prenat Diagn, 1998, 18(3):201-206.doi:10.1002/(sici)1097-0223(199803)18:33.0.co;2-w Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 30 Sep, 2025 Read the published version in Archives of Gynecology and Obstetrics → Version 1 posted Editorial decision: Revision requested 26 Aug, 2025 Reviewers agreed at journal 26 Jul, 2025 Reviewers agreed at journal 20 Jul, 2025 Reviews received at journal 11 Jul, 2025 Reviewers agreed at journal 21 Jun, 2025 Reviewers invited by journal 04 Jun, 2025 Editor assigned by journal 02 Jun, 2025 Submission checks completed at journal 30 May, 2025 First submitted to journal 30 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board 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-6781560","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":466557547,"identity":"f89e6136-b676-45f7-9f7c-33a639da5fac","order_by":0,"name":"Xiaoli pan","email":"","orcid":"","institution":"Women and Children’s Hospital of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoli","middleName":"","lastName":"pan","suffix":""},{"id":466557548,"identity":"d93c6e1a-442d-43cc-a0c5-6dd54ef0c226","order_by":1,"name":"Lixin Wen","email":"","orcid":"","institution":"The laboratory of Ningbo Yinzhou District Maternal and Child Health Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lixin","middleName":"","lastName":"Wen","suffix":""},{"id":466557549,"identity":"5b48ce54-9c85-45bd-bda3-39990d023a91","order_by":2,"name":"Yun Pan","email":"","orcid":"","institution":"Women and Children’s Hospital of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Pan","suffix":""},{"id":466557550,"identity":"13dbfffb-9565-4bdf-ba6d-1821d87d05e9","order_by":3,"name":"Shuqing Pan","email":"","orcid":"","institution":"Women and Children’s Hospital of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Shuqing","middleName":"","lastName":"Pan","suffix":""},{"id":466557551,"identity":"17bf9194-2c3e-4a72-9a2e-50f54bbeb415","order_by":4,"name":"Shanshan Wu","email":"","orcid":"","institution":"Women and Children’s Hospital of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Shanshan","middleName":"","lastName":"Wu","suffix":""},{"id":466557552,"identity":"e48b3d63-6f26-4e58-b0ca-9ac2d813ec51","order_by":5,"name":"Changshui Chen","email":"","orcid":"","institution":"Women and Children’s Hospital of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Changshui","middleName":"","lastName":"Chen","suffix":""},{"id":466557553,"identity":"4b35293c-d708-468d-abca-4e1121f68023","order_by":6,"name":"Haibo Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIie3QMQrCMACF4VcCcYl2jSCeISAUB8GrNAidFBw7CHaQdhA9i6OjReiU7jrZnkDddDNFnBRTN4f8cz6SF8Bm+8NahAEyAoanc1n44cxM6ItgNyaiUFkNAk2cJ6HtckFqkEYzvZTbgWZ5FsqIwk2WvuFhrRGXKgBx1sFBbjvgKt+Ytggu4/08JvAOUlEIPjGS3k0TMApvKjWsQ7zqFnDGPNQm/WqL4HTEfZUx4xbXVb3jXf+Y4CS93sJZ101W38lb7LfjNpvNZvvYA+aeQLxDD/QuAAAAAElFTkSuQmCC","orcid":"","institution":"Women and Children’s Hospital of Ningbo University","correspondingAuthor":true,"prefix":"","firstName":"Haibo","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-05-30 06:23:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6781560/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6781560/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00404-025-08193-2","type":"published","date":"2025-09-30T15:58:07+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84215915,"identity":"85f6a447-532b-4b50-8d7f-0ce2494ce1ef","added_by":"auto","created_at":"2025-06-09 10:39:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":32132,"visible":true,"origin":"","legend":"\u003cp\u003eThe NIPT results of the 700 pregnant women with borderline Z-score after re-construction or re-sampling.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6781560/v1/78e547d66f7d384910600938.png"},{"id":92884008,"identity":"009ad2f7-86ae-463b-a2fc-2eb87b008b84","added_by":"auto","created_at":"2025-10-06 16:12:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":605923,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6781560/v1/3b1b0e69-63fc-47ef-9084-71f228b3a199.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Borderline Z-scores in Non-Invasive Prenatal Screening: Does Its Presence Hold Clinical Significance?","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eNon-invasive prenatal testing (NIPT) is a screening method that analyzes cell-free fetal DNA (cffDNA) in the maternal peripheral blood through high-throughput sequencing. The sequencing data is compared with a model of normal pregnant women's data and risk assessment is performed to determine the risk of fetal chromosomal abnormalities. NIPT has high specificity and sensitivity for fetal chromosomal diseases, especially for trisomy 21, trisomy 18, and trisomy 13, making it an important part of prenatal screening \u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. For fetal chromosomal aneuploidies, results are typically expressed as Z-scores, indicating whether there is a statistically significant difference between the tested sample and normal pregnant women. The Z-score is a risk judgment value obtained after processing the sequencing data with GC correction and other treatments \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. The normal fetal Z-score range is -3 to +\u0026thinsp;3. When the Z-score is \u0026ge;\u0026thinsp;3, it suggests a high risk of