Functional and Compositional Analysis of the Fecal and Vaginal Microbiota in Vestibulodynia: An Explorative Case–Control Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript addresses an important and underexplored aspect of vestibulodynia pathophysiology by evaluating the vaginal and fecal microbiome compositions and functions in affected individuals compared to healthy controls. Overall, the study is methodologically sound and clearly written, with appropriate statistical analysis and a thoughtful discussion.

Strengths:

1- The use of both fecal and vaginal samples provides a comprehensive view of microbiome-host interaction.

2- Identification of functional differences (e.g., L-lactate dehydrogenase and fumarate reductase pathways) is a unique contribution to the field.

3-The association of Lactobacillus iners dominance with vestibulodynia and the inverse relationship with L. crispatus is biologically plausible and aligns with prior observations.

Major Comments:

The authors suggest potential clinical applications such as vaginal pH or PO₂ testing, but this remains speculative (please slight your conclusion)

Some microbiological terms are inconsistently used (e.g., "commensals" vs. “pathobionts”). Consider standardizing terminology and clearly defining what is meant by “pathobiont” in the context of this study.

Functional Interpretation Should Be More Cautious:
Functional inference using PICRUSt2 is based on predicted gene content and may not reflect actual metabolic activity. The discussion would benefit from a sentence acknowledging this limitation explicitly- include the limitation of the current study.

I have some questions regarding whether hormonal contraception, sexual activity, antibiotic use, or menstrual phase are controlled for or recorded. I think these variable can significantly alter the vaginal microbiota and should be discussed if not accounted for.

Relevant Literature Integration:
The authors may consider citing and discussing the findings from:
“Changes in the Vaginal Microbiota of Women With Secondary Localized Provoked Vulvodynia”, which provides complementary evidence of altered microbial patterns in vulvodynia. Integrating this work would help situate the current study within the growing body of literature addressing microbial signatures and their functional consequences in this population.

Minor Comments:

In the Results section, clarify the clinical significance of the observed correlations (e.g., B. leopoldii with Megasphaera). Are these correlations potentially causal or incidental?

The sample sizes are modest; a power analysis would be helpful, or at least acknowledgment of the sample size as a limitation.

Recommendation:
Minor revision. The manuscript is well-executed and offers valuable insights. Encouraging the inclusion of recent literature, particularly on microbial dynamics in localized provoked vulvodynia, would enhance the contextual depth and visibility of related findings.

Author Response

Major Comments:

Comment 1:

The authors suggest potential clinical applications such as vaginal pH or PO₂ testing, but this remains speculative (please slight your conclusion)

Response 1:

We agree that this part was probably too speculative, thus we have toned down the conclusion remarks (line 280)

Comment 2:

Some microbiological terms are inconsistently used (e.g., "commensals" vs. “pathobionts”). Consider standardizing terminology and clearly defining what is meant by “pathobiont” in the context of this study.

Response 2:

We completely agree with the reviewer that this term should have been clarified more extensively in the text. We have done as requested from line 117 to 120.

Comment 3:

Functional Interpretation Should Be More Cautious:
Functional inference using PICRUSt2 is based on predicted gene content and may not reflect actual metabolic activity. The discussion would benefit from a sentence acknowledging this limitation explicitly- include the limitation of the current study.

Response 3:

We are grateful to the reviewer for suggesting this. We have now added in the final discussion from line 272 to 276 as follows:” Our study has some limitations; the first one is the modest sample size of the investigation, which was designed as a single-center pilot study; another limitation is the fact that using the tool PICRUSt2, which bases its analysis on predicted gene content, our functional analysis is a functional inference, and might not reflect actual metabolic activity, but the metabolic potential of the microbiome.”

Comment 4:

I have some questions regarding whether hormonal contraception, sexual activity, antibiotic use, or menstrual phase are controlled for or recorded. I think these variable can significantly alter the vaginal microbiota and should be discussed if not accounted for.

Response 4:

We agree with the reviewer, and we added a new table (Table 1, while the former Table 1 has become Table 2) with anamnestic and clinical data that is discussed between line 75 to 79

Comment 5:

Relevant Literature Integration:
The authors may consider citing and discussing the findings from:
“Changes in the Vaginal Microbiota of Women With Secondary Localized Provoked Vulvodynia”, which provides complementary evidence of altered microbial patterns in vulvodynia. Integrating this work would help situate the current study within the growing body of literature addressing microbial signatures and their functional consequences in this population.

Response 5:

It is always important to integrate previous work with new research, thus we apologies if this article had not been included in our discussion. It has now been added in the main text as follows:” As an example, in the study published by Awad-Igbaria et al. in 2022 [16], the Ochrobactrum genus and Pseudomonadaceae family were identified as indicators for VBD, while in a contemporary study by Panzarella et al., Bifidobacterium longum, the bacterial genus Sneathia, and the bacterial family Leptotrichiaceae were found to be negatively correlated with VBD [9]; in a previous study from Murina et al., in 2020, Lactobacillus, Gardnerella, and Atopobium were indicated as the dominant genera and Lactobacillus gasseri as the dominant species in VBD [17].”  from line 225 to 231.

Minor Comments:

Comment 6:

In the Results section, clarify the clinical significance of the observed correlations (e.g., B. leopoldii with Megasphaera). Are these correlations potentially causal or incidental?

Response 6:

We thank the reviewer, because this observation allowed us to improve a section that needed clarification. We have added as follows: “Statistical correlations between bacterial species in the microbiota do not imply causation. Even so, correlation analysis is a valuable tool for identifying patterns and generating hypotheses regarding possible interactions in the microbiota, thus they can be considered as a starting point for further investigations.” From line 138 to line 141.

