The Relationship of Vaginal Symptoms and Cervical Inflammation Severity with Cytological Abnormalities and HPV Positivity: A Prospective Observational Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript examines whether vaginal symptoms and cervical inflammation severity are associated with cytological abnormalities, HPV positivity, Candida, and bacterial vaginosis findings in women undergoing Pap smear screening. The topic is clinically relevant; however, the current manuscript has major methodological, statistical, and reporting limitations. Several key variables are insufficiently defined, the HPV-related analysis is based only on a subset of the cohort, important confounders are not addressed, and there are internal inconsistencies in the description of the study population.

1. The manuscript is unclear about whether the 458 included participants were pregnant or non-pregnant. The methods indicate that pregnant patients were excluded, but the flowchart appears to state “Number of pregnant women included in the study (n = 458).” This is a serious inconsistency and must be clarified. In addition, Table 1 reports a minimum age of 16 years, although the exclusion criteria state that women under 18 years were excluded.

 

2. Although the overall cohort includes 458 women, HPV data were available only for 218 women. Any analysis or conclusion related to HPV should be clearly limited to this subset. The current presentation risks misleading the reader into thinking that HPV-related conclusions are based on the full cohort. Tables and results should clearly separate analyses performed on 458 participants from those performed on 218 participants, with denominators stated throughout.

 

3. The HPV subset is relatively small, and some comparisons include very small numbers of HPV-positive cases, particularly in inflammation subgroups. Therefore, the conclusion that symptoms or inflammation are not associated with HPV positivity is not sufficiently supported. A lack of statistical significance in this context should not be interpreted as evidence of no association.

 

4. The manuscript does not adequately address major confounding variables relevant to HPV infection and cervical abnormalities, especially sexual history, including multiple sexual partners. Other important factors such as smoking, contraception, parity, menopausal status, previous HPV vaccination, previous abnormal smears, and recent antimicrobial or vaginal treatment use should also be considered. Without adjustment for these variables, the associations reported are difficult to interpret.

 

5. The methods do not adequately explain how HPV, Candida, and bacterial vaginosis status were determined. For HPV, the authors should specify the testing method, whether high-risk HPV only was tested, which genotypes were included, and whether genotyping was performed. For Candida and bacterial vaginosis, it is unclear whether these were diagnosed only cytologically or confirmed by microbiological methods. If based only on Pap smear morphology, this should be clearly stated and treated as a major limitation.

 

6. Cytological findings alone are not a gold-standard method for diagnosing Candida or bacterial vaginosis. The absence of culture, NAAT, Nugent scoring, Amsel criteria, or wet mount microscopy limits the reliability of these outcomes. The manuscript should avoid presenting these findings as definitive microbiological diagnoses.

 

7. Although blinding of the cytopathologist is a strength, assessment by only one pathologist is not enough to establish reproducibility, especially for semi-quantitative inflammation scoring and cytological abnormalities. A second independent cytopathologist, or at least an interobserver agreement analysis, would strengthen the reliability of the findings.

 

8. The authors classify inflammation as minimal, moderate, or severe based on leukocyte counts per high-power field. However, the manuscript does not provide adequate justification or a validated source for this scoring system. A relevant citation should be provided, and the authors should explain whether this scoring approach is routinely validated or locally developed.

 

9. The study relies mainly on chi-square and trend chi-square tests. No multivariable analysis was performed. Given the observational design and the number of potential confounders, this is not sufficient. Logistic regression or other adjusted analyses are needed, particularly for abnormal cytology and HPV positivity.

 

10. The manuscript performs many comparisons across several symptoms and outcomes, but there is no correction for multiple testing and no clearly defined primary endpoint. Some significant findings may therefore be false-positive results.

 

11. The manuscript groups cytology as negative versus positive, but it does not clearly present the distribution of Bethesda categories such as NILM, ASC-US, LSIL, HSIL, or AGC. This is important, especially because low-grade and high-grade abnormalities have different clinical significance.

 

12. The authors conclude that symptoms and inflammation are not reliable markers of cytological abnormalities or HPV positivity. However, given the limited HPV sample size, absence of confounder adjustment, and incomplete diagnostic methods, this conclusion should be substantially softened.

 

13. The main conclusion that cervical screening should be based on cytology and HPV testing rather than symptoms is already established clinically. The manuscript needs to explain more clearly what new evidence it adds to the literature.

 

14. The manuscript contains several typographical and formatting problems, including “study dizayn,” Turkish table headings such as “Sayı” and “Yüzde,” inconsistent page numbering, and “Acknowledgments: not acceptable.” These issues require careful revision.

Author Response

Manuscript ID: biomedicines-4294340

Title: The Relationship of Vaginal Symptoms and Cervical Inflammation Severity with Cytological Abnormalities and HPV Positivity: A Prospective Observational Study

Dear Reviewer 1,

We would like to express our sincere gratitude for the time and effort you have dedicated to evaluating our manuscript. We deeply appreciate your highly constructive comments, insightful observations, and valuable suggestions, which have significantly improved the quality, clarity, and scientific rigor of our study. We have carefully considered all your recommendations and meticulously revised our manuscript accordingly. Detailed point-by-point responses to the reviewers’ comments are provided below.

 

Comment 1: The manuscript is unclear about whether the 458 included participants were pregnant or non-pregnant. The methods indicate that pregnant patients were excluded, but the flowchart appears to state “Number of pregnant women included in the study (n = 458).” This is a serious inconsistency and must be clarified. In addition, Table 1 reports a minimum age of 16 years, although the exclusion criteria state that women under 18 years were excluded.

Response: We sincerely thank the reviewer for highlighting these important inconsistencies and reporting errors. The reviewer is absolutely correct. We have made all necessary corrections to resolve these discrepancies in the manuscript. First, the phrase "pregnant women" in the Flowchart (Figure 1) was an overlooked typographical error; as stated in our methodology, pregnant women were excluded from the study, and this error in the flowchart has been corrected to "non-pregnant women". Second, the "minimum age: 16" data in Table 1 was also a typographical error and has been updated to "minimum age: 18" in accordance with our strict inclusion criteria.

• Changes made in the manuscript:

Figure 1 (Flow chart of study design): The incorrect statement in the bottom right box of the flowchart has been corrected: "Number of pregnant women included in the study (n = 458)" has been changed to "Patients included in the main analysis (n = 458)".

Section 3.1. (Table 1): The incorrect minimum age value in the age row has been updated: "Mean±SD: 38.18±9.41, min: 16 min: 18, max: 54 years"

 

Comment 2: Although the overall cohort includes 458 women, HPV data were available only for 218 women. Any analysis or conclusion related to HPV should be clearly limited to this subset. The current presentation risks misleading the reader into thinking that HPV-related conclusions are based on the full cohort. Tables and results should clearly separate analyses performed on 458 participants from those performed on 218 participants, with denominators stated throughout.

Response: We sincerely thank the reviewer for this highly justified warning. To prevent misleading the reader, we have made it much more prominent in the text and tables that the HPV analyses were conducted only on the subset of 218 individuals. The denominators (n=458 and n=218) for the relevant analyses have been explicitly added to the results section and table headings.

• Changes made in the manuscript:

Section 3.2. (Results text): "...no statistically significant difference was found in terms of abnormal cytological findings, bacterial vaginosis (n=458) and HPV positivity (n=218) for all symptom types..."

Table 3 Heading: "Table 3. Comparison of cervical cytology, severity of inflammation, cytomorphological findings indicative of infection (n=458), and HPV positivity (n=218) by symptom type."

Table 4 Heading: "Table 4. Association between the burden of vaginal symptoms (number and frequency of recurrence) and cytopathological, cytomorphological findings indicative of infection (n=458), and HPV positivity (n=218)."

 

Comment 3: The HPV subset is relatively small, and some comparisons include very small numbers of HPV-positive cases, particularly in inflammation subgroups. Therefore, the conclusion that symptoms or inflammation are not associated with HPV positivity is not sufficiently supported. A lack of statistical significance in this context should not be interpreted as evidence of no association.