chromosomal aneuploidies in the fetus, and the higher the Z-score, the higher the positive predictive value (PPV) \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn practical application, due to the inherent variability in experimental data, different NIPT technology platforms set a borderline Z-score threshold, also known as the \"gray zone\" for Z-scores, it cannot be definitively classified as high-risk or low-risk, leading to uncertainty results. If a borderline Z-score is not established, some normal fetuses may be misclassified as high-risk, resulting in an increased false-positive rate and unnecessary invasive prenatal diagnostic procedures. Conversely, some abnormal fetuses may be missed, leading to false-negative results. Research indicates that the borderline Z-score is a primary cause of NIPT experimental failure, and repeated testing can effectively reduce the false-positive rate associated with the borderline Z-score\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. This study conducted a retrospective analysis of samples that fell within the borderline Z-score range from January 2020 to January 2024, followed up on their pregnancy outcomes, and discussed the necessity of setting a Z-score threshold in the detection of cell-free fetal DNA in peripheral blood and the value of repeat testing.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Subjects\u003c/h2\u003e \u003cp\u003eData were collected from 83,443 pregnant women who underwent NIPT at Ningbo University Affiliated Women and Children's Hospital from January 2020 to January 2024. Inclusion criteria: voluntarily accepted NIPT testing and were at a gestational age of 12 to 26\u003csup\u003e+\u0026thinsp;6\u003c/sup\u003e weeks. Exclusion criteria: received allogeneic blood transfusion, transplantation surgery, etc., within the past year; received immunoglobulin treatment within 4 weeks; triplets or more; ultrasound indicated fetal structural abnormalities; had a clear history of chromosomal abnormalities or a family history of genetic diseases; coexistence with malignant tumors. For pregnant women at high risk of NIPT, invasive prenatal diagnosis was performed after informed consent was obtained.\u003c/p\u003e \u003cp\u003eAll pregnant women signed an informed consent form. This study has been reviewed and approved by the hospital's ethics committee (2024KYSL-068).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eResearch Methods\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eNIPT Testing\u003c/h2\u003e \u003cp\u003ePeripheral blood samples of 10 mL were collected from pregnant women using K- tubes, and plasma was separated within 72 hours. The extraction and construction of cell-free DNA libraries were performed using the Fetal Chromosomal Aneuploidy (T21, T18, T13) Detection Kit (based on the Combinatorial Probe-Anchor Synthesis Sequencing Method) and the MGISP-960 High-Throughput Automated Nucleic Acid Extraction and Library Preparation System. High-throughput sequencing was conducted using the Sequencing Reaction Universal Reagent Kit (based on the Combinatorial Probe-Anchor Synthesis Sequencing Method) and the MGISEQ-2000 sequencing platform. All reagents and instruments were procured from BGI Genomics Co., Ltd., Shenzhen, China. Following sequencing, the generated data were analyzed to determine the Z-scores for chromosomes 21, 18, and 13. A Z-score within the range of -3 to +\u0026thinsp;3 indicates a low-risk, while a Z-score of \u0026ge;\u0026thinsp;3 suggests a high-risk. Any other abnormalities were reported in an additional supplementary report format.\u003c/p\u003e \u003cp\u003eIn this study, the BGI HALOS Informatics Analysis System was employed, which has established a borderline Z-score based on accumulated experimental data. When the NIPT results showed a Z-score within the range of +\u0026thinsp;1.96 to +\u0026thinsp;3, the system flagged the sample as falling within the borderline Z-score zone, prompting a re-construction of the DNA library. If the re-constructed library results still indicated a borderline Z-score, the pregnant woman was notified via telephone to re-sampled for further verification. For cases where both blood draws consistently indicated a borderline Z-score, invasive prenatal diagnostic procedures were recommended.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePrenatal diagnosis\u003c/h3\u003e\n\u003cp\u003eFor pregnant women at high risk based on NIPT results, amniocentesis is performed under ultrasound guidance after informed consent, and 35ml of amniotic fluid is extracted for prenatal diagnosis (chromosomal karyotype analysis and/or CMA testing). Chromosomal karyotype analysis: Amniotic fluid cells are cultured, harvested, prepared, and analyzed for karyotype after G-banding, with karyotype descriptions based on the International System for Human Cytogenetic Nomenclature (ISCN2016). CMA testing uses the whole-genome CytoScantm 750k chip (Affymetrix, USA), and the ChAS4.0 (Chromosome Analysis Suite 4.0, Affymetrix) software is used to calculate and analyze the scanning image results, referring to international public databases including DGV, ClinGen, ClinVar, etc., to assess the clinical significance of copy number variations.