Dear Dr. Herraiz,

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Academic Editor

PLOS One

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“This study was funded by Spanish network RD21/0012/0024: Primary care interventions to prevent maternal and child chronic diseases of perinatal and developmental origin, Instituto de Salud Carlos III, Madrid, Spain, and financed by the European Union though the Next Generation EU funds, which finance the actions of the Recovery and Resilience Facility (RRF).”

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

Reviewer #1: Partly

Reviewer #2: Yes

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2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses??>

Reviewer #1: Partly

Reviewer #2: Yes

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3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable??>

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors described where all data underlying the findings will be made available when the study is complete??>

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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.requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

Reviewer #1: The aim of the protocol study is to determine whether genome-wide non-invasive prenatal testing (GW-NIPT) provides additional clinical value compared with standard NIPT for common fetal aneuploidies in pregnancies affected by placental dysfunction.

The topic is scientifically relevant, as placental dysfunction represents a complex clinical condition in which the interpretation of cfDNA-based screening results remains challenging.

Placental mosaicism is recognized as one of the biological causes of placental dysfunction and is frequently associated with rare chromosomal abnormalities. Given that cell-free DNA analyzed by NIPT predominantly originates from trophoblastic cells, GW-NIPT theoretically holds the potential to detect confined placental mosaicism (CPM). In this context, the biological rationale of the study is sound.

It is well documented that the rate of positive NIPT screening results for rare autosomal trisomies (RATs) and large structural chromosomal abnormalities, such as copy number variants (CNVs), is higher in populations classified as high-risk for chromosomal abnormalities based on first-trimester combined screening, compared with low-risk populations. However, it is also established that the vast majority of RAT findings detected by GW-NIPT—up to approximately 50%—represent false-positive results, most likely attributable to CPM rather than true fetal involvement. This significantly limits the clinical utility of GW-NIPT, particularly in the general obstetric population, and underscores the need for careful interpretation of expanded screening results.

The proposed study is inherently limited by its restriction to a selected population already classified as being at increased risk for chromosomal abnormalities. From a clinical perspective, this significantly constrains the generalizability of the results and limits the direct impact of the findings on routine prenatal screening strategies. Consequently, the study is unlikely to substantially modify current clinical practice regarding the use of GW-NIPT in the broader obstetric population.

Nevertheless, despite this limitation, the study may still provide additional information of scientific relevance. By focusing on pregnancies characterized by placental dysfunction, the protocol has the potential to contribute to a better understanding of the biological relationship between placental mosaicism, abnormal cfDNA findings, and adverse placental phenotypes. In this sense, the value of the study lies primarily in its ability to improve the interpretation of GW-NIPT results in complex clinical scenarios, rather than in demonstrating an immediate clinical advantage over standard NIPT.

Specific comments:

The protocol aims to evaluate the clinical value of GW-NIPT compared with standard NIPT in pregnancies at increased risk of placental dysfunction, with a particular focus on the following populations:

3. Pregnancies identified as high-risk at first-trimester screening based on abnormal placental and biochemical markers, including altered PAPP-A and/or free β-hCG levels, suggestive of early placental dysfunction; and

4. Pregnancies complicated by early-onset fetal growth restriction (FGR), a condition strongly associated with impaired placentation and adverse perinatal outcomes.

The study proposes to include 321 patients in the first group and 181 patients in the second group.

Given that cell-free fetal DNA analyzed by NIPT originates predominantly from placental trophoblasts, these high-risk pregnancies represent a biologically plausible cohort in which confined placental mosaicism may contribute to abnormal cfDNA findings. However, current evidence indicates that the ability of GW-NIPT to detect CPM is highly variable and influenced by multiple biological and technical factors, including fetal fraction (FF), the level and distribution of mosaicism within the placenta, and the chromosome involved. Importantly, pregnancies complicated by early-onset FGR or abnormal first-trimester placental markers frequently exhibit reduced fetal fraction, which further limits test performance. Low fetal fraction is a well-recognized cause of NIPT test failure (“no-call” results) or reduced analytical reliability.

As a consequence, despite the expanded analytical scope of GW-NIPT, the positive predictive value for detecting CPM remains low, particularly for rare autosomal aneuploidies and subchromosomal CNVs. In this context, the sample size proposed in the protocol appears insufficient to adequately assess the clinical utility of GW-NIPT in these selected high-risk populations, especially considering that the study intends to evaluate, for each pregnancy category:

• High-risk expanded NIPT results for RATs and CNVs;

• High-risk NIPT results for common aneuploidies;

• Low-risk NIPT results.

I would recommend revising the proposed sample size. In my opinion, the number of pregnancies to be included should be increased, as the current cohort appears insufficient to draw robust and clinically meaningful conclusions. A minimum of 500 and 600 patients should be included in the first and second study groups, respectively.

Furthermore, the protocol would benefit from the inclusion of the following clarifications:

4. A clear specification of the gestational age at which NIPT sampling is performed. Gestational timing critically affects fetal fraction and analytical reliability, particularly in pregnancies characterized by placental insufficiency, early-onset FGR, or abnormal first-trimester screening markers.

In addition, the protocol does not specify:

• how no-call or failed NIPT results will be managed,

• whether repeat blood sampling is planned following an initial test failure,

• nor how repeated GW-NIPT results will be handled in the final analysis.

5. A justification for the requirement of 20 mL of maternal blood instead of the standard 10 mL volume.

In conclusion, the proposed study is scientifically sound, and both the objectives and inclusion criteria are appropriate. However, expansion of the study cohort and clarification of the points outlined above are necessary. With these modifications, the protocol would be suitable for publication.

Reviewer #2: The proposed protocol is feasible and has high potential to generate clinically relevant results due to its methodological design, the relevance of the study population, and the use of advanced technologies. It is a prospective, multicenter, observational study, which allows real-time data collection. The participation of six hospitals in different regions of Spain increases the representativeness of the sample and the external validity of the findings.

The use of expanded NIPT based on whole-genome sequencing (VeriSeq™ NIPT Solution v2) is an innovative and clinically relevant element, as it enables the detection not only of common trisomies but also of rare autosomal trisomies and copy number variations (CNVs) greater than 7 Mb. This technology offers advantages over traditional invasive methods, such as amniocentesis, especially in cases where these are not feasible or are declined by the patient. The protocol includes a sample size calculations based on previous estimates of chromosomal abnormality and CPM prevalence in similar populations, ensuring 80% statistical power to detect significant differences. A

Finally, the study complies with ethical and regulatory standards, including approval by an ethics committee, informed consent, and data protection measures in accordance with the General Data Protection Regulation (GDPR). It also includes postnatal follow-up for six months, adding value to the clinical interpretation of the findings.

Taken together, these elements confirm that the protocol is feasible, and capable of providing relevant evidence to improve the interpretation and clinical use of expanded NIPT in pregnancies with suspected placental dysfunction.

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes: Isabel Hernandez GarcíaIsabel Hernandez GarcíaIsabel Hernandez GarcíaIsabel Hernandez García

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Protocol for a prospective observational cohort study to assess clinical applications of expanded noninvasive prenatal testing (NIPT) in pregnancies with placental dysfunction

PONE-D-25-50467R1

Dear Dr. Herraiz,

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.

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Kind regards,

María Teresa Llinás

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

Reviewer #1: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses??>

Reviewer #1: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable??>

Reviewer #1: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete??>

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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.requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

**********

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

Reviewer #1: The authors have addressed all the comments and observations in a precise and thorough manner, providing the necessary clarifications and revisions.

In light of the detailed responses and the improvements made, I believe the manuscript now meets the standards required for publication.

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..-->

Reviewer #1: Yes: Francesca SpinellaFrancesca SpinellaFrancesca SpinellaFrancesca Spinella

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