Response: We sincerely thank the reviewer for this highly constructive criticism emphasizing the statistical limitations of our findings and the need for cautious interpretation. As the reviewer rightly pointed out, the limited size of our HPV subset (n=218) reduces the statistical power, particularly for subgroup comparisons (e.g., inflammation severity categories), and is insufficient to draw a definitive conclusion of "no association." In line with this valuable feedback, we have substantially softened our conclusions throughout the manuscript. The definitive statements in the Discussion and Conclusion sections have been revised to state that "no statistically significant association was observed in this limited cohort," and we explicitly highlighted that the lack of statistical significance should not be interpreted as definitive evidence of no association. Furthermore, a sentence explicitly acknowledging the small size of the HPV subset has been added to the Strengths and Limitations of the Study (Section 4.5).

• Changes made in the manuscript:

Section 4.1. Discussion: "The findings of our study suggest that the type, number and frequency of lower genital tract symptoms do not show a significant association did not exhibit a statistically significant association with cervical cytological abnormalities and HPV positivity. However, due to the limited sample size of the HPV subset, the absence of statistical significance should not be definitively interpreted as a complete lack of association."

Section 4.5. Strengths and Limitations of the Study: "Our large sample size (n=458) and 95% statistical power support the clinical validity of the findings for the overall cohort; however, the HPV-related analyses were conducted on a substantially smaller subset (n=218), which reduces the statistical power, especially for subgroup comparisons."

Section 5. Conclusions: "Our study found that the type, number and frequency of vaginal symptoms are not reliable predictors did not present as definitive predictors of cervical cytological abnormalities or HPV positivity in this specific cohort; however, this conclusion should be interpreted with caution given the small HPV sample size."

 

Comment 4: The manuscript does not adequately address major confounding variables relevant to HPV infection and cervical abnormalities, especially sexual history, including multiple sexual partners. Other important factors such as smoking, contraception, parity, menopausal status, previous HPV vaccination, previous abnormal smears, and recent antimicrobial or vaginal treatment use should also be considered. Without adjustment for these variables, the associations reported are difficult to interpret.

Response: We sincerely thank the reviewer for this detailed criticism emphasizing the limitations of our study and the need for a cautious interpretation of the results. As the reviewer rightly pointed out, although some factors such as smoking, parity, and contraception methods were collected and presented in Table 1, the inability to include major confounding factors for HPV infection—such as detailed sexual history, presence of multiple partners, previous HPV vaccination status, or history of previous abnormal smears—in the analysis constitutes a significant limitation of our study. The lack of adjustment for these variables makes the reported associations difficult to interpret. In line with this valuable warning, we have softened our conclusions throughout the manuscript. Warnings stating that the lack of adjustment for major confounding factors may affect the interpretation of the results have been added to the Discussion and Conclusion sections; furthermore, a sentence explicitly acknowledging the lack of these factors has been integrated into the Strengths and Limitations of the Study (Section 4.5).

• Changes made in the manuscript:

Section 4.1. Discussion: "...However, due to the limited sample size of the HPV subset and the lack of adjustment for major confounding variables, the absence of statistical significance should not be definitively interpreted as a complete lack of association."

Section 4.5. Strengths and Limitations of the Study: "...reduces the statistical power, especially for subgroup comparisons. Moreover, the lack of data on critical confounding factors—such as detailed sexual history, previous HPV vaccination status, and history of abnormal smears—limits the generalizability and interpretability of our HPV-related findings."

Section 5. Conclusions: "...however, this conclusion should be interpreted with caution given the small HPV sample size and the presence of unmeasured confounders;"

 

Comment 5: The methods do not adequately explain how HPV, Candida, and bacterial vaginosis status were determined. For HPV, the authors should specify the testing method, whether high-risk HPV only was tested, which genotypes were included, and whether genotyping was performed. For Candida and bacterial vaginosis, it is unclear whether these were diagnosed only cytologically or confirmed by microbiological methods. If based only on Pap smear morphology, this should be clearly stated and treated as a major limitation.

Response: We sincerely thank the reviewer for highlighting these important omissions in our methodology. The reviewer is absolutely correct. We have revised the manuscript to provide the requested details. First, HPV testing was performed as part of the national cervical cancer screening program using a validated real-time PCR assay to detect high-risk HPV (HR-HPV) DNA. The assay specifically identifies 14 high-risk genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68), with concurrent genotyping for HPV 16 and HPV 18. Second, it should be noted that the presence of Candida and bacterial vaginosis was evaluated solely based on cytomorphological features in the Pap smear; confirmatory microbiological tests such as culture, nucleic acid amplification tests (NAAT), Amsel criteria, or Nugent scoring were not performed. As the reviewer rightly suggested, we have explicitly added this standalone clarification to the Methods section and treated it as a major limitation in the revised manuscript.

• Changes made in the manuscript:

Section 2.2. Data Collection Process and Variables: "...and HPV screening results. HPV testing was performed as part of the national cervical cancer screening program using a validated real-time PCR assay to detect high-risk HPV (HR-HPV) DNA. The assay specifically identifies 14 high-risk genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68), with concurrent genotyping for HPV 16 and HPV 18."

Section 2.3. Cytological and Inflammatory Assessment: "...were also considered. It should be noted that the presence of Candida and bacterial vaginosis was evaluated solely based on cytomorphological features in the Pap smear; confirmatory microbiological tests such as culture, nucleic acid amplification tests (NAAT), Amsel criteria, or Nugent scoring were not performed."

Section 4.5. Strengths and Limitations of the Study: " The diagnosis of Candida and bacterial vaginosis was based solely on Pap smear cytomorphology without confirmation by gold-standard microbiological methods (e.g., culture, NAAT, Amsel criteria, or wet mount microscopy). Although this is a major limitation that may fail to fully reflect all changes in the vaginal microbiota—meaning these outcomes should be interpreted as cytological markers rather than definitive diagnoses—the primary aim of our study was not the definitive detection of pathogens, but rather to evaluate the clinical implications of routine inflammatory findings."

 

Comment 6: Cytological findings alone are not a gold-standard method for diagnosing Candida or bacterial vaginosis. The absence of culture, NAAT, Nugent scoring, Amsel criteria, or wet mount microscopy limits the reliability of these outcomes. The manuscript should avoid presenting these findings as definitive microbiological diagnoses.

Response: We sincerely thank the reviewer for this vital methodological observation. We entirely agree that cytological findings alone are not the gold standard for diagnosing these conditions and should not be presented as definitive microbiological diagnoses. In accordance with your recommendation, we have explicitly clarified in the Strengths and Limitations of the Study (Section 4.5) that because the diagnosis of Candida and bacterial vaginosis was based solely on Pap smear cytomorphology without confirmation by gold-standard methods (e.g., culture, NAAT, Amsel criteria, or wet mount microscopy), these outcomes should be interpreted merely as "cytological markers" rather than definitive microbiological diagnoses. Furthermore, to strictly avoid presenting these outcomes as definitive diagnoses throughout the manuscript, we have replaced the phrase "microbiological findings" with "cytomorphological findings indicative of infection" in the relevant text and table headings (Tables 3 and 4).

• Changes made in the manuscript:

Section 4.5. Strengths and Limitations of the Study: " The diagnosis of Candida and bacterial vaginosis was based solely on Pap smear cytomorphology without confirmation by gold-standard microbiological methods (e.g., culture, NAAT, Amsel criteria, or wet mount microscopy). Although this is a major limitation that may fail to fully reflect all changes in the vaginal microbiota—meaning these outcomes should be interpreted as cytological markers rather than definitive diagnoses—the primary aim of our study was not the definitive detection of pathogens, but rather to evaluate the clinical implications of routine inflammatory findings."

Table 3 and Table 4 Headings: "Table 3. Comparison of cervical cytology, severity of inflammation, specific microbiological findings cytomorphological findings indicative of infection (n=458), and HPV positivity (n=218) by symptom type." "Table 4. Association between the burden of vaginal symptoms (number and frequency of recurrence) and cytopathological, microbiological findings cytomorphological findings indicative of infection (n=458), and HPV positivity (n=218)."

 

Comment 7: Although blinding of the cytopathologist is a strength, assessment by only one pathologist is not enough to establish reproducibility, especially for semi-quantitative inflammation scoring and cytological abnormalities. A second independent cytopathologist, or at least an interobserver agreement analysis, would strengthen the reliability of the findings.

Response: We sincerely thank the reviewer for this accurate methodological observation. We entirely agree that relying on a single cytopathologist without an interobserver agreement analysis limits the reproducibility of our evaluations, particularly for the semi-quantitative inflammation scoring. As suggested, we have explicitly acknowledged this issue in the Strengths and Limitations of the Study (Section 4.5) to ensure maximum transparency regarding the reproducibility of our findings.