\u003c/p\u003e\n\u003ch3\u003eStatistical Methods\u003c/h3\u003e\n\u003cp\u003eData were statistically analyzed using SPSS 19.0 software. Categorical data are represented as n, and inter-group comparisons were made using the chi-square test, with a P-value of less than 0.05 indicating statistical significance. Quantitative data are expressed as percentages.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eGeneral Situation of NIPT Test Failures\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong 83,443 pregnant women, the initial NIPT results indicated a borderline Z-score in 700 cases (0.84%, 700/83,443). Following library re-construction, 211 cases (0.25%, 211/83,443) remained within the borderline Z-score. After repeat blood sampling, 26 cases (0.03%, 26/83,443) still exhibited borderline Z-scores. Among the 700 cases requiring library re-construction, the most frequently observed borderline Z-scores were associated with T18 (37.9%, 265/700), followed by T21 (35.3%, 247/700) and T13 (22.7%, 159/700). Similarly, among the 206 cases requiring re-sampled, the most common borderline Z-scores were for T18 (45.1%, 93/206), followed by T21 (28.1%, 58/206) and T13 (18.4%, 38/206).Out of the 700 pregnant women, 34 cases (5.29%, 34/700) showed abnormal NIPT results. Further details are summarized in Table 1 and Figure 1.\u003c/p\u003e\n\u003cp\u003eTable 1. The distribution of borderline Z-score types among the 700 pregnant women after re-construction or re-sampling.\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"690\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 101px;\"\u003e\n \u003cp\u003eborderline Z-score types\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 60px;\"\u003e\n \u003cp\u003enegative after re-construction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003epositive\u0026nbsp;after re-construction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003eother chromosomal abnormalities after re-construction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"6\" valign=\"top\" style=\"width: 350px;\"\u003e\n \u003cp\u003eborderline after retest\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 47px;\"\u003e\n \u003cp\u003etotal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003enegative after re-sampled\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003epositive\u0026nbsp;re-sampled\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eother chromosomal abnormalities after re-sampled\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eborderline after re-sampled\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eno re-sampled\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003etotal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eT13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e117\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e159\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eT21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e181\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e247\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eT18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e163\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e265\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eT13、T18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eT13、T21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eT21、T18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eT13、T21、T18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e472\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e163\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e211\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e700\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003ePrenatal Diagnosis Situation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the 700 pregnant women, 65 cases were identified by NIPT as high-risk or other chromosomal abnormalities, no re-sampled, and experienced NIPT detection failure due to borderline Z-scores.. They were advised to seek prenatal diagnostic consultation. Among the 65 pregnant women, 31 underwent invasive prenatal diagnosis, 29 refused invasive prenatal diagnosis, and 5 were lost to follow-up. The prenatal diagnosis rates for cases with high-risk results, other chromosomal abnormalities, no re-sampled, and borderline Z-scores were 80%(16/20), 50%(7/14), 0%(0/5), and 23.1%(6/26), respectively（Table 2）.\u003c/p\u003e\n\u003cp\u003eTable 2. Prenatal Diagnosis Situation of 65 Pregnant Women\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"680\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 132px;\"\u003e\n \u003cp\u003eNIPT results\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 38px;\"\u003e\n \u003cp\u003en\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 293px;\"\u003e\n \u003cp\u003eunderwent prenatal diagnosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003edid not underwent prenatal diagnosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003eloss to follow-up\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003etrue positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eother chromosomal abnormalities\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eFalse positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003etotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003einduced abortion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003ebirth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003etotal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003ehigh-risk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eother chromosomal abnormalities\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eno re-sampled\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eborderline Z-scores\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e合计\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: a: In one case of a twin pregnancy, selective reduction was performed due to prenatal diagnosis ultrasound indicating abnormalities in one fetus, while the other fetus was born.