Dear Dr. Oltra,

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.

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PLOS ONE

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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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: 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

Reviewer #3: Yes

Reviewer #4: 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

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: The manuscript is important and relevant topic with clear implications. The authors present a well defined question, their methods appear sound and appropriate The data are adequately analysed, and the conclusions are generally supported by the findings.

Strengths of the manuscript:

The study addresses a significant gap in the literature and is timely.

Methodology is robust, with appropriate controls and sample size.

The manuscript is well written and structured logically.

The authors have provided access to the underlying data, in compliance with the PLOS data availability policy.

Areas for improvement:

While the data are available, the accompanying metadata or code used for analysis (if any) could be shared to enhance transparency and reproducibility.

In a few places, more detail regarding statistical methods or rationale for specific analytical decisions would strengthen the manuscript.

The discussion could be expanded slightly to more clearly situate the findings within the context of existing literature.

Overall, this is a technically sound manuscript that makes a valuable contribution to the field. I recommend acceptance with minor revisions to improve clarity and completeness in data sharing and discussion.

Please do acknowledge that the lack of protocol registration (line 74) is a limitation.

Was there a need for 2x2 tables or individual results reported? I do not recall seeing these.

Some inconsistency in abbreviations: e.g. "NPL" should be NLR (Negative Likelihood Ratio). Rather replace "NPL" with "NLR" consistently.

Check for citation styles I may have picked up the use of Vancouver and APA. please choose the one required by the journal. Superscript references (e.g., "22% (1)") are inconsistently formatted.

Please double check this. You wrote "RD" in some places instead of DR (e.g., line 268, 269, etc.). Correct “RD” to DR (Diabetic Retinopathy) to maintain consistency.

Reviewer #2: The study conducted by Martinez Santos et al performed a systemic review and meta-analysis on the different DR stages by screening countless published works. They then analyzed the 16 studies and found circulating miRNAs of interest for future diagnostic testing. The appreciate the Martinez Santos and colleagues for conducting important research in the field of diabetes. There are only a few minor suggestions prior to the manuscript being fully ready for publication.

Major:

- It would be nice to briefly touch upon previous efforts that have studies DR and the possible biomarkers that were identified, if any, and where they may have fallen short on to enhance the motivation behind this work. Perhaps including a brief paragraph diving into this between the paragraphs on line 60 and 61 might help with the flow.

Minor:

- Please provide citation for the claim of 92.6 million on line 44.

- On line 54, specifying what types of bodily fluids miRNAs can be found would be neat for potential readers to grasp at.

- On line 95, was gestational diabetes also excluded?

- For figure 3, it would be nice to see the 45 degree dashed line on panels C, F, I to show random chance and for visualization purposes.

- A side question, but I noticed in the supplements that sex and age was recorded. Were there any other potential features that were recorded by any chance?

Reviewer #3: Thank you for the opportunity to review the manuscript titled "microRNA-Diagnostic Test Accuracy (DTA) for diabetic retinopathy stage identification: A systematic review and meta-analysis" for consideration in PLOS ONE journal. This study aims to evaluate the diagnostic accuracy of circulating microRNAs (miRNAs) as biomarkers for distinguishing between different stages of diabetic retinopathy (DR), focusing on three specific comparisons: healthy controls versus diabetic retinopathy (CTL vs DR), type 2 diabetes mellitus without retinopathy versus diabetic retinopathy (T2DM vs DR), and non-proliferative diabetic retinopathy versus proliferative diabetic retinopathy (NPDR vs PDR).

1. Title & Abstract

Title Ambiguity (Page 1, Line 1): The title does not fully reflect the study's scope. It uses "microRNA-Diagnostic Test Accuracy (DTA)" and "stage identification" but fails to specify that it evaluates circulating miRNAs across distinct DR stages.

Inadequate Conciseness and Informativeness (Page 1, Line 1): While concise, the title omits critical details, such as the focus on circulating miRNAs and the specific staging comparisons.

Abstract Non-Compliance with PRISMA (Page 1, Lines 15-39): The abstract claims adherence to PRISMA-DTA guidelines but falls short. It lacks a detailed description of the search strategy (e.g., specific terms or dates), does not specify the number of reviewers involved in all processes, and omits explicit reporting of confidence intervals for all numerical results in a consistent manner.