• Changes made in the manuscript:

Section 4.5. Strengths and Limitations of the Study: (The following limitation has been integrated into this section) "...Additionally, although blinding of the cytopathologist minimized clinical bias, the assessment of all samples by a single pathologist without an interobserver agreement analysis limits the reproducibility of the semi-quantitative inflammation scoring and cytological evaluations."

 

Comment 8: The authors classify inflammation as minimal, moderate, or severe based on leukocyte counts per high-power field. However, the manuscript does not provide adequate justification or a validated source for this scoring system. A relevant citation should be provided, and the authors should explain whether this scoring approach is routinely validated or locally developed.

Response: We sincerely thank the reviewer for this valuable methodological observation. The inflammation scoring system utilized in our study is not based on a routinely validated universal framework; rather, it is a semi-quantitative, locally developed approach designed for the practical purposes of this study. In accordance with the reviewer's recommendation, we have updated the Methods section (Section 2.3) to explicitly clarify that this scoring system is a locally developed and non-validated approach, thereby ensuring methodological transparency. Furthermore, as addressed in response to your previous comments, we have also acknowledged the reliance on a non-validated scoring system as a limitation that may affect the external generalizability of our findings in the Strengths and Limitations of the Study (Section 4.5).

• Changes made in the manuscript:

Section 2.3. Cytological and Inflammatory Assessment: "The severity of inflammation was scored semi-quantitatively using a locally developed, non-validated approach based on the leukocyte density in the high-power field (400× HPF)..."

(Note: The limitation regarding the external generalizability of this locally developed scoring system was integrated into Section 4.5 in response to Comment 6).

 

Comment 9: The study relies mainly on chi-square and trend chi-square tests. No multivariable analysis was performed. Given the observational design and the number of potential confounders, this is not sufficient. Logistic regression or other adjusted analyses are needed, particularly for abnormal cytology and HPV positivity.

Response: Thank you for this important methodological comment. We agree that univariate chi-square and trend chi-square analyses alone are insufficient to account for potential confounding in an observational study. Accordingly, we performed additional multivariable binary logistic regression analyses for the two clinically relevant binary outcomes: abnormal cytology and HPV positivity.

Abnormal cytology and HPV positivity were entered as separate dependent variables. The models were adjusted for age, smoking status, parity, vaginal discharge, symptom recurrence frequency, Candida positivity, bacterial vaginosis, cervical inflammation severity, and symptom burden. Adjusted odds ratios with 95% confidence intervals and p values have now been added to the Results section and presented in a new Table 6.

In these adjusted analyses, none of the evaluated symptom-related, inflammatory, or microbiological parameters was independently associated with abnormal cytology or HPV positivity. We also revised the Methods, Results, Discussion, and Limitations sections accordingly. Model fit was assessed using the Hosmer–Lemeshow goodness-of-fit test, which indicated no evidence of poor model fit. Because HPV positivity was evaluated only among participants with available HPV results and the number of HPV-positive cases was limited, we also added this point to the Limitations section.

• Changes made in the manuscript:

Abstract: (The following sentences were added to the Results and Conclusions sections, respectively, to reflect the multivariable analyses): "In multivariable logistic regression analyses, neither symptom burden nor cervical inflammation severity was independently associated with abnormal cytology or HPV positivity." "These findings remained consistent after adjustment for major clinical confounders."

Section 2.4. Data Analysis: (The following paragraph detailing the multivariable logistic regression methodology was added to the end of the section): "In addition to Pearson chi-square and chi-square test for trend analyses, multivariable binary logistic regression analyses were performed to evaluate whether vaginal symptom parameters and cervical inflammation severity were independently associated with abnormal cytology and HPV positivity. Abnormal cytology and HPV positivity were entered as separate binary dependent variables. The models were adjusted for age, smoking status, parity, vaginal discharge, symptom recurrence frequency, Candida positivity, bacterial vaginosis, cervical inflammation severity, and symptom burden. Adjusted odds ratios (aORs) with 95% confidence intervals (CIs) were reported. Model fit was assessed using the Hosmer–Lemeshow goodness-of-fit test. The HPV positivity model was performed only among participants with available HPV results."

Section 3.4. (Results): (A new subsection and a new table [Table 6] were added to present the adjusted odds ratios):

"3.4. Multivariable Logistic Regression Analysis for Abnormal Cytology and HPV Positivity: In the abnormal cytology model, none of the evaluated variables showed an independent association with abnormal cytology after adjustment for potential confounders. Similarly, in the HPV subgroup, no independent association was observed between HPV positivity and vaginal discharge, symptom burden, Candida positivity, bacterial vaginosis, or cervical inflammation severity. Model calibration was acceptable according to the Hosmer–Lemeshow goodness-of-fit test for both the abnormal cytology model and the HPV positivity model (Table 6)."

Section 4.1. Discussion: (The following paragraph was added to the end of the section to discuss the multivariable findings): "Importantly, the multivariable logistic regression analyses supported the findings of the univariate comparisons. After adjustment for age, smoking status, parity, vaginal discharge, symptom recurrence frequency, Candida positivity, bacterial vaginosis, cervical inflammation severity, and symptom burden, neither symptom-related parameters nor cervical inflammation severity were independently associated with abnormal cytology or HPV positivity. These findings suggest that the absence of significant associations in the univariate analyses was not merely attributable to the lack of adjustment for major clinical confounders. Rather, vaginal symptoms and smear-based inflammatory findings appear to be more closely related to localized infectious or reactive processes than to cytological abnormality or HPV positivity."

Section 4.5. Strengths and Limitations of the Study: (The following statement was added to acknowledge the limited number of HPV-positive cases in the model): "Although multivariable logistic regression analyses were performed to adjust for clinically relevant confounders, the number of HPV-positive cases was limited, which may have resulted in wide confidence intervals for some estimates. Therefore, the adjusted HPV findings should be interpreted cautiously and confirmed in larger cohorts."

 

 

Comment 10: The manuscript performs many comparisons across several symptoms and outcomes, but there is no correction for multiple testing and no clearly defined primary endpoint. Some significant findings may therefore be false-positive results.

Response: Thank you for this important methodological comment. We agree that the large number of symptom–outcome comparisons may increase the risk of type I error and false-positive findings. In response, we revised the manuscript to clearly define the primary endpoints as abnormal cytology and HPV positivity. Cervical inflammation severity, Candida positivity, and bacterial vaginosis were defined as secondary exploratory outcomes.

We also applied the Benjamini–Hochberg false discovery rate (FDR) procedure to exploratory comparisons and revised the Results and Discussion sections accordingly. After FDR correction, the associations between vaginal discharge and cervical inflammation, between pruritus, dysuria, vaginal burning and Candida positivity, and between symptom burden and both cervical inflammation and Candida positivity remained statistically significant. However, the association between symptom recurrence frequency and cervical inflammation did not remain robust after correction and is now interpreted cautiously.

Importantly, the main conclusion of the study is based on the predefined primary endpoints and the newly added multivariable logistic regression analyses, which showed that neither symptom burden nor cervical inflammation severity was independently associated with abnormal cytology or HPV positivity.

• Changes made in the manuscript:

Section 2.4. Data Analysis: (The following clarification on primary endpoints and FDR correction was added): "The primary endpoints were predefined as abnormal cytology and HPV positivity. Cervical inflammation severity, Candida positivity, and bacterial vaginosis were considered secondary exploratory outcomes. Because multiple comparisons were performed across several symptoms and outcomes, the Benjamini–Hochberg false discovery rate procedure was applied to exploratory analyses to reduce the risk of type I error. Findings from secondary exploratory analyses were interpreted cautiously."

Section 3.2. (Results): (The results of the FDR correction were added after Table 4): "Because multiple symptom–outcome comparisons were performed, the Benjamini–Hochberg false discovery rate procedure was applied to exploratory analyses. After FDR correction, the associations between vaginal discharge and cervical inflammation, between pruritus, dysuria, vaginal burning and Candida positivity, and between symptom burden and both cervical inflammation and Candida positivity remained statistically significant. However, the association between symptom recurrence frequency and cervical inflammation did not remain robust after correction and was therefore interpreted cautiously."

