Dear Dr. Cheng,

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.

Please submit your revised manuscript by Jun 14 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.

• 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'.
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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,

Gary S. Stein

Academic Editor

PLOS ONE

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“This work was supported by the Municipal Health Commission of Nantong (No. MSZ2023057).”

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6. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: This study employed bioinformatics approaches to uncover the differential expression of oxidative stress-responsive genes in RM, and establishes a diagnostic model and provides insights into immune-modulation therapies for RM. However, the study was only based on bioinformatic analyses using publicly available data, and experimental validation on clinical cohorts, cell lines and animal models is recommended. Moreover, the figures in the manuscript are vague and some figure legends are difficult to be recognized, such as figure 10 and figure 11. So the quality of the figures should be improved.

Reviewer #2: This study systematically analyzed the expression characteristics of oxidative stress-related genes in RM by integrating multiple public gene expression datasets using bioinformatics methods, identifying 18 differentially expressed genes and further selecting 6 core genes to construct a high-precision diagnostic model (AUC=0.975). Functional analysis revealed that these genes participate in RM pathogenesis by regulating hypoxia, epithelial-mesenchymal transition, and Wnt signaling pathways, while immune infiltration analysis further demonstrated their significant correlations with neutrophils and CD8+ T cells. Overall, this represents a well-designed and rigorously executed study that offers new perspectives for understanding the molecular mechanisms of RM.

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

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Reviewer #1: No

Reviewer #2: No

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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

Dear Dr. Cheng,

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.

Please submit your revised manuscript by Oct 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.

• 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 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,

Gary S. Stein

Academic Editor

PLOS ONE

Journal Requirements:

1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 

2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

Reviewer #5: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: 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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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Reviewer #1: (No Response)

Reviewer #2: I recommend the acceptance of this paper. Strengths: appropriate methods, clear writing, useful findings. Limitations are acknowledged appropriately.

Reviewer #3: (No Response)

Reviewer #4: Minor Revisions: The abstract contains a sentence structure issue: UCN2 was positively associated with T. cells. CD8+ T cells are inversely associated with monocytes. This phrasing is confusing and requires revision for improved clarity (such as: "UCN2 demonstrated positive correlation with CD8+ T cells, which in turn showed negative correlation with monocytes"). Also, verify that gene symbols are formatted in italics consistently across the entire manuscript.

Although the computational analysis demonstrates technical rigor, the findings remain theoretical predictions. The authors' response to the first reviewer's concerns is inadequate. For work submitted as a "Research Article," some degree of experimental confirmation is a reasonable expectation.

I suggest implementing a major revision requirement that includes experimental corroboration of the central discoveries. The basic expectation should involve:

Quantitative RT-PCR verification of the six key genes' expression levels using a separate, small collection of clinical specimens (recurrent miscarriage patients compared to control endometrial or placental samples).

Alternatively, confirmation could be achieved through analysis of an independent publicly accessible dataset not previously utilized in the model development or validation phases.

If experimental validation cannot be provided by the authors, the manuscript would be better positioned for submission to a specialized computational biology publication or should be explicitly recharacterized as predictive research or a methodological contribution.

The fundamental analytical framework demonstrates excellent quality and deserves publication. Nevertheless, the manuscript lacks completeness in its current state. Incorporating experimental validation is essential to elevate these promising computational predictions to findings with biological and clinical significance. The authors should receive the opportunity to supply this validation prior to making a final publication decision.

Reviewer #5: As a reviewer, here's a detailed critique of the article "Identification of oxidative stress-responsive genes in recurrent miscarriage and their role in disease pathogenesis."

This effectively outlines the study's scope and key findings, presenting a compelling case for the research's relevance. However, as with most articles, it has both strong points and areas that would require further clarification in the full manuscript.

1. Strengths

1.1. The research addresses recurrent miscarriage (RM), a major issue in reproductive health, and connects it to oxidative stress, a poorly understood but increasingly relevant factor. The subject matter is highly pertinent and of great interest to the scientific community.