Comment 7:

The sample sizes are modest; a power analysis would be helpful, or at least acknowledgment of the sample size as a limitation.

Response 7:

Thanks for highlighting this; we have now addressed this point as a limitation of our study from line 278 to 279.

Comment 8:

Recommendation:
Minor revision. The manuscript is well-executed and offers valuable insights. Encouraging the inclusion of recent literature, particularly on microbial dynamics in localized provoked vulvodynia, would enhance the contextual depth and visibility of related findings.

Response 8:

Thanks for your kind revision, we have done as requested.

Reviewer 2 Report

Comments and Suggestions for Authors

Very interesting work that should provide answers for patients with vulvodynia, such as chronic pain.
In this sense, it would have been important to know the history of gynecological pathology (specifically, recurrent vulvovaginal infections) in both groups. Because the current findings in the flora of women with vulvodynia could be the cause of this increased sensitivity, not the cause.

Author Response

Comment 1:

Very interesting work that should provide answers for patients with vulvodynia, such as chronic pain.
In this sense, it would have been important to know the history of gynecological pathology (specifically, recurrent vulvovaginal infections) in both groups. Because the current findings in the flora of women with vulvodynia could be the cause of this increased sensitivity, not the cause.

Response 1:

We are thankful to the reviewer for highlighting this. Some of the patients suffered from recurrent vaginal infections indeed. Their clinical data have been added in the new Table 1 and are discussed between line 75 and 79.

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for the opportunity to review the manuscript “Functional and compositional analysis of the fecal and vaginal microbiota in vestibulodynia: an exploratory case-control study“
The study provides a dual analysis (vaginal and fecal microbiota) in vestibulodynia (VBD), which is relatively new. The use of 16S rRNA sequencing and PICRUSt2 improves the depth of microbial and functional profiling. The integration of diversity indices, correlation analyses and LEfSe is methodologically sound and comprehensive.
Statistical analysis is correctly applied PERMANOVA, Kendall's Tau with FDR correction and LEfSe. You could include effect sizes (e.g. Cohen's d, η²) where appropriate.
The discussion of K function is pertinent.
The biological relevance of the enzymes K00945 and K01265 in the context of VBD is perhaps a bit speculative. There is much data in the literature linking these enzymes of mucosal or immune functions.
Heat maps and LEfSe graphs are appropriate
Discussions are well structured, integrate previous literature and identify contradictory findings. Highlights CST-specific associations that are insufficiently explored in VBD.
Cite figures in a consistent format (e.g., “Figure A2, right panels” could be confusing).
Make sure figure numbers and legends match the final layout.
Otherwise the manuscript is well structured, correct and interesting.
With these minor changes I recommend publication.

Author Response

Comment 1:

The study provides a dual analysis (vaginal and fecal microbiota) in vestibulodynia (VBD), which is relatively new. The use of 16S rRNA sequencing and PICRUSt2 improves the depth of microbial and functional profiling. The integration of diversity indices, correlation analyses and LEfSe is methodologically sound and comprehensive.
Statistical analysis is correctly applied PERMANOVA, Kendall's Tau with FDR correction and LEfSe. You could include effect sizes (e.g. Cohen's d, η²) where appropriate.

Response 1:

We are grateful to the reviewer for their thorough and detailed revision. We could have added the R2 to the PERMANOVA, but it came out as non-significant. Regarding the LEfSe analysis, the LDA gives a proxy of the effect size of the associations in its score, but in the Kendall’s Tau it is the coefficient itself that gives the measure of the effect size of the correlation that has been calculated; thus, we did not add other calculations apart from the FDR correction.

Comment 2:

The discussion of K function is pertinent.
The biological relevance of the enzymes K00945 and K01265 in the context of VBD is perhaps a bit speculative. There is much data in the literature linking these enzymes of mucosal or immune functions.

Response 2:

We thank the reviewer for pointing this out. It is indeed true that the K00945 and K01265 enzymes can be linked to mucosal and immune functions in the eucaryotic cells. Even if our work has been shaped around bacterial metabolism, the reviewer’s comment has pushed us to improve this part and to try to link it to the general picture more thoroughly; thus, we have addressed this topic in the conclusion of the manuscript by adding:” The cytidine monophosphate kinase is a crucial enzyme in the pyrimidine nucleotide salvage pathway, which is pivotal in rapidly dividing cells, such as activated immune cells, in the intestinal mucosa, but also in the bacterial cells; a higher bacterial functional contribution for this enzyme could be an indication of a higher bacterial growth in the patients’ intestine coupled with a higher scavenging of nucleotide precursors from the gut lumen. We hypothesize that this behavior could indirectly alter the intestinal immune and mucosal functionality in the long term.” from line 242 to 248.

Regarding K01265, we have added instead:” K01265 is a fundamental enzyme in bacterial metabolism, since it is actively involved in the protein maturation process. A higher level of this enzyme in the control group could be associated with a sound metabolic activity in the healthy intestinal microbiota.” from line 252 to 257.

Comment 3:

Heat maps and LEfSe graphs are appropriate
Discussions are well structured, integrate previous literature and identify contradictory findings. Highlights CST-specific associations that are insufficiently explored in VBD.
Cite figures in a consistent format (e.g., “Figure A2, right panels” could be confusing).

Response 3:

We thank the reviewer for addressing this point. The citation of some figures could have been confusing in some parts; thus, we have modified the writing accordingly in the manuscript. We hope that by clearing the text from unnecessary indications (the figures already have descriptions of their sections inside their figure legends), the readability would be improved.

Comment 4:

Make sure figure numbers and legends match the final layout.

Response 4:

Done as requested

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors addressed my comments and concerns and have revised the manuscript accordingly.