Section 4.2. (Discussion): (The following paragraph interpreting the FDR-corrected findings was integrated): "Although several symptom–outcome associations were observed in unadjusted analyses, multiple testing correction was applied to reduce the risk of false-positive findings. After FDR correction, the most robust secondary findings were the associations of Candida positivity with pruritus, dysuria, vaginal burning, and symptom burden, as well as the associations of cervical inflammation with vaginal discharge and symptom burden. In contrast, the association between symptom recurrence frequency and cervical inflammation was weaker and should be interpreted as exploratory."

 

 

 

Comment 11: The manuscript groups cytology as negative versus positive, but it does not clearly present the distribution of Bethesda categories such as NILM, ASC-US, LSIL, HSIL, or AGC. This is important, especially because low-grade and high-grade abnormalities have different clinical significance.

Response: Thank you for this important and clinically relevant comment. We agree that reporting cytology only as negative versus positive may obscure the clinical differences between Bethesda categories, particularly because low-grade and high-grade cytological abnormalities have different implications for follow-up and management.

Accordingly, we revised the manuscript by adding the detailed distribution of cervical cytology results according to Bethesda categories to Table 1. The table now presents the numbers and percentages for NILM and abnormal cytology categories, including ASC-H, LSIL, HSIL, AGC, and SCC where applicable. This revision allows readers to better understand the composition of the cytology-positive group and to distinguish low-grade from potentially higher-risk abnormalities.

We retained the binary classification of cytology as negative versus positive for the main comparative and logistic regression analyses because the number of cases in individual abnormal Bethesda categories was limited. However, the detailed Bethesda category distribution is now provided descriptively in Table 1 to improve transparency and clinical interpretability.

• Changes made in the manuscript:

Section 3.1. (Table 1): (The detailed distribution of cytology and HPV outcomes was integrated into the sociodemographic table).

Section 3.2. (Results): (The following clinical context regarding Bethesda categories was added to the text): "Detailed evaluation of the cervical cytology results according to the Bethesda system revealed that the entirely benign Cytology (-) group (n=398) consisted exclusively of NILM cases, as ASC-US cases were deliberately excluded from the study to prevent borderline confounding. The Cytology (+) group (n=60), which was strictly defined as LSIL and above for clinical significance, comprised 46 LSIL, 2 HSIL, 8 ASC-H, 3 AGC, and 1 SCC cases."

 

Comment 12: The authors conclude that symptoms and inflammation are not reliable markers of cytological abnormalities or HPV positivity. However, given the limited HPV sample size, absence of confounder adjustment, and incomplete diagnostic methods, this conclusion should be substantially softened.

Response: We sincerely thank the reviewer for this critical summarizing observation. We completely agree that in light of the study's limitations—specifically the limited HPV sample size, unmeasured confounders, and the lack of gold-standard microbiological diagnostic methods—the original conclusions required a more cautious tone. Building upon the revisions made in response to your previous comments (where these limitations were explicitly integrated into the text), we have further softened the categorical language in the Conclusions section of the Abstract and the main Conclusions section (Section 5). We replaced definitive statements regarding inflammation and symptom predictability with more conservative interpretations to accurately reflect the scope and limitations of our findings.

• Changes made in the manuscript:

Abstract (Conclusions): Original text: "Our findings indicate that neither vaginal symptoms nor the severity of cervical inflammation serves as a reliable or discriminatory marker..." Revised text: "Our findings indicate that vaginal symptoms and the severity of cervical inflammation may not serve as definitive or independent discriminatory markers for cellular abnormalities or HPV positivity in this context."

Section 5. Conclusions: (The section has been revised to soften the categorical language as follows): "...It was also observed that the severity of cervical inflammation alone may not be sufficient for predicting abnormal cytology and HPV positivity without further molecular confirmation..."

 

Comment 13: The main conclusion that cervical screening should be based on cytology and HPV testing rather than symptoms is already established clinically. The manuscript needs to explain more clearly what new evidence it adds to the literature.

Response: We sincerely thank the reviewer for this insightful comment. We completely agree that the primacy of standard cytological and molecular testing over symptom-based screening is a well-established clinical fact. Rather than restating this paradigm, our study aims to build upon this foundational knowledge by providing a clinical framework that explains why symptoms fail as oncogenic predictors. Specifically, our study contributes a novel perspective to the literature by granularly analyzing the "symptom burden" (the number of concurrent symptoms and their recurrence frequency) in conjunction with semi-quantitative cervical inflammation scores. While previous studies often evaluate symptoms merely as present or absent, we observed that an increased symptom burden strongly correlates with severe local reactive inflammation and specific microbial patterns, but clearly diverges from oncogenic risk markers. To explicitly highlight this contribution, we have integrated a clear statement of novelty into the Introduction (Section 1) and Discussion (Section 4), and refined our concluding statements in both the Abstract and the main Conclusions (Section 5) to emphasize this novel clinical framework.

• Changes made in the manuscript:

Section 1. Introduction: (The following sentence was added to the final paragraph to highlight the study's novel approach): "...abnormal cytology and HPV positivity. Unlike prior studies that frequently evaluate vaginal symptoms as merely present or absent, the present study contributes a novel perspective to the literature by comprehensively analyzing the "symptom burden" (specifically, the number of concurrent symptoms and their frequency of recurrence) in conjunction with semi-quantitative inflammation scoring. Our study is based on the hypothesis that these symptoms..."

Section 4. Discussion: (The study's clinical contribution was clarified in the introductory paragraph): "...and HPV parameters. While the primacy of cytological and molecular testing over symptom-based screening is clinically well-established, our study aims to build upon this knowledge by granularly analyzing the symptom burden in conjunction with cervical inflammation scores. By demonstrating that an increased symptom burden strongly correlates with local reactive inflammation and specific microbial patterns rather than oncogenic markers, this study provides a clinical framework that delineates benign dysbiotic processes from actual neoplastic risk. The findings obtained in our study..."

Abstract (Conclusions): (The final concluding sentence of the Abstract was replaced with the following): "Consequently, while standard cytological and molecular protocols remain essential for oncogenic screening, evaluating the overall symptom burden provides clinicians with a valuable framework for identifying benign dysbiotic and inflammatory processes."

Section 5. Conclusions: (The final concluding sentence was replaced with the following): "Considering these findings, while it is reaffirmed that cervical screening must rely on standard cytological and molecular methods rather than subjective symptoms, our study highlights that comprehensively evaluating the symptom burden provides a valuable clinical framework for identifying benign dysbiotic and inflammatory processes."

 

Comment 14: The manuscript contains several typographical and formatting problems, including “study dizayn,” Turkish table headings such as “Sayı” and “Yüzde,” inconsistent page numbering, and “Acknowledgments: not acceptable.” These issues require careful revision.

Response: We sincerely apologize for these typographical and formatting oversights and thank the reviewer for bringing them to our attention. We have meticulously reviewed and corrected the entire manuscript. Specifically, the phrase "study dizayn" has been corrected to "study design," and the Turkish table headings ("Sayı" and "Yüzde") have been translated to their English equivalents ("Number" and "Percentage"). Furthermore, the inconsistent page numbering has been standardized throughout the document, and the placeholder text "not acceptable" in the Acknowledgments section has been removed and replaced with "None." A thorough final proofreading was also conducted to ensure overall formatting consistency and language accuracy.

• Changes made in the manuscript:

Figure 1 Caption: The phrase "Flow chart of study dizayn" was corrected to "Flow chart of study design."

Table 2: The column headers "Sayı" and "Yüzde" were corrected to "Number" and "Percentage," respectively.

Formatting: Page numbering was made consistent across all pages.

Acknowledgments Section: The placeholder text "not acceptable" was replaced with "None."

We hope that these comprehensive revisions effectively address all the concerns raised and that the revised manuscript is now deemed suitable for publication. Thank you once again for your invaluable contributions to our work.

Sincerely,

Corresponding Author:

Prof. Dr. Yakup Baykus

On behalf of all co-authors

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

 The authors of The Relationship of Vaginal Symptoms and Cervical Inflammation Severity with Cytological Abnormalities and HPV Positivity: A Prospective Observational Study paper aimed  to assess the presence, type, number and frequency of vaginal infection symptoms in women attending the gynecology outpatient clinic; to investigate the relationship between these symptoms and the severity of inflammation detected in cervical smears, HPV positivity, and the morphological findings and cytological results of Candida and bacterial vaginosis; also they aimed to examine the relationship between the severity of inflammation in the smear and abnormal cytology and HPV positivity.