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrenatal Diagnosis Results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the pregnant women who underwent prenatal diagnostic consultation, 5 cases of chromosomal abnormalities were detected, including 1 case of trisomy 13 mosaicism,1case of trisomy 21 mosaicism, 1 case of Klinefelter syndrome, and 2 cases of Copy Number Variation (CNV) abnormalities. Among these, 1 of the CNV abnormalities was classified as a variant of uncertain clinical significance (VUS), the pregnant woman chose to continue the pregnancy, and no abnormalities were observed postnatally. The remaining 4 pregnant women opted for induced abortion. Among the pregnant women who did not undergo prenatal diagnosis, 1 case with a borderline trisomy 18 at 16 weeks of gestation experienced an unexplained miscarriage, and 1 case with borderline trisomy 18 in a twin pregnancy chose fetal reduction due to abnormal findings on prenatal diagnostic ultrasound(NT 5.3 mm, omphalocele)in one of the fetuses. In summary, the fetal abnormality rate was 0.9%(6/700).Follow-up did not identify any false-negative cases. For details, see Table 3.\u003c/p\u003e\n\u003cp\u003eTable 3. Prenatal Diagnosis Results of 6 Pregnant Women\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"739\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003ecase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003cp\u003e(years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eGestational age(weeks)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eclinical indicators\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eborderline Z-score types\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003eFirst Z-score\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eZ-score after re-construction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eNIPT results\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003eprenatal diagnosis results\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003efollow-up\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e18\u003csup\u003e+4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eabnormal MOM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eT13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e2.727\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e3.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eT13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003earr[hg19]15q21.1(44,927,065-45,904,982)x3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003ebirth\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e13\u003csup\u003e+3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003evoluntary request\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eT21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e2.912\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e3.570\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eT21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003e46，XN，1qh+,rob(21:21)[17]/46,XN,1qh+[40]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003einduced abortion\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003ematernal age\u0026gt;35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eT13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e2.146\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e3.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eT13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003e47,X?,+13[34]/46,X?[4]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003einduced abortion\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e14\u003csup\u003e+2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003ematernal age\u0026gt;35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eT21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e2.997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.558\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eXXY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003e47，XXY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003einduced abortion\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e21\u003csup\u003e+4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eserological screening borderline risk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eT21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e2.877\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003edetection failure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003earr[hg19]3q29(195,703,616-197,356,334)x1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003einduced abortion\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eIVF