1.2. The abstract clearly states that a bioinformatics approach was used, analyzing data from the Gene Expression Omnibus (GEO). This sets expectations for the type of study and its data-driven nature.

1.3. The findings are presented in a clear and organized manner. The abstract lists key discoveries, including the identification of: 1)18 oxidative stress-responsive differentially expressed genes (OSRDEGs). 2) Six key hub genes (ARRB2, BMF, SORCS2, STK3, UCN2, and VIPR1) proposed as potential biomarkers. 3)Associated biological pathways via GSEA (e.g., hypoxia, EMT). 4) A mRNA-miRNA interaction network, highlighting STK3's central role. 5) Immune cell infiltration correlations, linking specific genes to immune cell types.

1.4. The study goes beyond theoretical findings and directly mentions its potential for developing a diagnostic model and guiding immune-modulation therapies. This highlights the practical value of the research.

2. Weaknesses and Key Questions for the Authors

2.1. Missing Experimental Validation:

This is the most significant limitation. The study is purely computational (bioinformatics). There is no mention of experimental validation using techniques like RT-qPCR, Western blot, or immunohistochemistry to confirm the differential expression of the identified hub genes in a separate cohort of patient samples. Without this, the findings remain hypothetical and require further verification.

2.2. Details on Results and Mechanistic Roles:

The research identifies hub genes, but it offers minimal insight into their potential mechanistic roles in the pathogenesis of RM. For instance, what is the proposed function of STK3 and its interaction with miRNAs in the context of recurrent miscarriage?

Some of the GSEA pathways mentioned (e.g., "EMT in breast tumors," "Wnt signaling in liver cancer progenitors") seem to be directly from the database output rather than being contextualized for RM. The authors should briefly explain in the full paper why these specific pathways are relevant to the disease.

In addition, there are several ambiguous phrases.

The sentence "UCN2 was positively associated with T. cells. CD8+ T cells are inversely associated with monocytes" is slightly ambiguous. A reviewer would wonder if UCN2 is positively associated with both T cells and CD8+ T cells, or if this is a separate finding. The phrasing could be clearer.

Reviewer's Recommendation

Overall, this is a promising study that lays important groundwork for future research. The article is well-structured and presents novel findings.

However, for publication in a reputable journal such as PLOS One, the authors would need to:

1. Include experimental validation of at least a few of the key hub genes (e.g., using patient samples).

2. Expand on the mechanistic roles of the identified genes and their potential contribution to the disease pathophysiology.

3. This study generates excellent hypotheses, but a strong paper would need to show from correlation (bioinformatics) to causation (experimental validation).

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

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Reviewer #5: Yes:  Dr. Abolfazl Akbari (Physiologist)

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[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

Identification of oxidative stress-responsive genes in recurrent miscarriage and their role in disease pathogenesis

PONE-D-25-04005R2

Dear Dr. Cheng,

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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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,

Gary S. Stein

Academic Editor

PLOS ONE

Reviewer's Responses to Questions

Comments to the Author

Reviewer #4: All comments have been addressed

Reviewer #5: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #4: Yes

Reviewer #5: (No Response)

**********

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

Reviewer #4: Yes

Reviewer #5: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #4: Yes

Reviewer #5: (No Response)

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

Reviewer #4: Yes

Reviewer #5: (No Response)

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Reviewer #4: The authors have been very receptive and have made a significant revision, which has made the manuscript very strong. Addition of IHC data is a direct response to the most significant weakness, which will bring the work to a new level of bioinformatic research with proven biological significance. No additional revisions are needed.

Reviewer #5: It should be noted that bioinformatics studies sometimes provide forward-looking opportunities to improve basic studies, however, they cannot be a suitable substitute for them.

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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 #4: Yes:  Jonah Bawa Adokwe PhD

Reviewer #5: Yes:  Dr. Abolfazl Akbari (Physiology)

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