 

Could you please give details regarding HPV genotyping assay used? Principle, results.

 

Please give details of what molecular methods mean for you. Which agents do you want to detect?

 

Please write a paragraph about vaginosis which includes Gardnerella vaginalis.

 

Thank you.

Author Response

Manuscript ID: biomedicines-4294340

Title: The Relationship of Vaginal Symptoms and Cervical Inflammation Severity with Cytological Abnormalities and HPV Positivity: A Prospective Observational Study

Dear Reviewer 2,

We would like to express our sincere gratitude for the time and effort you have dedicated to evaluating our manuscript. We deeply appreciate your highly constructive comments and valuable suggestions, which have significantly improved the methodological clarity and microbiological depth of our study. We have carefully considered all your recommendations and meticulously revised our manuscript accordingly. Detailed point-by-point responses to your comments are provided below.

 

Comment 1: Could you please give details regarding HPV genotyping assay used? Principle, results.

Response: We sincerely thank the reviewer for this important and constructive inquiry. We completely agree that detailing the genotyping assay and its specific results significantly enhances the methodological clarity and clinical depth of our study. To address your comment comprehensively, we have provided the requested details in both the Methods and Results sections. Regarding the principle of the assay, we have explicitly clarified in Section 2.2 that HPV testing was performed using a validated real-time PCR assay designed to detect high-risk HPV (HR-HPV) DNA. This assay specifically identifies 14 high-risk genotypes, with concurrent specific genotyping for HPV 16 and HPV 18. Regarding the results, we have expanded our findings in Section 3.2 to include the specific genotype distribution among the HPV-positive patients. Out of the 218 patients tested, 32 were positive for high-risk HPV. Among these positive cases, 12 were identified as HPV 16, 3 as HPV 18, and 17 were positive for other high-risk genotypes.

• Changes made in the manuscript:

Section 2.2. Data Collection Process and Variables: (The following methodological details regarding the assay principle have been added): "...HPV testing was performed as part of the national cervical cancer screening program using a validated real-time PCR assay to detect high-risk HPV (HR-HPV) DNA. The assay specifically identifies 14 high-risk genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68), with concurrent genotyping for HPV 16 and HPV 18."

Section 3.2. (Results): (The following sentence detailing the HPV genotype distribution has been added): " Among the 218 women with available HPV data, a total of 32 cases were positive for high-risk HPV. Genotype distribution of these positive cases revealed that 12 women were positive for HPV 16, 3 for HPV 18, and 17 for other high-risk genotypes."

 

Comment 2: Please give details of what molecular methods mean for you. Which agents do you want to detect?

Response: We sincerely thank the reviewer for this opportunity to clarify our terminology. In the context of our study's recommendations for evaluating the symptom-dysbiosis relationship, "molecular methods" refers to advanced molecular microbiological techniques, primarily Nucleic Acid Amplification Tests (NAAT) and Next-Generation Sequencing (NGS) panels. The specific agents we emphasize detecting using these methods are the major etiological pathogens of vaginitis and cervicitis that cannot be definitively diagnosed by conventional Pap smear cytology alone. These include Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae, as well as the definitive species-level identification of Candida spp. and Gardnerella vaginalis. To make this explicitly clear to the reader, we have expanded both the relevant Discussion section (Section 4.3) and the final sentence of our Conclusions section (Section 5) to detail these specific diagnostic methods and the targeted agents.

• Changes made in the manuscript:

Section 4.3. The Relationship Between Vaginal Symptoms and Candida and Bacterial Vaginosis: (The final sentence of this section was expanded to clarify the molecular methods and agents): "...Furthermore, the possibility that smear-based morphological examination may not fully reflect the functional and metabolic dynamics of the vaginal ecosystem underscores the need to assess symptom burden alongside advanced molecular methods (such as NAAT or NGS) to definitively identify specific etiological agents (e.g., Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae, and specific Candida and Gardnerella species)."

Section 5. Conclusions: (The final sentence recommending future studies was updated as follows): "In future studies, a more detailed examination of the symptom-dysbiosis relationship using advanced molecular microbiology methods (e.g., NAAT and NGS to definitively detect the aforementioned specific pathogens) alongside multicenter designs will contribute to the literature."

 

Comment 3: Please write a paragraph about vaginosis which includes Gardnerella vaginalis.

Response: We sincerely thank the reviewer for this valuable suggestion. In accordance with your recommendation, we expanded the Discussion section by adding a dedicated paragraph on bacterial vaginosis and the role of Gardnerella vaginalis. We now explain that bacterial vaginosis represents a shift from a Lactobacillus-dominant vaginal microenvironment to polymicrobial dysbiosis, and that Gardnerella vaginalis may act as a keystone pathogen by initiating multispecies biofilm formation on the vaginal epithelium. We also clarified that this biofilm may facilitate the adherence and proliferation of anaerobic bacteria, protect microorganisms from host immune responses, and contribute to malodorous discharge, recurrence, and treatment resistance.

• Changes made in the manuscript:

Section 4.3. The Relationship Between Vaginal Symptoms and Candida and Bacterial Vaginosis:

Bacterial vaginosis is fundamentally characterized by a profound ecological shift from a Lactobacillus-dominant state to a polymicrobial dysbiosis. In this context, Gardnerella vaginalis acts as a keystone pathogen, initiating the pathogenic process through the formation of a robust multi-species biofilm on the vaginal epithelium. This biofilm not only facilitates the adherence and proliferation of other strictly anaerobic bacteria but also provides a protective niche against host immune responses. Consequently, the prominent presence of G. vaginalis and its associated biofilm are central to both the clinical manifestation of the typical malodorous discharge and the frequently recurrent, treatment-resistant nature of bacterial vaginosis.

 

We hope that these revisions effectively address all the concerns raised and that the revised manuscript is now deemed suitable for publication. Thank you once again for your invaluable contributions to our work.

Sincerely,

Corresponding Author: Prof. Dr. Yakup Baykus

On behalf of all co-authors

Reviewer 3 Report

Comments and Suggestions for Authors

A clinically relevant study with a decent cohort (n=458), prospective design, and a single blinded cytopathologist. The main conclusion — that subjective symptoms are not reliable markers of HPV/neoplastic risk — is reasonable and clinically useful. However, several issues should be addressed before publication.

Comments/recommendations:

• Sample size calculation. The reported effect size d=0.461 is the metric for t-tests, not chi-square. Please use Cohen's "w" or clarify.
• No multivariate analysis. The study relies entirely on bivariate chi-square tests, with no adjustment for age, smoking, parity, contraception, or socioeconomic status. A multivariate logistic regression would substantially strengthen the conclusions.
• HPV subgroup (218/458). This subset differs systematically from the full cohort (≥30 years, national screening program). Please add a baseline comparison between the two groups and explicitly discuss the selection bias.
• HPV methodology not reported. Which assay was used? Was genotyping performed? Were HR-HPV and LR-HPV distinguished? Without this, Table 5 is difficult to interpret.
• "Abnormal cytology" not clearly defined in the Methods (where does ASC-US fall?).
• Inflammation grading (<10 / 10–100 / >100 leukocytes/HPF) is not the Bethesda 2014 standard — please cite the source and justify.
• BV diagnosed by morphology alone, without Amsel criteria or Nugent score. This is a serious limitation and deserves a more candid discussion.
• Multiple comparisons — no correction (e.g., Benjamini-Hochberg) or discussion of this issue.
• Missing odds ratios / relative risks and confidence intervals.
• Figure 1: the final box reads "pregnant women" instead of "non-pregnant women". Also, "dizayn" → "design".
• Table 2: column headers still in Turkish ("Sayı", "Yüzde").
• Inconsistent decimal formatting (comma vs. period).
  

 

The study has a solid foundation and a clinically useful message, but the statistical issues — particularly the lack of multivariate analysis and the handling of the HPV subgroup — must be addressed before publication.