twins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eT18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e2.014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.541\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003edetection failure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 136px;\"\u003e\n \u003cp\u003eNT 5.3 mm, omphalocele\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003efetal reduction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTrue Positive Detection Rates for Different\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eBorderline Z-score Types\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing invasive prenatal diagnosis and/or prenatal diagnostic ultrasound as the gold standard, the true positive detection rates for different types of borderline Z-score were analyzed. The results showed that the true positive rates for T13,T21,and T18 borderline Z-score were 1.3%,1.2%,and 0.4%,respectively,with positive predictive values(PPVs)of 40%,30%,and 6.7%,respectively.No true positives were detected in the other cases(Table 4).\u003c/p\u003e\n\u003cp\u003eTable 4. True Positive Detection Rates for Different Borderline Z-score Types\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"586\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 85px;\"\u003e\n \u003cp\u003eborderline Z-score types\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 123px;\"\u003e\n \u003cp\u003enumber of individuals in the borderline zone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 123px;\"\u003e\n \u003cp\u003enumber of individuals in the borderline zone after retest\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 94px;\"\u003e\n \u003cp\u003eunderwent prenatal diagnosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003etrue positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 66px;\"\u003e\n \u003cp\u003ePPV（%）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003en\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e%\u0026nbsp;\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eT13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e159\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eT21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e247\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eT18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e265\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e6.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eother \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003etotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e206\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e19.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: PPV = True Positives / Number of Prenatal Diagnostic Procedures; a: True Positive Rate = True Positives / Number of Critical Zone Individuals (initial); b: Individuals with two or more types of critical zone categories.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe National Health and Family Planning Commission issued the \"Technical Specifications for Prenatal Screening and Diagnosis of Cell-Free Fetal DNA in Maternal Peripheral Blood,\" which states that the failure rate of NIPT should not exceed 5%\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. An increasing number of domestic and international scholars have conducted analyses and discussions on the reasons of NIPT failure\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. The NIPT testing process is lengthy and complex, susceptible to various influencing factors, including sample-related issues such as hemolysis and coagulation, as well as experimental procedural issues such as DNA extraction and library construction failures. Therefore, in practical clinical practice, NIPT testing often requires library reconstruction and/or re-sampled to reduce the failure rate. The borderline Z-score is a common reason for library re-construction and/or re-sampled in NIPT.\u003c/p\u003e \u003cp\u003eThe Technical Specifications for Prenatal Screening and Diagnosis of Cell-Free Fetal DNA in Maternal Peripheral Blood defines the serum screening risk value between the high-risk cutoff and 1/1000 as the borderline risk\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. The chromosomal abnormality rate of the serum screening borderline risk group is significantly higher than that of the low-risk group\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Therefore, the author believes that a borderline Z-score may be established in NIPT. In this study, among the 700 pregnant women whose initial NIPT results indicated a borderline Z-score, 34 cases with abnormal results were detected, with a screening positive rate of 5.29% (34/700). Following prenatal diagnosis, a total of 6 fetal abnormalities were detected (including 1 case with prenatal diagnosis ultrasound indicating abnormalities and 5 cases with invasive prenatal diagnosis indicating chromosomal abnormalities), with a fetal abnormality rate of 0.9% (6/700). Without the establishment of a borderline Z-score, these abnormal fetuses might have been missed. This underscores the necessity of setting a borderline Z-score zone in NIPT to reduce the rate of missed screenings. Pan Shuqing et al.