Incomplete Key Findings (Page 1, Lines 29-34): The abstract reports pooled sensitivity and specificity but does not include confidence intervals consistently (e.g., "77% (95% CI 0.070-82)" uses an unconventional format). It also omits the number of participants per comparison and specific miRNAs analyzed.

2. Introduction

Unclear Research Questions and Objectives (Page 10, Lines 42-69): The introduction states the goal of evaluating miRNA diagnostic accuracy (Line 68) but does not explicitly define the research questions or detail the three comparisons (CTL vs DR, T2DM vs DR, NPDR vs PDR) until later sections.

Insufficient Background (Page 10, Lines 43-52): The background mentions DR prevalence and diagnostic tool limitations but lacks detail on the clinical significance of staging DR (e.g., NPDR vs PDR progression).

Weak Rationale (Page 10, Lines 61-69): The rationale for this meta-analysis is underdeveloped. It notes the need for minimally invasive methods (Line 51) but does not explain why existing miRNA studies are insufficient or how this review fills a specific gap.

Undefined Knowledge Gap (Page 10, Lines 61-63): The introduction hints at a lack of "comprehensive evaluations" but does not clearly articulate the specific knowledge gap this study addresses, such as inconsistencies in prior miRNA research or unmet clinical needs.

3. Methods

Poorly Defined Eligibility Criteria (Page 12, Lines 84-96): The criteria mention human participants and fundus fluorescein angiography as the gold standard but lack specificity on DR stage definitions, miRNA types (e.g., single vs. panels), or exclusion of studies with incomplete data.

Vague Study Selection and Data Extraction (Page 12, Lines 97-103): The process mentions two reviewers and a third for arbitration but lacks detail on how disagreements were resolved (e.g., consensus or voting) or what specific data were extracted (e.g., miRNA thresholds).

Inadequate Risk of Bias Tool Application (Page 13, Lines 105-109): The use of QUADAS-2 is noted, but there’s no explanation of how it was applied (e.g., domain-specific scoring) or how results influenced the analysis.

Unclear Statistical Methods (Page 13, Lines 110-125): The methods mention a bivariate random-effects model and heterogeneity tests but do not justify the model choice, detail heterogeneity exploration beyond, or explain meta-regression variables.

4. Results

Opaque PRISMA Flow Diagram (Page 14, Lines 127-136): The flow diagram (Figure 1) is referenced, but exclusions like "objective different from the study" (Line 134) are vague.

Incomplete Study Characteristics (Page 15, Line 155; Table 1): Table 1 lists studies but omits critical details, such as specific miRNAs per study or diagnostic thresholds.

Unjustified Risk of Bias Reporting (Page 16, Lines 156-167): The risk of bias summary (Figure 2) notes patient selection concerns but lacks specific examples or impact analysis, undermining its justification and usefulness.

Weak Meta-Analysis Reporting (Page 16-17, Lines 172-218): Results report sensitivity and specificity but inconsistently present confidence intervals (e.g., "77% (95% CI: 0.70-0.82)" vs. "84% (79-87)"). High heterogeneity is noted but not adequately explored or mitigated, questioning robustness.

5. Discussion

Limited Contextualization (Page 24, Lines 326-340): The discussion references Ma et al. (2022) but does not deeply compare findings or explain how this study advances prior work.

Inadequate Limitation Discussion (Page 25, Lines 369-384): Limitations mention demographic bias and study design but overlook key issues like variability in miRNA detection methods or lack of standardized thresholds.

Unclear Implications (Page 25, Lines 386-394): Clinical and research implications are vague (e.g., "integrated with other clinical assessments" lacks specificity).

6. Conclusion

Unjustified Claims (Page 26, Lines 387-390): The conclusion asserts "high diagnostic accuracy" despite moderate-to-high heterogeneity and low-to-moderate evidence quality (Page 22, Lines 316-318).

Lack of Specificity (Page 26, Lines 391-394): It calls for future studies but does not specify research directions (e.g., which miRNAs or methods).

7. Transparency & Reproducibility

Unregistered Study (Page 11, Line 73): The lack of registration (e.g., PROSPERO) deviates from best practices and reduces transparency.

Reviewer #4: 1) Grammatical and Orthographic Comments

- The manuscript is generally well-written and clear. I did not find systemic grammatical errors or awkward phrases in the text. Some minor suggestions:

Consistency: The manuscript sometimes switches between percentage formats (e.g., “77 %” and “77%”). Choose one and apply it uniformly (journal style guides typically prefer “77%” without a space).

