PONE-D-24-35956The alteration of uterine microbiota participated in the activation of the decidual inflammatory response in early spontaneous abortionPLOS ONE

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.

After a thorough review, we would like to commend you on the quality of your work and its potential contribution to the study of early spontaneous abortion. Your study presents changes in uterine microbiota as a possible cause to early spontaneous abortion.

We believe that it is very close to being ready for publication. However, the peer-reviewers have identified a few minor revisions that would further strengthen the paper and improve its clarity. The peer-reviewed comments are attached herewith.

We are confident that addressing these points will help enhance the overall quality of the manuscript.

Please submit your revised manuscript by Dec 23 2024 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 appreciate your attention to these points and look forward to receiving your revised manuscript. Should you have any questions or need further clarification on any of the revisions, please do not hesitate to contact us.

We look forward to receiving your revised manuscript.

Kind regards,

Sameer Timilsina, MD

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. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.  When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 3. Thank you for stating the following financial disclosure: "This research was funded by the National Natural Science Foundation of China (No.82101717)." Please state what role the funders took in the study.  If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.""  If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 4. 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

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: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

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: Yes

**********

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: Yes

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: The study provides valuable insights into the relationship between uterine microbiota and inflammatory responses in early spontaneous abortion (ESA). However, some clarifications and adjustments will improve the manuscript’s clarity and robustness.

Figures and Tables:

Figures are informative, but some (e.g., Figures 3 and 4) are dense and could benefit from being split into sub-panels for clarity. Explain the Bayesian test with BH correction in the figure legend.

Methods:

Contamination Control: Provide more details on how contamination from the lower genital tract was controlled during 16S rRNA gene sequencing.

Cytokine Assays: Clarify the use of negative controls in cytokine detection.

Patient Selection: Specify the randomization method used for selecting ESA and AA patients.

Study Design:

Confounders: Clarify if lifestyle, diet, or other factors that affect microbiota were controlled.

Longitudinal Studies: Consider a longitudinal design to track microbiota and cytokine changes over time.

Mechanistic Studies: Future work could explore how microbial shifts drive inflammatory responses.

Statistics:

Multiple Comparisons: Clarify if corrections (e.g., Benjamini-Hochberg) were applied for multiple tests.

Missing Data: Explain how missing data were handled, especially in microbial sequencing and cytokine profiles.

Confounding Variables: Describe whether multivariate analyses were performed to control for confounders.

Results and data accessibility:

Patient-Level Data: Include more granular patient-level data in supplementary materials to strengthen the correlation between microbiota and inflammatory responses.

Outliers: Clarify how outliers in cytokine levels or microbiota data were handled.

References:

Include recent studies on reproductive tract microbiota to provide context, and reference the bioinformatics tools (e.g., USEARCH, Silva database) more thoroughly.

Title:

Consider simplifying the title.

Conclusions:

Emphasize the need for future mechanistic studies to explain how microbiota shifts influence inflammation in ESA.

Final Recommendation:

This is an important study. Clarifying the methods, refining figure presentation, and addressing statistical concerns will enhance the manuscript.

Overall Rating:

Significance:High

Clarity: Moderate (requires some clarification)

Reproducibility: Good (with minor adjustments)

Statistical Soundness:** Moderate (requires clarification)

Reviewer #2: This is a well-written manuscript with good quality research demonstrated. I would like to request the authors for a minor revision:

1. Immunological interaction in the decidual environment is crucial in terms of pregnancy success. The concept has been clearly elucidated. One factor is the HLA-G expression in the trophoblastic cells and the detection of soluble isoforms can guide predicting pregnancy outcomes (pre-eclampsia is one of the causes of spontaneous abortion). Below is a study worth citing:

Bhattarai A, Shah S, Dahal K, Neupane R, Thapa S, Neupane N, Barboza JJ, Shrestha A, Sah R, Apostolopoulos V. Biomarker role of maternal soluble human leukocyte antigen G in pre-eclampsia: A meta-analysis. Immun Inflamm Dis. 2024 Apr;12(4):e1254. doi: 10.1002/iid3.1254. PMID: 38639563; PMCID: PMC11027746.

Overall, I have no significant comments to the authors. When interrogating in the introduction, I would like to request authors to mention that their objective was to answer those questions, rather than simply making interrogations.

Congratulations on the good work!

**********

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: Yes:  Dr Manim Amatya

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.

Attachments

Attachment

Submitted filename: Manuscript_PLOSONE_0910.docx

Attachment

Submitted filename: Figures.docx

The alteration of uterine microbiota participated in the activation of the decidual inflammatory response in early spontaneous abortion

PONE-D-24-35956R1

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.

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. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

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,

Sameer Timilsina, MD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for submitting your manuscript to PLOS One. After peer-review we would like to inform you that your manuscript has been accepted for publication. Congratulations!!

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

**********

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: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

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: 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 #2: 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: (No Response)

Reviewer #2: (No Response)

**********

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:  Dr Manim Amatya, MD Pathology, Grande International Hospital, Dhapasi, Kathmandu, Nepal

Reviewer #2: No

**********