\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e analyzed over 50,000 NIPT screening cases from this laboratory, reporting screening positive and fetal abnormal rates of 0.6% (326/54957) and 0.2% (135/54957), respectively, which are significantly lower than those observed in this study. Similarly, Xiang L et al. \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e and Liu Jing et al. \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e analyzed NIPT screening data from the general population, and their screening positive and fetal abnormal rates were significantly lower than those in this study. These findings indicate that the screening positive rate and fetal abnormality rate in cases with initial NIPT results indicating a borderline Z-score are higher than those in the general population, which is consistent with the studies by Luo Yanmei et al. \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, Chan N et al. \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, and Lu Y et al. \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis study analyzed NIPT-related data from over 80,000 pregnant women and identified 700 cases (0.84%, 700/83,443) with initial borderline Z-scores (Z-scores between 1.96 and 3), which is lower than the 2.62% (160/6110) reported by Qi Hong et al. \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e and the 1.64% (663/40311) reported by Luo Yanmei et al\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. This may be due to different sequencing platforms and differences in the definition range of the borderline Z-score, resulting in a significant difference in the detection failure rate (borderline Z-score). After reconstruction, 211 cases were indicated as the borderline Z-score, indicating a repeat sampling rate (borderline Z-score) of 0.25% (211/83,443), which is consistent with the aforementioned studies\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. After re-sampled, NIPT results indicated that 26 cases failed due to the borderline Z-score, accounting for 0.03% (26/83443). This demonstrates that library reconstruction and/or re-sampled can greatly reduce the NIPT detection failure rate (borderline Z-score), decreasing it from 0.84\u0026ndash;0.03%, possibly because as the gestational age increases, the fetal concentration increases, enhancing the accuracy of NIPT detection. Ultimately, among the 700 pregnant women, 635 cases (90.71%, 635/700) received low-risk results, 34 cases (4.86%, 34/700) indicated abnormal NIPT results, 26 cases indicated NIPT detection failure, and 5 pregnant women did not cooperate with re-drawing blood. Follow-up did not find any false negatives. It can be seen that reconstruction and/or re-drawing can screen out most normal pregnant women, effectively reducing unnecessary invasive prenatal diagnoses and alleviating the psychological burden on pregnant women.\u003c/p\u003e \u003cp\u003eIn addition, this study found that the true positive detection rate varies for different borderline Z-score types. The true positive rate for the T21 borderline is the highest (1.3%), followed by the T13 borderline (1.2%) and the T18 borderline (0.4%), with the positive predictive value (PPV) also decreasing accordingly, indicating that the T18 borderline has the highest \"false positive rate.\" When two or more borderline types coexist, no true positives were detected, mostly caused by data fluctuations. This may guide clinical genetic counseling for such populations. Additionally, this study found that when NIPT results indicate abnormal results, pregnant women have good compliance with prenatal diagnosis, but they do not pay enough attention to the borderline Z-score, with 80.6% (25/31) of pregnant women not choosing prenatal diagnosis (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Therefore, for pregnant women who fail detection due to the borderline Z-score, prenatal genetic counseling should be strengthened, actively guiding pregnant women to undergo further examinations to prevent missed screenings.\u003c/p\u003e \u003cp\u003eMitotic or meiotic non-disjunction errors can lead to the formation of mosaicism, resulting in the existence of two or more cell lines in the fetus and placenta\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Placental mosaicism is the main cause of false negatives and false positives in NIPT\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Among the 5 cases of fetal chromosomal abnormalities, 2 were mosaic types, including 1 case of translocation-type trisomy 21 mosaicism with a mosaic ratio of 40%, and another case of trisomy 13 mosaicism with a mosaic ratio of 43%. Both cases had two cell lines in the fetus, possibly due to meiotic errors forming a triploid zygote, and then through mitotic in subsequent mitosis, losing the extra chromosomes, ultimately leading to the coexistence of normal diploid and abnormal triploid\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Both cases initially showed borderline Z-score, and after library reconstruction, the Z-score exceeded 3, showing that multiple NIPT results with Z-scores near 3 may indicate the possibility of fetal and placental mosaicism. The other 3 cases were other chromosomal abnormalities, possibly due to abnormal chromosome fragments affecting the calculation of Z-scores. In addition, 1 case of twins with one abnormality leading to a borderline Z-scores was found. A 34-year-old pregnant woman transplanted two fresh embryos, and at 13 weeks of pregnancy, ultrasound indicated one twin with NT 5.3mm, dotted nasal bone, and omphalocele. It is speculated that the abnormal fetus may be trisomy 18, and the abnormal fragments released by the abnormal fetus neutralize with the normal fragments of the normal fetus, reducing the concentration of abnormal fragments and resulting in a borderline Z-score for trisomy 18 in NIPT.