Some long sentences in the Introduction (lines 43–51) could be broken for clarity; e.g., “This makes them less accessible, especially in remote or low-resource areas.” could be split for better readability.

Minor repetition: In lines 55–60, there is redundancy explaining miRNAs’ importance; consider condensing to maintain focus.

Typo-like inconsistency: lines like “overall evidence certainly was graded via GRADE” (line 26) sound off. Perhaps intended “certainty was graded”; a correction improves precision.

2) Scientific Rigor

Strengths:

- The methodology follows PRISMA-DTA and QUADAS-2 rigorously.

- Clear definition of comparison groups (CTL vs DR, T2DM vs DR, NPDR vs PDR) is excellent and adds strength.

- Subgroup and meta-regression analyses address potential heterogeneity sources robustly.

- Sensitivity analysis and publication bias assessment with Deeks’ funnel plot are thorough.

Weaknesses:

- Protocol registration: The authors state the protocol was not registered. While not mandatory, protocol registration (e.g., PROSPERO) increases transparency.

- Risk of bias: High risk in patient selection and absence of randomized trials limits robustness; the authors acknowledge this but could suggest solutions.

- Applicability: Most data from Chinese cohorts; limits generalizability to other ethnicities. Consider highlighting this limitation more prominently.

- Diagnostic thresholds: Lack of consistent cut-offs across studies weakens translation to clinical practice.

Some references (e.g., [33]) are relevant but recent studies post-2022 could enrich the context.

3) Scientific Impact

The study fills an important gap by stratifying meta-analysis across clinically distinct DR stages, which previous works have not done in detail. This approach gives clinicians better guidance on miRNAs’ diagnostic utility at specific DR stages.

The work will interest endocrinologists, ophthalmologists, and biomarker researchers; potential for real-world impact if validated prospectively.

4) Questions to Authors

- Given the predominance of Chinese populations, how do you propose validating these findings across ethnically diverse cohorts?

- How do differences in miRNA normalization strategies (e.g., U6 vs miR-16-5p) influence diagnostic performance in your dataset?

- Can you discuss practical steps to standardize pre-analytical miRNA handling and measurement to improve reproducibility?

- Since your sensitivity analysis shows individual studies’ influence, how confident are you that these outliers don’t bias the conclusions?

5) Additional Comments

- Figures and tables are well presented; figures showing forest plots, SROC curves, Fagan’s nomograms, and meta-regressions are clear and informative.

- The inclusion of a detailed GRADE assessment is commendable, but the presentation of certainty ratings (Tables 5–7) could benefit from more narrative discussion in the Results or Discussion.

- The Discussion excellently compares results with prior meta-analyses but could expand on how novel your subgroup stratification truly is compared to Ma et al. (2022).

Overall Recommendation:

The manuscript demonstrates rigorous methods and meaningful results but should address generalizability limitations more explicitly and discuss how preanalytical standardization could improve clinical adoption. Minor editorial corrections on consistency and sentence clarity are recommended.

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Reviewer #1: Yes:  Ameer Steven-Jorg Hohlfeld

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Dear Dr. Oltra,

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 04 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,

Yalong Dang

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.

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Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: 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 #2: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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

Reviewer #2: Yes

Reviewer #4: Yes

Reviewer #5: I Don't Know

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

The PLOS Data policy

Reviewer #2: Yes

Reviewer #4: (No Response)

Reviewer #5: Yes

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

Reviewer #2: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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Reviewer #2: Thank you for addressing all comments. There are no further questions regarding the manuscript, and I give my recommendations for publication.

Reviewer #4: (No Response)

Reviewer #5: In general, the manuscript is well written and scientifically sound. The authors thoroughly revised the manuscript to address the insightful comments from the reviewers and deepened the discussion raised by them. In addition, I appreciate the effort of the authors in providing the raw data in the Excel format and the STATA code used for the analyses, which is in line with contemporary best practices that promote transparency and reproducibility.

Nevertheless, there are inconsistencies that need to be resolved in order to further improve clarity and precision.