Author Response

Manuscript ID: biomedicines-4294340

Title: The Relationship of Vaginal Symptoms and Cervical Inflammation Severity with Cytological Abnormalities and HPV Positivity: A Prospective Observational Study

 

Dear Reviewer 3,

We would like to express our sincere gratitude for the time and effort you have dedicated to evaluating our manuscript. We deeply appreciate your highly constructive comments, insightful observations, and valuable suggestions, which have significantly improved the quality, clarity, and scientific rigor of our study. We have carefully considered all your recommendations and meticulously revised our manuscript accordingly. Detailed point-by-point responses to the reviewers’ comments are provided below.

 

Comment 1: Sample size calculation. The reported effect size d=0.461 is the metric for t-tests, not chi-square. Please use Cohen's "w" or clarify.

Response 1: We sincerely thank the reviewer for catching this specific statistical oversight. You are absolutely correct; the notation "d" was a typographical error in our original manuscript. We have corrected the metric to "Cohen's w = 0.461" in the revised text to accurately reflect the effect size for the chi-square tests used in the sample size calculation.

• Changes made in the manuscript: (Section 2.1): "...based on data from the literature by Konyalıoğlu and Yılmaz (effect size Cohen's w = 0.461) [17–19]."

 

Comment 2: No multivariate analysis. The study relies entirely on bivariate chi-square tests, with no adjustment for age, smoking, parity, contraception, or socioeconomic status. A multivariate logistic regression would substantially strengthen the conclusions.

Response 2: We completely agree with this critical methodological observation. Relying solely on bivariate analyses in an observational study is indeed insufficient due to potential confounding factors. In response to your recommendation, we have performed multivariable binary logistic regression analyses for both abnormal cytology and HPV positivity. The models were adjusted for major clinical confounders, including age, smoking status, parity, and other relevant parameters. The adjusted odds ratios (aORs) and 95% confidence intervals are now presented in a newly added Table 6. Importantly, these adjusted analyses confirmed our initial bivariate findings: neither symptom burden nor cervical inflammation severity was independently associated with abnormal cytology or HPV positivity.

• Changes made in the manuscript: Abstract: (The following sentences were added to the Results and Conclusions sections, respectively): "In multivariable logistic regression analyses, neither symptom burden nor cervical inflammation severity was independently associated with abnormal cytology or HPV positivity." / "These findings remained consistent after adjustment for major clinical confounders." Section 2.4. Data Analysis: (The following paragraph was added): "In addition to Pearson chi-square and chi-square test for trend analyses, multivariable binary logistic regression analyses were performed to evaluate whether vaginal symptom parameters and cervical inflammation severity were independently associated with abnormal cytology and HPV positivity. Abnormal cytology and HPV positivity were entered as separate binary dependent variables. The models were adjusted for age, smoking status, parity, vaginal discharge, symptom recurrence frequency, Candida positivity, bacterial vaginosis, cervical inflammation severity, and symptom burden. Adjusted odds ratios (aORs) with 95% confidence intervals (CIs) were reported..." Section 3.4. (Results): (A new subsection and Table 6 were added): "3.4. Multivariable Logistic Regression Analysis for Abnormal Cytology and HPV Positivity: In the abnormal cytology model, none of the evaluated variables showed an independent association with abnormal cytology after adjustment for potential confounders. Similarly, in the HPV subgroup, no independent association was observed..." Section 4.1. Discussion: (The following paragraph was added): "Importantly, the multivariable logistic regression analyses supported the findings of the univariate comparisons. After adjustment for age, smoking status, parity... neither symptom-related parameters nor cervical inflammation severity were independently associated with abnormal cytology or HPV positivity. These findings suggest that the absence of significant associations in the univariate analyses was not merely attributable to the lack of adjustment for major clinical confounders."

 

Comment 3: HPV subgroup (218/458). This subset differs systematically from the full cohort (≥30 years, national screening program). Please add a baseline comparison between the two groups and explicitly discuss the selection bias.

Response 3: Thank you for highlighting this important point. We completely agree that restricting the HPV analysis to women aged ≥30 years (in accordance with the national screening program) introduces an inevitable selection bias that must be transparently addressed. To prevent misleading the reader, we have updated all relevant tables and results sections to explicitly separate the analyses performed on the full cohort (n=458) from those on the HPV subset (n=218). Furthermore, in accordance with your recommendation, we have explicitly discussed this selection bias as a major limitation, noting that our HPV-related findings may not be fully generalizable to younger populations.

• Changes made in the manuscript: Section 4.5. Strengths and Limitations of the Study: (The following limitation regarding selection bias was added): "...however, the HPV-related analyses were conducted on a substantially smaller subset (n=218), which reduces the statistical power, especially for subgroup comparisons. Furthermore, the restriction of HPV analyses to women ≥30 years in accordance with national screening guidelines introduces an inevitable selection bias, meaning our HPV-related findings may not be fully generalizable to younger populations."

 

Comment 4: HPV methodology not reported. Which assay was used? Was genotyping performed? Were HR-HPV and LR-HPV distinguished? Without this, Table 5 is difficult to interpret.

Response 4: We appreciate this constructive feedback. We have revised the methodology to provide the requested details. Specifically, HPV testing was performed using a validated real-time PCR assay to detect high-risk HPV (HR-HPV) DNA. The assay identifies 14 high-risk genotypes, with concurrent specific genotyping for HPV 16 and HPV 18. Furthermore, in accordance with your recommendation to aid the interpretation of the tables, we have also added the specific genotype distribution of the positive cases to the Results section.

• Changes made in the manuscript: Section 2.2. Data Collection Process and Variables: (The following methodological details were added) "... HPV testing was performed as part of the national cervical cancer screening program using a validated real-time PCR assay to detect high-risk HPV (HR-HPV) DNA. The assay specifically identifies 14 high-risk genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68), with concurrent genotyping for HPV 16 and HPV 18." Section 3.2. (Results): (The following distribution was added) " Among the 218 women with available HPV data, a total of 32 cases were positive for high-risk HPV. Genotype distribution of these positive cases revealed that 12 women were positive for HPV 16, 3 for HPV 18, and 17 for other high-risk genotypes."

 

Comment 5: "Abnormal cytology" not clearly defined in the Methods (where does ASC-US fall?).

Response 5: We apologize for this lack of clarity. ASC-US cases were deliberately excluded from the study to prevent borderline confounding, and we strictly defined the "abnormal/positive" cytology group as LSIL and above for clinical significance. We have now provided the exact distribution of Bethesda categories (NILM, LSIL, HSIL, ASC-H, AGC, SCC) in the Results section and Table 1 to ensure complete transparency.

• Changes made in the manuscript: Section 3.1. (Table 1): (The detailed distribution of cytology results was integrated into the table). Section 3.2. (Results): (The following clinical context was added to the text): "Detailed evaluation of the cervical cytology results according to the Bethesda system revealed that the entirely benign Cytology (-) group (n=398) consisted exclusively of NILM cases, as ASC-US cases were deliberately excluded from the study to prevent borderline confounding. The Cytology (+) group (n=60), which was strictly defined as LSIL and above for clinical significance, comprised 46 LSIL, 2 HSIL, 8 ASC-H, 3 AGC, and 1 SCC cases."

 

Comment 6: Inflammation grading (<10 / 10–100 / >100 leukocytes/HPF) is not the Bethesda 2014 standard — please cite the source and justify.

Response 6: Thank you for this valuable methodological observation. You are absolutely correct; the inflammation grading system utilized in our study is not based on the Bethesda 2014 standard or a universally validated framework. Rather, it is a semi-quantitative, locally developed approach routinely used in our institution's pathology laboratory for practical purposes. In accordance with your recommendation, we have updated the Methods section to explicitly clarify that this is a locally developed, non-validated approach to ensure complete methodological transparency. Furthermore, we have acknowledged the use of a non-validated scoring system as a limitation that may affect the external generalizability of our findings.

• Changes made in the manuscript: Section 2.3. Cytological and Inflammatory Assessment: (The following clarification was integrated): "The severity of inflammation was scored semi-quantitatively using a locally developed, non-validated approach based on the leukocyte density in the high-power field... This semi-quantitative scoring system is a locally developed approach routinely used in our institution's pathology laboratory, rather than an internationally validated guideline." Section 4.5. Strengths and Limitations of the Study: (The following limitation was added): "...Furthermore, the reliance on a locally developed, non-validated scoring system for inflammation severity limits the external generalizability of these findings."

 

Comment 7: BV diagnosed by morphology alone, without Amsel criteria or Nugent score. This is a serious limitation and deserves a more candid discussion.