\u003c/p\u003e \u003cp\u003eIn summary, among pregnant women with initial NIPT results indicating borderline Z-scores, both the screening positive rate and fetal abnormality rate are higher than those in the general population. Therefore, it is necessary to establish a borderline Z-score in NIPT to reduce the rate of missed screenings. Additionally, library reconstruction and/or re-drawing can significantly decrease the NIPT detection failure rate caused by the borderline Z-score, effectively identifying the majority of normal pregnancies and reducing unnecessary invasive prenatal diagnoses. For pregnant women who fail detection due to the borderline Z-score, active guidance should be provided to encourage further diagnostic testing to prevent missed screenings\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXiaoli Pan wrote the main manuscript text .Wen prepared all figures.Yun pan and Shuqing Pan collected data.Wu、Chen and Li reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eXiang L, Zhu J, Deng K, et al. Non-invasive prenatal testing for the detection of trisomies 21, 18, and 13 in pregnant women with various clinical indications: A multicenter observational study of 1,854,148 women in China [J]. Prenat Diagn, 2023, 43(8):1036-1043. doi: 10.1002/pd.6312.\u003c/li\u003e\n\u003cli\u003eBayefsky MJ, Caplan AL, Hoskins IA. Evaluating Expanded Noninvasive Prenatal Screening [J]. Obstet Gyneco, 2022, 139(6):1009-1011.doi: 10.1097/AOG.0000000000004809.\u003c/li\u003e\n\u003cli\u003eLiu J, Zhao JH, Chu W, et al. Retrospective analysis for 424 330 first-line screening results of non-invasive prenatal testing in Hebei province[J]. Chin J Obstet Gynecol,2022,57(12):900-906.doi:10.3760/cma.j.cn112141-20220711-00453.\u003c/li\u003e\n\u003cli\u003eBalslev-Harder M, Richter SR, Kjaergaard S, et al. Correlation between Z - score，fetal fraction and sequencing reads in Non - Invasive Prenatal Testing［J］. Prenat Diagn, 2017,37（9）：943-945. doi: 10.1002/pd.5116.\u003c/li\u003e\n\u003cli\u003eMo J, Ren JQ, Yang LQ, et al. Clinical evaluation of true and false positive Z values among high-risk cases screened by non-invasive prenatal testing[J]. Chin J Med Genet,2022,39(11):1187-1191.DOI:10.3760/cma.j.cn511374-20220120-00046.\u003c/li\u003e\n\u003cli\u003eLuo YM, Hu HM, Zhang R, et al. Analysis of factors related to non-invasive prenatal testing failure and the feasibility of repeat testing [J].Chin J Med Genet,2020,37(6):603-608.doi:10.3760/cma.j.issn.1003-9406.2020.06.002.\u003c/li\u003e\n\u003cli\u003eQi H, Zhu JJ, Zeng W, et al. The application value of repeat testing in non-invasive prenatal testing for pregnant women[J].Journal of Developmental Medicine(Electronic Edition),2019,7(3):182-187,195.doi:10.3969/j.issn.2095-5340.2019.03.005.\u003c/li\u003e\n\u003cli\u003eNational Health and Family Planning Commission of the People\u0026apos;s Republic of China. Technical specifications for non-invasive prenatal testing using cell-free fetal DNA in maternal peripheral blood[Z].2016.\u003c/li\u003e\n\u003cli\u003eSuzumori N, Sekizawa A, Takeda E, et al. Classification of factors involved in nonreportable results of noninvasive prenatal testing (NIPT) and prediction of success rate of second NIPT [J]. Prenat Diagn, 2019, 39(2): 100-106. doi: 10.1002/pd.5408. \u003c/li\u003e\n\u003cli\u003eZhao GY, Dai P, Gao SS, et al. The value of re-sampling and retesting in cases of non-invasive prenatal testing failure due to low fetal cell-free DNA concentration[J].Chin J Med Genet,2022,39(2):135-138.doi:10.3760/cma.j.cn511374-20201119-00813.\u003c/li\u003e\n\u003cli\u003eGu CH. The Value of Combined Maternal Serum Screening and Ultrasound Soft Markers in Fetal Chromosomal Diagnosis[D].Zhengzhou University,2022.\u003c/li\u003e\n\u003cli\u003ePan SQ, Pan XL, Ge LS, et al. The Application Value of Non-Invasive Prenatal Testing in Prenatal Screening[J].Zhejiang Medical Journal,2024,46(17):1881-1884.\u003c/li\u003e\n\u003cli\u003eChan N, Smet ME, Sandow R, et al. Implications of failure to achieve a result from prenatal maternal serum cell-free DNA testing: a historical cohort study [J]. BJOG, 2018, 125(7): 848-855. doi: 10.1111/1471-0528.15006.\u003c/li\u003e\n\u003cli\u003eLu Y, Linpeng S, Ding S, et al. Retrospective analysis of the risk factors associated with failure in obtaining effective noninvasive prenatal test results and pregnancy outcomes: a case-control study [J]. Expert Rev Mol Diagn, 2022, 22(3):387-394. doi: 10.1080/14737159.2022.2049245.\u003c/li\u003e\n\u003cli\u003eZhang HY, Fu ML, Wang W. Analysis of biological causes of false positives and false negatives in non-invasive prenatal genetic testing for chromosomal aneuploidies[J].Chinese Journal of Prenatal Diagnosis(Electronic Edition),2017,9(3):48-58.DOI:10.13470/j.cnki.cjpd.2017.03.010.\u003c/li\u003e\n\u003cli\u003eFu ML. Research on the Causes of False Negatives and False Positives in Non-Invasive Prenatal Genetic Testing and the Establishment of a Laboratory Investigation Protocol[D].South China University of Technology,2020.