Title: Blood Metabolites, Mitochondrial Biofunction, and Cervical Cancer: A Bidirectional
Mendelian Randomization Study
Version: 3 Date: 01 Jul 2025
Reviewer's report:
"PEER REVIEWER ASSESSMENTS:
OBJECTIVE - Full research articles: is there a clear objective that addresses one or several
testable research questions? (Brief or other article types: is there a clear objective?)
No - there are minor issues
DESIGN - Is the current approach (including controls and analysis protocols) appropriate for
the objective?
No - there are minor issues
EXECUTION - Are the experiments and analyses performed with sufficient technical rigor to
allow confidence in the results?
No - there are major issues
STATISTICS - Is the use of statistics in the manuscript appropriate?
No - there are issues with the statistics in the study
INTERPRETATION - Is the current interpretation/discussion of the results reasonable and not
overstated?
No - there are major issues
OVERALL MANUSCRIPT POTENTIAL - Has the author addressed your concerns sufficiently
for you to now recommend the work as a technically sound contribution? If not, can further
revisions be made to make the work technically sound?
Maybe - with major revisions
PEER REVIEWER COMMENTS:
GENERAL COMMENTS: This study offers a timely and innovative application of
bidirectional two-sample Mendelian randomization to explore causal relationships between
blood metabolites, mitochondrial biofunction, and cervical cancer. The methodology is
sound and strengthens causal inference, addressing limitations of observational studies. The
identification of multiple metabolite categories and mitochondrial pathways associated with
cervical cancer adds depth and novelty to the research. However, the presentation of results is
dense and would benefit from clearer structure and synthesis of key findings. Some technical
explanations in the discussion detract from the focus on biological relevance and should be
better integrated or streamlined. The connection between specific metabolites and cervical
cancer pathogenesis is not consistently articulated and requires more focused interpretation.
The limitations are acknowledged, but more detail on MR-specific biases would enhance
transparency. While the study is grounded in strong analytical rigor, additional narrative
synthesis would improve accessibility for broader readership. Overall, the work makes a
valuable contribution to cancer epidemiology and metabolic research and offers a solid
foundation for future experimental and clinical studies.
For the sake of clarity, I declare that I am a reviewer for the revised version of the manuscript,
although I was not a reviewer for the first version. The authors addressed the previous reviewers'
comments.
REQUESTED REVISIONS:
I recommend to take into account the following suggestions:
- Introduction: The introduction is lengthy and contains several long sentences that may reduce
readability and clarity. Consider breaking complex sentences into shorter ones and focusing each
paragraph on a single idea to improve flow.
- Introduction: The phrase ""On other hand"" is grammatically incorrect. Correct it to ""On the
other hand"" to maintain proper formal English usage.
- Introduction: The description of blood metabolites lacks specificity in its connection to cervical
cancer. Provide a clearer transition or direct evidence linking blood metabolites specifically to
cervical cancer to strengthen the rationale.
- Introduction: Some phrases, such as “a power ATP,” are awkward and unclear. Rephrase as
“ATP, which provides energy” or similar to improve clarity and precision.
- Introduction: The explanation of mitochondrial function overlaps with basic biology and
may be too detailed for an introduction. Focus more on its specific implications in cancer
development to maintain relevance.
- Introduction: The phrase “biometabolisms” is nonstandard and confusing. Replace with
“biological metabolic processes” or simply “metabolic processes” for clarity.
- Introduction: The reference to ""previous research"" and ""some studies"" is vague and
weakens credibility. Name specific studies or summarize key findings to strengthen the evidence
base.
- Introduction: The final paragraph could better emphasize the novelty and importance of the
proposed MR approach. Make the benefits of bidirectional two-sample MR in this context more
explicit and concise.
- Introduction: The conclusion of the introduction is too general about the aim of the study.
Clearly define the hypotheses and expected outcomes to improve focus.
- Introduction: The sentence structure throughout is often repetitive and wordy. Vary the
sentence structure and eliminate redundancy (e.g., avoid repeating phrases like “the occurrence
and development of diseases” multiple times) to improve readability.
- Methods: The objective of the study is only briefly stated and not clearly defined in the opening
paragraph. Add a more concise and specific statement of the research aim to clarify the study’s
purpose upfront.
- Methods: The description of datasets lacks information about population overlap. Clarify
whether there is any sample overlap between exposure and outcome GWAS datasets to address
potential bias in two-sample MR.
- Methods: The threshold for selecting instrumental variables (P<1×10##) is relatively liberal
and may introduce weak instruments. Justify this threshold more robustly or consider presenting
sensitivity analyses with stricter thresholds.
- Methods: The explanation of quality control measures lacks detail on how linkage
disequilibrium (LD) pruning was implemented. Briefly describe the tool or procedure used for
LD clumping to enhance reproducibility.
- Methods: The mention of F-statistics as a measure of instrument strength is appropriate, but
results of F-statistics are not provided. Include a summary of average or range of F-values to
demonstrate instrument validity.
- Methods: The reverse MR analysis is introduced only at the end of the methods, despite being
essential to the study. Move the reverse MR explanation closer to the study aim to emphasize
its role and rationale.