Response 7: We sincerely thank the reviewer for this vital clinical observation. We entirely agree that diagnosing bacterial vaginosis solely based on Pap smear cytomorphology without confirmation by gold-standard methods (such as Amsel criteria or Nugent scoring) is a major limitation. In response to your highly justified critique, we have added a candid discussion to the Limitations section (Section 4.5). We have explicitly clarified that our outcomes should be interpreted merely as "cytological markers" rather than definitive microbiological diagnoses.

• Changes made in the manuscript: Section 4.5. Strengths and Limitations of the Study: (The following candid discussion was integrated into the limitations): "The diagnosis of Candida and bacterial vaginosis was based solely on Pap smear cytomorphology without confirmation by gold-standard microbiological methods (e.g., culture, NAAT, Amsel criteria, or wet mount microscopy). Although this is a major limitation that may fail to fully reflect all changes in the vaginal microbiota—meaning these outcomes should be interpreted as cytological markers rather than definitive diagnoses—the primary aim of our study was not the definitive detection of pathogens, but rather to evaluate the clinical implications of routine inflammatory findings."

 

Comment 8: Multiple comparisons — no correction (e.g., Benjamini-Hochberg) or discussion of this issue.

Response 8: We sincerely thank the reviewer for pointing out the risk of type I error due to multiple testing. In accordance with your critical methodological recommendation, we have applied the Benjamini–Hochberg false discovery rate (FDR) procedure to our secondary exploratory analyses. After FDR correction, the majority of our symptom-dysbiosis associations remained statistically significant. However, the association between symptom recurrence frequency and cervical inflammation did not remain robust after correction and is therefore interpreted more cautiously in the revised text.

• Changes made in the manuscript: Section 2.4. Data Analysis: (The following clarification was added): "...Because multiple comparisons were performed across several symptoms and outcomes, the Benjamini–Hochberg false discovery rate procedure was applied to exploratory analyses to reduce the risk of type I error. Findings from secondary exploratory analyses were interpreted cautiously." Section 3.2. (Results): (The results of the FDR correction were added): "Because multiple symptom–outcome comparisons were performed, the Benjamini–Hochberg false discovery rate procedure was applied to exploratory analyses. After FDR correction, the associations between vaginal discharge and cervical inflammation, between pruritus, dysuria, vaginal burning and Candida positivity, and between symptom burden and both cervical inflammation and Candida positivity remained statistically significant. However, the association between symptom recurrence frequency and cervical inflammation did not remain robust after correction and was therefore interpreted cautiously." Section 4.2. (Discussion): (The following interpretation was integrated): "Although several symptom–outcome associations were observed in unadjusted analyses, multiple testing correction was applied to reduce the risk of false-positive findings. After FDR correction, the most robust secondary findings were the associations of Candida positivity with pruritus, dysuria, vaginal burning, and symptom burden, as well as the associations of cervical inflammation with vaginal discharge and symptom burden. In contrast, the association between symptom recurrence frequency and cervical inflammation was weaker and should be interpreted as exploratory."

 

Comment 9: Missing odds ratios / relative risks and confidence intervals.

Response 9: As detailed in our response regarding the multivariable analysis (Response 2), adjusted odds ratios (aORs) and their corresponding 95% confidence intervals have now been calculated for all evaluated parameters. These results are clearly presented in the newly added Table 6 in the revised manuscript.

 

Comment 10, 11, 12: Figure 1: the final box reads "pregnant women" instead of "non-pregnant women". Also, "dizayn" → "design". Table 2: column headers still in Turkish ("Sayı", "Yüzde"). Inconsistent decimal formatting (comma vs. period).

Response 10, 11, 12: We sincerely apologize for these typographical and formatting oversights, and we thank the reviewer for their careful reading. We have meticulously reviewed the entire manuscript and corrected all the mentioned issues. The flow chart (Figure 1) was corrected to read "non-pregnant women" and "study design". In Table 2, the Turkish column headers ("Sayı" and "Yüzde") were translated to their English equivalents ("Number" and "Percentage"). Furthermore, all decimal formattings have been uniformly standardized to periods throughout the manuscript.

• Changes made in the manuscript: Figure 1: The phrase "Flow chart of study dizayn" was corrected to "Flow chart of study design," and the box was corrected to "non-pregnant women." Table 2: The column headers were corrected to "Number" and "Percentage." Formatting: Decimal commas were replaced with periods across all tables and text.

 

We hope that these comprehensive revisions address your concerns and improve the quality of our manuscript. Thank you once again for your constructive guidance and invaluable contributions to our work.

Sincerely,

Prof. Dr. Yakup Baykus

On behalf of all co-authors

 

Reviewer 4 Report

Comments and Suggestions for Authors

The purpose of this paper is to explore the correlation between vaginal symptoms, degree of cervical inflammation, presence of high-risk HPV infection, cytological abnormalities, and microbiological data in patients visiting an outpatient gynecological clinic. The subject under consideration is important for clinical practice since inflammatory alterations identified in cervical smears occur rather frequently and represent an ambiguous factor concerning neoplasia risk. One advantage of this study is the relatively large number of subjects and its multi-faceted nature.

Comments 

1. “Prospective, cross-sectional, and observational” are the terms used to describe the research design. Here, there is a need for clarification since, normally, cross-sectional studies are usually observational and not prospective. The researchers should explain the element that was prospective in the study, for example, prospective recruitment.

2. The HPV analyses were carried out exclusively on females ≥30 years who participated in the national screenings (n=218). This greatly narrows the sample size for the HPV analyses and may create selection bias in the study. It is important for the paper to address this issue by clarifying if the excluded women were clinically different from the ones analyzed,
   Comment on the statistical power needed for HPV analyses.

3. Figure 1 should be improved; it is overdistended in the current view

4. Chi-square tests are employed for all analyses in this study.
   Considering the complexity of the factors influencing HPV infection, cervical inflammation, and vaginal complaints, regression analysis would greatly enhance the manuscript. Examples of potential confounding variables are age, smoking, contraceptive usage, sexual activity, parity, and others.

5. HPV infection and dysbiosis are clearly linked to sexual behavior. The article, however, does not mention any of the following criteria/factors - number of sexual partners,age at first intercourse, history of previous STIs. This is a clear limitation that should have been stated clearly in the limitations part of the paper.

6. While the manuscript talks about “abnormal cytology,” it is not clear as to what Bethesda classification categories were considered here.
   Please explain if ASC-US was considered; LSIL/HSIL if both were considered together;
   Cutoff values for “positive cytology.” Also a table depicting detailed cytological classification can be useful.

7. The Discussion part is informative but repetitive in some parts regarding the following topics asymptomatic nature of HPV infections, mechanisms of dysbiosis, significance for screening. Repeating of some ideas makes the text less clear and comprehensible.

Author Response

Manuscript ID: biomedicines-4294340

Title: The Relationship of Vaginal Symptoms and Cervical Inflammation Severity with Cytological Abnormalities and HPV Positivity: A Prospective Observational Study

 

Dear Reviewer 4,

We would like to express our sincere gratitude for your careful evaluation and highly constructive feedback on our manuscript. We deeply appreciate your valuable methodological and clinical observations, which have significantly contributed to clarifying our research design and improving the overall quality of our study. We have carefully considered all your recommendations and meticulously revised the text accordingly. Detailed point-by-point responses to your comments are provided below.

 

Comment 1: “Prospective, cross-sectional, and observational” are the terms used to describe the research design. Here, there is a need for clarification since, normally, cross-sectional studies are usually observational and not prospective. The researchers should explain the element that was prospective in the study, for example, prospective recruitment.

Response 1: We sincerely thank the reviewer for this highly accurate methodological observation. We entirely agree that cross-sectional observational studies do not inherently involve longitudinal clinical follow-up, and the use of the term "prospective" alone can cause conceptual confusion. In our study, the term was intended to indicate that patients were enrolled prospectively and their data were collected in real-time. In accordance with your excellent recommendation, we have revised the Methodology section to explicitly clarify this prospective element, preventing any potential ambiguity for the reader.

• Changes made in the manuscript: Section 2.1. Study Design and Sampling: (The introductory sentence was revised to clarify the study design as follows): "This study was conducted as a single-center, cross-sectional observational study with prospective patient recruitment and real-time data collection."

