Cell-free DNA screening for trisomies 21, 18, and 13: Clinical application and accuracy evaluation

Xiangmei Sun; Yuyi Ying; Caiping Chen; Xiaoliang Shi

doi:10.1371/journal.pone.0332081

Peer Review History

Original SubmissionMay 28, 2025
2 Jul 2025 Decision Letter - Yee Gary Ang, Editor PONE-D-25-27034Cell-free DNA screening for trisomies 21, 18, and 13: clinical application and accuracy evaluationPLOS ONE Dear Dr. Shi, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== We managed to get 2 reviewers to review and they have suggested some ways to improve the manuscrpt Please see and consider resubmitting if possible ============================== Please submit your revised manuscript by Aug 16 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Yee Gary Ang, MBBS MPH Academic Editor PLOS ONE Journal requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. For studies involving third-party data, we encourage authors to share any data specific to their analyses that they can legally distribute. PLOS recognizes, however, that authors may be using third-party data they do not have the rights to share. When third-party data cannot be publicly shared, authors must provide all information necessary for interested researchers to apply to gain access to the data. (https://journals.plos.org/plosone/s/data-availability#loc-acceptable-data-access-restrictions) For any third-party data that the authors cannot legally distribute, they should include the following information in their Data Availability Statement upon submission: 1) A description of the data set and the third-party source 2) If applicable, verification of permission to use the data set 3) Confirmation of whether the authors received any special privileges in accessing the data that other researchers would not have 4) All necessary contact information others would need to apply to gain access to the data Additional Editor Comments (if provided): [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: No ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Methodological Clarity • Details of the NIPS method (BGISEQ-2000) and validation (IPD) are well-described. However, the inclusion and exclusion criteria (e.g., what defines a "high-risk" group beyond Z-score) should be clarified. o The manuscript lacks a clear description of the control population used for generating Z-scores, which is central to their interpretation. o No analysis of fetal fraction, which is a critical confounder in NIPS. o Limited discussion of false negatives—despite being a real concern in NIPS. Statistical analysis • Confidence intervals are missing from some PPV estimates and AUCs—these should be included. Discussion: Expand comparison with other large cohort studies and meta-analyses Referencing - References are adequate and up-to-date, though there is inconsistent formatting. Language: Use professional English editing service before resubmission. Reviewer #2: Xiangmei Sun and collaborators report how z-score value could be a good predictor for NIPT results and how it correlate with PPV at high score, mainly for T21 and T18. This data is well known and this study help to corroborate this association highlighting its utility. The paper is well written and statistical analysis are well conducted. Some minor amendements are required before publication: - please choose if using NIPS or NIPT, maybe NIPT is more widely recognized - in table 3 the values recorded in this study should be filled together with the previous publications in order to have a better view of the comparison among studies - Data availability statement is missing, it is not clear where the data may be found - please check the punctuation that is often misplaced All the limitations of this study are well explained in the discussion section ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0332081.r001
Revision 1
6 Aug 2025 Author Response We would like to thank both reviewers for their valuable comments. We feel this has helped us to improve our manuscript substantially. We have revised the manuscript accordingly, with main revisions highlighted. We also revised the manuscript according to the formatting requirements of your journal of PLOS ONE. The detailed response to each comment is listed below in red. Reviewer #1: 1)“Details of the NIPS method (BGISEQ-2000) and validation (IPD) are well-described. However, the inclusion and exclusion criteria (e.g., what defines a "high-risk" group beyond Z-score) should be clarified.” Response:We sincerely appreciate the reviewer's valuable comment regarding the clarification of inclusion/exclusion criteria. In response, we have explicitly stated in the “Methods:Study subjects” section that "The inclusion criteria comprised all pregnant women who underwent NIPT and tested positive during the study period, while those with negative results were excluded."This revision provides clear study population parameters while maintaining consistency with our reported results. 2)“The manuscript lacks a clear description of the control population used for generating Z-scores, which is central to their interpretation.” Response:As suggested,we have added this in the new manuscript.More details were in the “Methods: Noninvasive prenatal Testing” section. 