\u003c/li\u003e\n\u003cli\u003eSirchia SM, Garagiola I, Colucci G, et al. Trisomic zygote rescue revealed by DNA polymorphism analysis in confined placental mosaicism [J]. Prenat Diagn, 1998, 18(3):201-206.doi:10.1002/(sici)1097-0223(199803)18:3\u0026lt;201::aid-pd245\u0026gt;3.0.co;2-w\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-gynecology-and-obstetrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arch","sideBox":"Learn more about [Archives of Gynecology and Obstetrics](https://www.springer.com/journal/404)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/arch/default.aspx","title":"Archives of Gynecology and Obstetrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"NIPT, borderline Z- score, prenatal diagnosis, positive rate, library construction","lastPublishedDoi":"10.21203/rs.3.rs-6781560/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6781560/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eThis study aimed to investigate the utility of repeated testing for the detection of fetal cell-free DNA (cfDNA) from maternal peripheral blood in cases with borderline Z-scores, and to analyze the associated pregnancy outcomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003eA retrospective analysis was conducted on 83,443 pregnant women who voluntarily underwent non-invasive prenatal testing (NIPT) at the Affiliated Women and Children's Hospital of Ningbo University between January 2020 and January 2024. Pregnant women whose initial NIPT results indicated borderline Z-scores were subsequently followed up.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eAmong 83,443 pregnant women undergoing NIPT, 700 cases (0.84%) initially showed borderline Z-scores. After retesting, this number decreased to 211 cases (0.25%), and further decreased to 26 cases (0.03%) after re-sampling. Among the initial 700 cases, 34 exhibited abnormal NIPT results, corresponding to a positive rate of 5.29%. Subsequent prenatal diagnosis confirmed a total of 6 fetal abnormalities, including one case of trisomy 13 mosaicism, one case of trisomy 21 mosaicism, one case of 47,XXY, two cases of copy number variations (CNVs), and one case with B-ultrasound findings indicating an abnormality (nuchal translucency [NT] of 3.5 mm and omphalocele). The overall rate of fetal abnormalities was 0.9%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e Initial NIPT outcomes suggested elevated screening positive rates and a higher incidence of fetal abnormalities among cases with borderline Z-scores compared to the general population. Establishing a defined borderline Z-score threshold in NIPT protocols is crucial to mitigate the risk of missed screenings. Implementing reconstruction and/or re-sampling procedures significantly reduces the failure rate attributed to borderline Z-scores, facilitating the accurate identification of most of pregnancies with normal fetal development and decreasing the need for unnecessary invasive prenatal diagnostic interventions. Pregnant women with detection failures due to borderline Z-scores should be actively counseled and encouraged to pursue prenatal diagnosis.\u003c/p\u003e","manuscriptTitle":"Borderline Z-scores in Non-Invasive Prenatal Screening: Does Its Presence Hold Clinical Significance?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-09 10:39:12","doi":"10.21203/rs.3.rs-6781560/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-26T20:55:58+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"28549256076535929563837667147129070504","date":"2025-07-26T07:09:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7862277293988585660941330454919672536","date":"2025-07-20T22:39:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-11T22:23:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254735683499502394140890072805085510039","date":"2025-06-21T10:16:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-04T07:49:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-02T08:50:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-30T13:34:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Gynecology and Obstetrics","date":"2025-05-30T06:13:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-gynecology-and-obstetrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arch","sideBox":"Learn more about [Archives of Gynecology and Obstetrics](https://www.springer.com/journal/404)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/arch/default.aspx","title":"Archives of Gynecology and Obstetrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8254ee46-1911-45b7-a54b-9343f7eba909","owner":[],"postedDate":"June 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T16:07:43+00:00","versionOfRecord":{"articleIdentity":"rs-6781560","link":"https://doi.org/10.1007/s00404-025-08193-2","journal":{"identity":"archives-of-gynecology-and-obstetrics","isVorOnly":false,"title":"Archives of Gynecology and Obstetrics"},"publishedOn":"2025-09-30 15:58:07","publishedOnDateReadable":"September 30th, 2025"},"versionCreatedAt":"2025-06-09 10:39:12","video":"","vorDoi":"10.1007/s00404-025-08193-2","vorDoiUrl":"https://doi.org/10.1007/s00404-025-08193-2","workflowStages":[]},"version":"v1","identity":"rs-6781560","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6781560","identity":"rs-6781560","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}