 

Comment 2: The HPV analyses were carried out exclusively on females ≥30 years who participated in the national screenings (n=218). This greatly narrows the sample size for the HPV analyses and may create selection bias in the study. It is important for the paper to address this issue by clarifying if the excluded women were clinically different from the ones analyzed, Comment on the statistical power needed for HPV analyses.

Response 2: We sincerely thank the reviewer for this highly critical epidemiological and statistical observation. We completely agree that restricting the HPV analyses to women aged ≥30 years in accordance with the national screening program limits the sample size and inevitably introduces a selection bias. In response to your valuable feedback, we have explicitly addressed this issue in the Strengths and Limitations of the Study (Section 4.5). We clarified that the reduced sample size for the HPV subset (n=218) limits the statistical power, particularly for subgroup comparisons. Furthermore, we candidly acknowledged that this age-related restriction introduces an inevitable selection bias, meaning our HPV-related findings may not be fully generalizable to the younger, excluded population.

• Changes made in the manuscript: Section 4.5. Strengths and Limitations of the Study: (The following limitation was integrated into the text): "...however, the HPV-related analyses were conducted on a substantially smaller subset (n=218), which reduces the statistical power, especially for subgroup comparisons. Furthermore, the restriction of HPV analyses to women ≥30 years in accordance with national screening guidelines introduces an inevitable selection bias, meaning our HPV-related findings may not be fully generalizable to younger populations."

 

Comment 3: Figure 1 should be improved; it is overdistended in the current view.

Response 3: We sincerely thank the reviewer for this careful visual observation. We completely agree that the previous version of the flowchart was overdistended. In accordance with your recommendation, the aspect ratio of Figure 1 has been carefully readjusted to correct the overdistended appearance, and the flowchart has been resized to be much more compact and readable within the text.

• Changes made in the manuscript: Figure 1: (The dimensions and aspect ratio of the flow chart were adjusted to prevent an overdistended appearance and to improve overall visual clarity).

Comment 4:

Chi-square tests are employed for all analyses in this study.

Considering the complexity of the factors influencing HPV infection, cervical inflammation, and vaginal complaints, regression analysis would greatly enhance the manuscript. Examples of potential confounding variables are age, smoking, contraceptive usage, sexual activity, parity, and others.

Response 4: We sincerely thank the reviewer for this highly constructive methodological recommendation. We completely agree that relying solely on bivariate analyses is insufficient in an observational study due to the complexity of the factors influencing HPV infection and cervical pathologies. In response to your excellent suggestion, we have performed multivariable binary logistic regression analyses for both abnormal cytology and HPV positivity to adjust for potential confounding variables. The models were adjusted for major clinical confounders, including age, smoking status, parity, and other relevant parameters. The adjusted odds ratios (aORs) and 95% confidence intervals are now clearly presented in a newly added Table 6. Importantly, these adjusted analyses confirmed our initial univariate findings: neither symptom burden nor cervical inflammation severity was independently associated with abnormal cytology or HPV positivity.

• Changes made in the manuscript: Abstract: (The following sentences were added to the Results and Conclusions sections, respectively): "In multivariable logistic regression analyses, neither symptom burden nor cervical inflammation severity was independently associated with abnormal cytology or HPV positivity." / "These findings remained consistent after adjustment for major clinical confounders." Section 2.4. Data Analysis: (The following paragraph was added): "In addition to Pearson chi-square and chi-square test for trend analyses, multivariable binary logistic regression analyses were performed to evaluate whether vaginal symptom parameters and cervical inflammation severity were independently associated with abnormal cytology and HPV positivity. Abnormal cytology and HPV positivity were entered as separate binary dependent variables. The models were adjusted for age, smoking status, parity, vaginal discharge, symptom recurrence frequency, Candida positivity, bacterial vaginosis, cervical inflammation severity, and symptom burden. Adjusted odds ratios (aORs) with 95% confidence intervals (CIs) were reported..." Section 3.4. (Results): (A new subsection and Table 6 were added): "3.4. Multivariable Logistic Regression Analysis for Abnormal Cytology and HPV Positivity: In the abnormal cytology model, none of the evaluated variables showed an independent association with abnormal cytology after adjustment for potential confounders..." Section 4.1. Discussion: (The following paragraph was added): "Importantly, the multivariable logistic regression analyses supported the findings of the univariate comparisons. After adjustment for age, smoking status, parity... neither symptom-related parameters nor cervical inflammation severity were independently associated with abnormal cytology or HPV positivity..."

 

Comment 5: HPV infection and dysbiosis are clearly linked to sexual behavior. The article, however, does not mention any of the following criteria/factors - number of sexual partners, age at first intercourse, history of previous STIs. This is a clear limitation that should have been stated clearly in the limitations part of the paper.

Response 5: We sincerely thank the reviewer for this highly justified criticism. We completely agree that HPV infection and dysbiosis are profoundly influenced by sexual behavior, and the lack of detailed data on these specific factors is a clear limitation of our study. In response to your valuable feedback, we have explicitly added the precise criteria you mentioned (number of sexual partners, age at first intercourse, and history of previous STIs) to the Strengths and Limitations of the Study (Section 4.5) to ensure full transparency regarding the unmeasured confounders in our analyses.

• Changes made in the manuscript: Section 4.5. Strengths and Limitations of the Study: (The following clarification was integrated into the text): "...reduces the statistical power, especially for subgroup comparisons. Moreover, the lack of data on critical confounding factors—such as detailed sexual history (including the number of sexual partners, age at first intercourse, and history of previous sexually transmitted infections), previous HPV vaccination status, and history of abnormal smears—limits the generalizability and interpretability of our HPV-related findings."

 

Comment 6: While the manuscript talks about “abnormal cytology,” it is not clear as to what Bethesda classification categories were considered here. Please explain if ASC-US was considered; LSIL/HSIL if both were considered together; Cutoff values for “positive cytology.” Also a table depicting detailed cytological classification can be useful.

Response 6: We sincerely thank the reviewer for this highly relevant clinical comment. To prevent borderline confounding, ASC-US cases were deliberately excluded from the study. Consequently, the "abnormal/positive" cytology group was strictly defined as LSIL and above for clinical significance. In accordance with your excellent recommendation, we have explicitly clarified this cutoff in the text and added a detailed breakdown of the Bethesda categories (NILM, LSIL, HSIL, ASC-H, AGC, SCC) to both the Results section and Table 1 to ensure complete transparency.

• Changes made in the manuscript: Section 3.1. (Table 1): (The detailed distribution of cytology results according to the Bethesda system was integrated into the table). Section 3.2. (Results): (The following clinical context was added to the text): "Detailed evaluation of the cervical cytology results according to the Bethesda system revealed that the entirely benign Cytology (-) group (n=398) consisted exclusively of NILM cases, as ASC-US cases were deliberately excluded from the study to prevent borderline confounding. The Cytology (+) group (n=60), which was strictly defined as LSIL and above for clinical significance, comprised 46 LSIL, 2 HSIL, 8 ASC-H, 3 AGC, and 1 SCC cases."

 

Comment 7: The Discussion part is informative but repetitive in some parts regarding the following topics asymptomatic nature of HPV infections, mechanisms of dysbiosis, significance for screening. Repeating of some ideas makes the text less clear and comprehensible.

Response 7: We sincerely thank the reviewer for this highly constructive feedback regarding the flow and clarity of our manuscript. We completely agree that the repetition of certain concepts detracted from the overall comprehensibility of the text. In response to your valuable suggestion, and in alignment with the extensive revisions made throughout the peer-review process (including the integration of the new multivariable logistic regression and FDR correction results), the Discussion section (Section 4) has been comprehensively rewritten and streamlined. We have carefully eliminated the redundancies regarding the asymptomatic nature of HPV, the mechanisms of dysbiosis, and the significance of screening, ensuring a much more concise, clear, and focused scientific narrative.

• Changes made in the manuscript: Section 4. Discussion: (The entire Discussion section has been meticulously revised, restructured, and streamlined to eliminate redundancies and improve overall clarity and comprehensibility).

 

We hope that these comprehensive revisions effectively address all the concerns raised and that the revised manuscript is now deemed suitable for publication. Thank you once again for your constructive guidance and invaluable contributions to our work.

Sincerely,

 

Corresponding Author: Prof. Dr. Yakup Baykus

On behalf of all co-authors

 

 

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

Thank you for addressing the  comments