- Methods: The description of MetaboAnalyst 6.0 lacks explanation of which parameters or
metabolite sets were used. Provide more detail on how the metabolic pathway analysis was
carried out (e.g., enrichment method, significance thresholds).
- Methods: The statistical methods section does not report how missing or invalid SNPs were
handled. Clarify the criteria for SNP exclusion and any imputation or harmonization strategies
applied to the data.
- Methods: The decision rules for interpreting MR results (e.g., exclusion of inconsistent results)
are somewhat vague. Specify exactly how results were deemed consistent across MR methods
to enhance transparency.
- Methods: The methods section is dense and contains multiple nested clauses that affect
readability. Consider breaking down long paragraphs into clearly labeled sub-sections for easier
comprehension and reference.
- Results: The overview paragraph lacks clarity on how many metabolites and mitochondrial
functions were initially tested versus retained after exclusions. Specify the initial versus final
counts more explicitly to enhance transparency.
- Results: The sentence “Preliminary identification of 25 blood metabolites...” lacks a subject and
is grammatically incomplete. Rephrase to “We preliminarily identified 25 blood metabolites...”
to ensure proper sentence structure.
- Results: The method of categorizing metabolites into types is introduced without explanation of
criteria or source. Clarify the classification rationale or reference relevant biochemical categories
for consistency and credibility.
- Results: The results in each metabolic category are listed without context or interpretation.
Include a brief summary statement after each category to help readers understand the biological
or clinical relevance of the associations.
- Results: There is inconsistent punctuation and spacing in reported odds ratios (e.g., “1.39
95%CI” vs. “1.39, 95% CI”). Standardize formatting to improve readability and adherence to
reporting guidelines.
- Results: The description of subgroup analyses is vague and lacks detail on how overlap across
datasets was assessed. Provide clearer methodology or definitions used to determine overlap
across CC subtypes.
- Results: The mitochondrial biofunction results are underdeveloped compared to the metabolite
results. Expand briefly on the biological significance of these findings to balance the
presentation.
- Results: The final comments mention sensitivity analyses but do not describe how many
exposures passed all robustness checks. Summarize how many of the 28 total exposures were
fully robust across all MR methods to reinforce confidence in the results.
- Results: The entire section is densely packed with numerical results, making it difficult to
interpret key messages. Add 1–2 summary sentences at the end of each subsection to synthesize
patterns or implications for cervical cancer research.
- Discussion:  The phrase ""SNPs was employed as an IVs"" is grammatically incorrect. Revise
to “SNPs were employed as IVs” to maintain proper subject-verb agreement.
- Discussion:  The introduction to the Discussion repeats basic MR methodology rather than
focusing on findings. Consider shortening the MR method explanation and focusing more
quickly on the interpretation of your study’s results.
- Discussion:  The logic for choosing IVW as the primary method is too technical and might
distract from the interpretation of findings. Move the rationale for IVW to the Methods or
Supplementary section to streamline the Discussion.
- Discussion:  The paragraph divisions are inconsistent and overly dense, especially under
mitochondrial function and metabolite categories. Break down long paragraphs into shorter,
focused ones for each key point or finding to improve readability.
- Discussion:  Some metabolite descriptions (e.g., C10:1, 13-HODE) lack context for clinical
significance. Briefly explain the biological or clinical relevance for each metabolite to reinforce
their importance.
- Discussion:  The description of linolenic acid's effect overlaps with multiple mechanisms
without clear organization. Separate these mechanisms into distinct sentences or subpoints to
clarify how each contributes to the antitumor effect.
- Discussion:  The discussion of bile acid metabolism does not fully explain why some bile acids
are protective while others are harmful. Add a sentence summarizing the dual roles of bile acids
and suggest why specific bile acids may behave differently.
- Discussion:  The amino acid section introduces several hypotheses without linking back to
cervical cancer consistently. Explicitly relate each metabolite’s mechanism to cervical cancer
pathogenesis to maintain relevance.
- Discussion:  The hormone metabolism section is notably underdeveloped compared to
others. Expand on the possible links between the identified hormone metabolite and cervical
carcinogenesis to ensure consistent depth.
- Discussion:  The caffeine metabolism section raises the idea of concentration-dependent
effects without evidence. Acknowledge the speculative nature of this hypothesis and suggest
experimental designs to test it.
- Discussion:  The subgroup analysis is mentioned briefly and lacks supporting data or
interpretation. Provide at least one example of how the subtypes differed to make this point
more informative.
- Discussion:  The paragraph synthesizing mitochondrial function and blood metabolites lacks
flow and jumps between abstract concepts. Restructure it to follow a logical progression from
function to impact to implication.
- Discussion:  The limitations paragraph is well-placed but lacks detail on the specific biases
inherent in MR (e.g., weak instrument bias, population stratification). Elaborate on how these
might affect your findings.
- Discussion: Several citations in the discussion are used to make critical points but are placed
at the end of long paragraphs. Cite studies closer to the specific claims they support to improve
traceability and academic rigor.
- Conclusion: The conclusion repeats content from earlier in the Discussion nearly verbatim.
Rewrite the conclusion to be more concise and focused on clinical implications and future
research directions."