1) Abstract section (pdf file, revised manuscript, pages 53-54):

a) First sentence (lines 17-18): To improve clarity and specificity, in addition to avoid redundancy (“This systematic review and meta-analysis”), I suggest the purpose of the study be modified to: “To evaluate the diagnostic accuracy of circulating miRNAs in distinguishing between different DR stages in type 2 diabetes mellitus.”

b) On line 20, it is stated that that “no external funding was received”. However, in the submission form and in the “Funding information” (pages 79-80), the authors report that “The present work received internal funds from Centro de Investigación Traslacional SanAlberto Magno (CITSAM, UCV) and external funds from Agencia Estatal de Investigación Española (PID2020-117875GB-10), Instituto de Salud Carlos III (ISCIII, PI21/00083) and the European Union research fund, HORIZON MSCA 2021-DN-01-01_RETORNA 101073316 and Generalitat Valenciana ACIF 2023-128-001.” Which statement is correct?

2) Introduction section (pdf file, revised manuscript, page 55):

The first sentence (page 55, lines 73-74) states that “Diabetic retinopathy (DR) is one of the leading causes of vision loss among working-age adults worldwide (1).” That is true. However, this claim is supported by the paper by Lundeen et al. (Prevalence of Diabetic Retinopathy in the US in 2021. JAMA ophthalmology. 2023;141(8):747-54), and US is not “worldwide”.

3) Introduction section (pdf file, revised manuscript, pages 55-57):

Reviewer 3 states in comment #2.3: “The rationale for this meta-analysis is underdeveloped. It notes the need for minimally invasive methods (Line 51) but does not explain why existing miRNA studies are insufficient or how this review fills a specific gap.” In the rebuttal letter, the authors responded that “In response, we would like to highlight that the Introduction now includes a clear rationale supported by two key points. First, it states that while numerous studies have evaluated the diagnostic potential of circulating miRNAs in DR, there is marked heterogeneity in sample types, analytical methods (e.g., RT-qPCR, microarrays, NGS), and target miRNAs.”

a) I did not find this statement in the introduction of the revised manuscript.

b) What is the second key point supporting a clear rationale?

4) Methods section, “Eligibility criteria” subsection (pdf file, revised manuscript, page 58, lines 137-138):

Reviewer 2 asked in comment #3: “Was gestational diabetes also excluded?” In the rebuttal letter, the authors responded that they have revised the sentence from: “We excluded studies that focused only on type 1 diabetes” to: “We excluded studies that focused on other types of diabetes, such as type 1 or gestational diabetes.” This is implied in the revised sentence but not explicitly stated.

5) Results section (pdf file, revised manuscript, page 63):

Table 1 is detailed and informative. However, I suggest adding a column with the total number of individuals included in each study. If necessary, due to space restrictions, the “Method” column can be removed because this information is already in the text.

6) Results section, “Subgroup Analysis and Meta-Regression in CTL vs DR, T2DM vs DR, and NPDR vs PDR Comparisons” subsection (pdf file, revised manuscript, page 66, lines 308-309):

The authors assert that “studies conducted in China demonstrated higher diagnostic performance across all three comparison groups, as reflected by AUC.” I do not know if this is the case for CTL vs DR and T2DM vs DR. In the comparison between CTL and DR (Table 2), only one study was not performed in China, which hinders its interpretation. Regarding the comparison between T2DM and DR (Table 3), the difference in the AUC value was 0.01.

7) Results section (pdf file, revised manuscript, page 70, lines 371-376):

The text refers to the paper by Shaker et al. (reference #26) as if there were more than one study: “Cook’s distance analysis pinpointed 371 Shaker et al. (miR-20b and miR-17-3p)(26) as influential studies (Figure 7G). Yet, removing these studies caused only marginal changes: pooled sensitivity declined from 81.0% to 78.9%, and specificity increased from 80.0% to 81.1%. Interestingly, heterogeneity (I²) rose notably from 74.12% to 97.71%, suggesting that these studies contributed to controlled variance across studies. Outlier assessment confirmed no excessive deviations (Figure 7H).” Is it correct (“two studies”)?

8) Regarding I² (heterogeneity estimates), is it really necessary to use two decimal places to express this metric (Results section)?

9) Limitations section (pdf file, revised manuscript, page 75, lines 481-483):

“Fourth, pre-analytical and analytical heterogeneity was substantial across studies. Variations in the biological sample type , normalization strategies, and miRNA isolation and detection platforms contributed significantly to inconsistency in results.”