3)“No analysis of fetal fraction, which is a critical confounder in NIPS.” Response:We appreciate the reviewer’s insightful comment. We fully agree that fetal fraction (FF) is a critical confounder in NIPS analysis, and its inclusion would strengthen our study. However, due to the high proportion of missing FF data in our cohort, we intentionally excluded it from the primary analysis to avoid statistical bias. To indirectly address this limitation, we stratified the NIPS testing gestational age into three groups (�16周, 17–20, and >20 weeks), as FF is known to correlate positively with gestational age[1-3]. No significant intergroup differences in PPV were observed. In future studies, we plan to expand the sample size and incorporate additional covariates (e.g., FF, maternal CNVs) for a more comprehensive analysis. References 1.Gamisch A, Hess J, Mustafa-Korninger ME. Diurnal Effects on the Fraction of Fetal Cell-Free DNA in Maternal Plasma.Prenat Diagn. 2025;45(8):979-87. https://doi.org/10.1002/pd.6836 PMID: 40533246. 2.Deng C, Liu J, Liu S, Liu H, Bai T, Jing X, et al. Maternal and fetal factors influencing fetal fraction: A retrospective analysis of 153,306 pregnant women undergoing noninvasive prenatal screening. Front Pediatr. 2023;11:1066178. https://doi.org/10.3389/fped.2023.1066178 PMID: 37114008. 3.Mousavi S, Shokri Z, Bastani P, Ghojazadeh M, Riahifar S, Nateghian H. Factors affecting low fetal fraction in fetal screening with cell-free DNA in pregnant women: a systematic review and meta-analysis. BMC Pregnancy Childbirth. 2022;22(1):918. https://doi.org/10.1186/s12884-022-05224-7 PMID: 36482322. 4)“Limited discussion of false negatives—despite being a real concern in NIPS.Expand comparison with other large cohort studies and meta-analyses.” Response:We thank the reviewer for this important point. Additional discussion on false negatives and comparisons with other studies have been added (Line 257-269). “In a nationwide Chinese multicenter study encompassing nearly 2 million pregnancies, noninvasive prenatal testing (NIPT) demonstrated positive predictive values (PPVs) of 69.77% for T21, 47.24% for T18, and 22.36% for T13. The analysis revealed 26 false-negative cases, corresponding to a false-negative rate of 1.4 per 100,000 pregnancies[25].In a large-scale retrospective cohort study of 282,911 pregnancies screened NIPT, the test demonstrated robust performance for common aneuploidies, with PPVs of 86.81% for T21, 56.81% for T18, and 18.18% for T13. The false-negative rate was 2.47 per 100,000 pregnancies, representing 7 undetected cases of trisomy[26].The findings highlight that despite NIPT's robust performance in detecting common aneuploidies, NIPT-negative pregnancies still require ultrasound follow-up and genetic counseling, with invasive diagnostic testing recommended if abnormalities are detected.” 5)“Confidence intervals are missing from some PPV estimates and AUCs—these should be included.” Response:As suggested, we have added confidence intervals for the PPV estimates and AUCs. 6)“References are adequate and up-to-date, though there is inconsistent formatting.” Response:We appreciate the reviewer's feedback. We have now standardized all references according to the journal's formatting guidelines. 7)“Use professional English editing service before resubmission.” Response:Thank you for the suggestion! We have proofread the manuscript and tried to correct grammatical errors and rephrase certain sentences. Reviewer #2: 1)“please choose if using NIPS or NIPT, maybe NIPT is more widely recognized.” Response:We appreciate this constructive comment. As recommended, we have uniformly adopted 'NIPT' throughout the revised manuscript. 2)“in table 3 the values recorded in this study should be filled together with the previous publications in order to have a better view of the comparison among studies.” Response:Thank you for the suggestion! As recommended, we have integrated the data from our current study with relevant published findings in Table 3 to enable direct comparison across studies. 3)“Data availability statement is missing, it is not clear where the data may be found.” Response:As suggested, we have added Data availability statement in the revised manuscript.The datasets for this study can be found in the this link: https://figshare.com/s/d3fd982f9b830a52ec4f. 4)“please check the punctuation that is often misplaced.” Response: We have proofread the manuscript as suggested. We tried our best to improve the manuscript and made some changes highlighted with different colors in revised paper. We appreciate for Editors/Reviewers’ warm work earnestly, and hope the correction will earn your recognition and approval. Once again, thank you very much for your comments and suggestions. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0332081.r002
26 Aug 2025 Decision Letter - Yee Gary Ang, Editor Cell-free DNA screening for trisomies 21, 18, and 13: clinical application and accuracy evaluation PONE-D-25-27034R1 Dear Dr. Shi, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support . If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Yee Gary Ang, MBBS MPH Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: (No Response) Reviewer #3: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #3: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #3: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #3: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Thank you for your revisions. The manuscript is now clearer, more coherent, and easier to understand. The study’s limitations have also been acknowledged to guide further research. Reviewer #3: The authors have successfully addressed all concerns, and the manuscript has been revised accordingly. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy . Reviewer #1: No Reviewer #3: No ******** https://doi.org/10.1371/journal.pone.0332081.r003
Formally Accepted
Acceptance Letter - Yee Gary Ang, Editor PONE-D-25-27034R1 PLOS ONE Dear Dr. Shi, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Yee Gary Ang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0332081.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .