PONE-D-24-57732Telomere length as a biomarker for fetal fraction prediction in non‑invasive prenatal testingPLOS ONE

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Reviewers' comments:

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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

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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: No

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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: The authors explore the hypothesis that telomere length could serve as a novel biomarker for predicting fetal fraction (FF) in non-invasive prenatal testing (NIPT). While the integration of telomere-related metrics into applied models (e.g., seqFF and comboFF) provides modest improvements, the overall impact appears limited compared to established genetic-based methodologies. Nevertheless, this research offers valuable insights into potential supplementary approaches for FF estimation. I would like to share some comments and suggestions to enhance the manuscript's clarity and rigor.

Major Comments:

-The correlation coefficients reported (e.g., telomere content alone vs. best-performing model) suggest that telomere metrics provide only incremental improvements when integrated with conventional tools. Consider elaborating on the practical implications of these modest gains (e.g., Would the improved prediction accuracy significantly impact clinical outcomes or test reliability?).

-The manuscript would benefit from a deeper insights of the biological variability of telomere length across populations (e.g., potential influences of maternal health, genetic background, or environmental factors on telomere dynamics). Furthermore, a discussion is necessary on whether the observed telomere-derived signals are consistent across diverse maternal-fetal conditions (e.g., healthy fetuses vs affected).

-Although the models incorporate telomere metrics and maternal age, the manuscript does not sufficiently address potential biases or limitations (e.g., the exclusion of pregnancies with female fetuses; technical variability in telomere measurement; the impact of maternal BMI or other confounding factors). Including a limitations section to discuss these points would strengthen the study.

-Indicate as supporting data will be shared to improve transparency and reproducibility.

Minor suggestions:

-Line 23: placental trophoblast is the main but not sole source of cffDNA. A lesser source is apoptosis of erythroblasts in the fetal circulation, which generates cfDNA that can cross the placenta and enter the maternal circulation (PMID: 11113889; 12200465).

-Ensure consistent use of terms like “telomere content,” “TelomereHunter features,” and “fetal fraction prediction models” throughout the manuscript.

-The Introduction is effective but could provide more context on the limitations of current FF estimation methods, highlight the potential gaps that telomere metrics aim to fill.

-In Discussion, emphasize how the findings align or diverge from prior research on telomere dynamics in prenatal diagnostics.

Reviewer #2: The accuracy of methods for quantifying fetal DNA fraction in maternal plasma samples during pregnancy is maximized by two main factors, fetal sex (M) and sequencing coverage (>10M reads). In the absence of these requirements, further criteria for defining FF require robust statistical processing dependent on data variability. The search for new models must overcome this last problem. This is not the case for telomere sequences which, although decreasing with age, do not easily distinguish placenta from maternal lymphocytes due to the innumerable inter and intra-individual variables. It is therefore not surprising that the authors did not draw favorable conclusions in their work, nor does it appear useful to share such data.

Reviewer #3: This study investigates the potential utility of telomere content as an additional biomarker to enhance the estimation of fetal fraction (FF) in non-invasive prenatal testing.

The manuscript is clearly written, methodologically sound, and well-structured.

The results suggest that telomere length may serve as a “complementary tool” for refining established FF prediction methods.

Although the inclusion of telomere-related features yielded only modest improvements in FF prediction accuracy, the study provides valuable insights and highlights the importance of integrating multiple biomarkers to advance FF estimation models.

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Reviewer #1: Yes:  Ludovico Graziani

Reviewer #2: No

Reviewer #3: No

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Telomere length as a biomarker for fetal fraction prediction in non‑invasive prenatal testing

PONE-D-24-57732R1

Dear Dr. Holesova,

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.

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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: All comments have been addressed

Reviewer #2: All comments have been addressed

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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 #2: (No Response)

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: (No Response)

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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 #2: (No Response)

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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 #2: (No Response)

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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: (No Response)

Reviewer #2: (No Response)

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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: Yes:  Ludovico Graziani

Reviewer #2: No

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