It is known that these factors affect the quantification of miRNA expression. But are the authors referring to the results of their analyses? Does “detection platforms” mean the method of quantifying microRNAs? I did not find any mention to the miRNA isolation method used in the previous studies in the manuscript. Moreover, as per Table 1 and S3 Table, all but one study measured the miRNA levels by RT-qPCR. In sum, did the authors perform the meta-analysis considering these two factors (miRNA isolation and detection platforms)?

10) Editing and typo errors:

a) Abstract (pdf file, page 53, lines 20-23): “We searched PubMed, CENTRAL, Scopus, Web of Science, ScienceDirect and ClinicalTrials (up to 15 January 2025) for diagnostic test accuracy studies of circulating miRNAs in three clearly defined groups: […]”. However, in the Methods section, “Search strategy” subsection (page 57, lines 114-116), it is stated that the last update of this review was on January 20, 2025. Which date is correct?

b) Percentages (sensitivity and specificity) remain formatted with a space between the numeral and the percentage symbol in the Abstract section (pdf file, page 54).

c) Reviewer 4 states in comment #1.4: “lines like “overall evidence certainly was graded via GRADE” (line 26) sound off. Perhaps intended “certainty was graded”; a correction improves precision.” In the rebuttal letter, the authors responded that the abstract has been revised to state “overall evidence certainty was graded via GRADE”. The word “certainly” was removed, but “certainty” was not added. Thus, the revised sentence states that “[…] overall evidence was graded via GRADE.” (pdf file, page 53, line 29).

d) NLR is written as NPL in the caption of Figure 4 (pdf file, page 66, line 302).

e) Results section (pdf file, page 69, lines 357-358): “PDR” is spelled as “DR”.

f) In the caption of Figure 7 (pdf file, page 70, lines 385-386), the Fig. 7E refers to both the goodness-of-fit and the bivariate normality, while the Fig. 7B does not exist: “Figure 7. Sensitivity analysis. Goodness-of-fit (A,E,I), Bivariate normality (E,F,J) Influence analysis (C,G,K) and Outlier detection (D,H,L) across: CTL vs DR, T2DM vs DR and NPDR vs PDR.”

g) Discussion section (pdf file, page 72, lines 416-418): The first paragraph ends with a sentence with missing terms: “Furthermore, the study lacks a formal assessment of risk of bias using tools such as QUADAS-2, nor does it explore heterogeneity or undermining both its methodological robustness and reproducibility.” Explore heterogeneity and what else?

h) Discussion section (pdf file, page 73, lines 426-430): “By stratifying the analysis into three distinct and clinically relevant comparison groups—healthy controls CTL vs DR, T2DM , T2DM vs DR, and NPDR vs PDR, we were able to reduce inter-study heterogeneity and increase the precision and relevance of pooled diagnostic accuracy estimates comparing with Ma et al (2022).” The term “T2DM” before “T2DM vs DR” should be removed.

i) Discussion section (pdf file, page 73, lines 439-441): The following sentence needs to be reworded for clarity and completeness: “miR-21, also was found in 3 studies (15, 18, 34) has been implicated in retinal angiogenesis and inflammation in the diabetic context(38).”

j) “Prisma” is spelled as “Prima” in the title of S2 Table (pdf file, page 80, line 603).

l) S4 Table is numbered as S5 Table in the manuscript (pdf file, page 80, line 605).

m) Should not supplementary tables be mentioned in the text (accompanying the respective methods or results)?

n) S3 Table (“Extraction data”) presents some Spanish words (“Casos y controles”, “Controles”, “Egipto”, “Cuantitative”, and “Cualitative”). Moreover, a term is missing from the “Controls (Healthy and T2DM)” column specifying who the 70 control subjects are in the study by Wan et al., 2017.

o) Bibliography (pdf file, page 83, lines 769-772): The text citing the study by Théry et al. (reference #55) was both added and removed during the manuscript review, but the reference remains in the Bibliography.

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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 #2: No

Reviewer #4: No

Reviewer #5: Yes:  Kátia Gonçalves dos Santos

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Dear Dr. Oltra,

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 Nov 08 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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We look forward to receiving your revised manuscript.

Kind regards,

Yalong Dang

Academic Editor

PLOS ONE

Journal Requirements:

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. 

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

Reviewer #5: (No Response)

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

Reviewer #5: Yes

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

Reviewer #5: I Don't Know

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

The PLOS Data policy

Reviewer #5: Yes

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

Reviewer #5: Yes

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Reviewer #5: The manuscript was revised to address the comments raised in the previous submission (R1). However, the submitted manuscript (R2) does not present some of the modifications stated in the rebuttal letter and some errors (that did not exist) were introduced into the new version of the manuscript (PONE-D-25-27197_R2.pdf file). After I started writing my comments, I found out that the “clean manuscript” (pages 15 to 39) is not identical to the “manuscript with track changes” (pages 48 to 73), which makes it difficult to follow what has been modified (or not) in the revised manuscript. The comments I provide below are based on the manuscript included on pages 48 to 78. Finally, I have to say that I did not check the figures and the supplementary tables in the revised manuscript.

1) Abstract section (revised manuscript with track changes, page 48):

In the response to the comment #1a, the authors state in the rebuttal letter that they “have revised the first sentence of the Abstract accordingly to improve clarity and avoid redundancy. The updated sentence now reads: “Purpose: To evaluate the diagnostic accuracy of circulating miRNAs in distinguishing between different diabetic retinopathy stages in type 2 diabetes mellitus.”

Although this detail does not compromise the manuscript, the purpose remains the same. Instead, the second sentence (first one of the Methods) was modified (revised manuscript with track changes, page 48, lines 18-20).

2) Abstract section (revised manuscript with track changes, page 48):

An error was introduced in the sentence “We searched PubMed, CENTRAL, Scopus, Web of Science, ScienceDirect and ClinicalTrials (up to 20 January 2025) for diagnostic test accuracy studies […]” (lines 21-27) by deleting the following terms: “type 2 diabetes mellitus without retinopathy versus diabetic retinopathy”. Now, the sentence is incomplete.

3) Introduction section (revised manuscript with track changes, page 49):

In response to the comment #2, the reference by Lundeen et al. was replaced by the paper by Thomas et al. However, the study by Lundeen et al. remains in the Bibliography (page 70) as the reference #2. Therefore, all references from Thomas et al. onward should be renumbered.

4) Results section (revised manuscript with track changes, page 56):

In response to the comment #5, the authors added a column with the total N in Table 1 described on page 22, but not on that presented on page 56. I think this is not a problem, as the Journal uses the clean version of the manuscript to typeset the proof.

5) Results section (revised manuscript with track changes, page 57):

In response to the comment #8, the authors decided to report I² as an integer percentage (throughout the manuscript). However, the following sentence still has the I² estimates with two decimals between the parentheses and needs to be corrected (lines 203-206): “The pooled estimates from the random‐effects model showed a summary sensitivity of 77% (70–82), with an I² of 47% (1.39–92.75), and a summary specificity of 84% (77–89), with an I² of 62% (31.26–93.30), indicating moderate heterogeneity (Fig 3A-B).”

6) Editing and typo errors:

In response to the comment #10m, the authors added the following sentences to the Methods section: “The complete extraction dataset is provided in S3 Table.” and “Additional details are provided in S4, S5, and S6 tables.” These additions were made to the clean manuscript (pages 19 and 21), but not to the manuscript with track changes.

7) Funding section:

In the response to the comment #1b, the authors state in the rebuttal letter that “no funds from these grants were specifically allocated or used for the conduct of this systematic review and meta-analysis. To avoid any possible misunderstanding, we have removed the Funding section from the manuscript and clarified that this work received no dedicated external financial support.”

However, the Funding section was neither removed from the manuscript nor corrected according to this statement (revised manuscript with track changes, page 69).

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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.

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Reviewer #5: Yes:  Kátia Gonçalves dos Santos

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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

Circulating microRNAs as biomarkers for diabetic retinopathy stage identification: a DTA systematic review and meta-analysi s

PONE-D-25-27197R3

Dear Dr. Oltra,

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,

Yalong Dang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #5: All comments have been addressed

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

Reviewer #5: Yes

**********

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

Reviewer #5: I Don't Know

**********

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

The PLOS Data policy

Reviewer #5: Yes

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

Reviewer #5: Yes

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Reviewer #5: All the remaining inconsistencies in the manuscript were solved. The only thing I noticed on a quick revision is that the following sentence is now duplicated in the Abstract section: “Data were synthesized via a bivariate random-effects meta-analysis, with subgroup analyses, meta-regression, and sensitivity tests to explore heterogeneity”.

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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 #5: Yes:  Kátia Gonçalves dos Santos

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