Managing Vulvovaginal Atrophy, in Postmenopausal Women, Without Hormones: Efficacy of a Hyaluronic Acid Vaginal Moisturizer

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate the opportunity to review this manuscript, which aims to evaluate the efficacy and safety of Provagin®, a vaginal moisturizer composed of hyaluronic acid (HA) for vaginal mucosa application in postmenopausal women with mild or moderate vaginal dryness.

In my opinion, there are several problems with the study design. Even within the context of a medical device clinical investigation designed to demonstrate safety and performance, the fact that the study was not pre-registered, is single-blind, lacks a comparison group, has no randomisation, is single-centre, and has a very small sample size greatly undermines the reliability of the results.

In the Introduction section, the perimenopausal definition "perimenopause (the 12-month period since the last menstruation and is characterized as hormonal fluctuations)" does not align with the standard perimenopausal definition, as clearly established by the STRAW criteria.

In the second paragraph, the sentence "Non-The pharmacological treatments" appears to be a typographical error.

In the third paragraph, the reference used to support the statement "its use is established as safe and effective as vaginal estrogens [12]" corresponds to a single-centre study in 20 patients followed for 8 weeks and published in a journal not indexed in PubMed. It would be advisable to replace it with one of the references cited in the Discussion section.

In the Material and Methods section, it is not clearly stated how many gynaecologists assessed the patients. Was it a single gynaecologist or several?

In the Discussion section, the authors do not discuss why pH does not change despite the increase in Lactobacillus and the use of lactic acid as an excipient in the product, or how pruritus, dryness, and dyspareunia improve in the absence of changes in parameters such as pH and epithelial mucosal integrity. Perhaps vaginal colouration and rugosity could have been parameters to evaluate. Why were changes in the vulva not assessed either, given that patients applied the product to that area?

 

Comments on the Quality of English Language

The manuscript contains several typographical errors

Author Response

Comments1 : I appreciate the opportunity to review this manuscript, which aims to evaluate the efficacy and safety of Provagin®, a vaginal moisturizer composed of hyaluronic acid (HA) for vaginal mucosa application in postmenopausal women with mild or moderate vaginal dryness.

In my opinion, there are several problems with the study design. Even within the context of a medical device clinical investigation designed to demonstrate safety and performance, the fact that the study was not pre-registered, is single-blind, lacks a comparison group, has no randomisation, is single-centre, and has a very small sample size greatly undermines the reliability of the results.

Response1: We acknowledge the reviewer’s concerns regarding the study design (single-arm, single-center). We agree that randomized, controlled, multicenter studies with larger sample sizes provide the highest level of clinical evidence. However, we respectfully note that the present investigation was designed as an exploratory clinical study intended primarily to evaluate the safety and performance of the device under real-use conditions, rather than to establish definitive comparative efficacy.

We also would like to clarify that the study was registered in the ClinicalTrials.gov database under registration number NCT06564883, which is cited in line 69, page 02. We have updated the Limitations section to acknowledge that the lack of a control group limits the ability to definitively attribute results to the investigational product alone, framing these as preliminary but clinically meaningful observations.

Comments 2: In the Introduction section, the perimenopausal definition "perimenopause (the 12-month period since the last menstruation and is characterized as hormonal fluctuations)" does not align with the standard perimenopausal definition, as clearly established by the STRAW criteria.

Response 2: We have revised the definition in the Introduction to align with the STRAW criteria, clarifying the transition period. Lines 36 and 37.

Comments 3: In the second paragraph, the sentence "Non-The pharmacological treatments" appears to be a typographical error.

In the third paragraph, the reference used to support the statement "its use is established as safe and effective as vaginal estrogens [12]" corresponds to a single-centre study in 20 patients followed for 8 weeks and published in a journal not indexed in PubMed. It would be advisable to replace it with one of the references cited in the Discussion section.

Response 3: In response to the reviewer's suggestion, we have replaced the original reference with a more robust and clinically relevant study: "Hyaluronic acid injection to treat symptoms of vulvovaginal atrophy and improve sexual function in postmenopausal women: A 52-week long-term follow-up." This study included 115 postmenopausal women, provided long-term follow-up over 52 weeks, and is indexed in PubMed, thereby offering stronger clinical evidence regarding the safety and effectiveness of hyaluronic acid-based treatments for vulvovaginal atrophy. Lines 54 and 55, page 02.

Comments 4: In the Material and Methods section, it is not clearly stated how many gynaecologists assessed the patients. Was it a single gynaecologist or several?

Response 4: We have clarified in the Methods section that all gynecological assessments were performed by a single qualified gynecologist . Lines 107 and 108, page 03.

This approach was adopted to ensure consistency across all visits and to reduce inter-examiner variability, which is particularly relevant in the clinical evaluation of genitourinary syndrome of menopause and vaginal dryness. In this condition, clinical signs such as vulvovaginal mucosal appearance, epithelial integrity, irritation, and local dryness may involve a degree of subjective clinical interpretation. Therefore, assessment by the same examiner throughout the study helped standardize the evaluation of vulvovaginal findings over time and strengthened the internal consistency of the clinical assessments.

Comments 5: In the Discussion section, the authors do not discuss why pH does not change despite the increase in Lactobacillus and the use of lactic acid as an excipient in the product, or how pruritus, dryness, and dyspareunia improve in the absence of changes in parameters such as pH and epithelial mucosal integrity. Perhaps vaginal colouration and rugosity could have been parameters to evaluate. Why were changes in the vulva not assessed either, given that patients applied the product to that area?

Response 5: We thank the reviewer for this important observation and agree that the relationship between vaginal pH, Lactobacillus abundance, and symptom improvement deserves further discussion. To address this point, we have expanded the Discussion section to clarify that symptom improvement may occur independently of significant changes in vaginal pH (lines 310-313; 315-320, page 11).

We would like to clarify that the clinical evaluation was not limited to the vagina. As described in the Methods section (lines 102 and 103, page 03), visual examinations of the vulva, vagina, and cervix were conducted at each visit by the same qualified gynecologist, enabling the monitoring of vulvovaginal clinical signs over the course of the study.

Reviewer 2 Report

Comments and Suggestions for Authors

1. Study design limits attribution of efficacy - the principal limitation of this study is its single-arm, before-and-after design without a placebo, comparator, or standard-of-care control group. Although symptom improvements were observed, it is impossible to determine the extent to which these changes are attributable to the investigational product rather than placebo effects, regression to the mean, increased participant attention, behavioral modifications during the study period, or natural symptom fluctuation.

This concern is particularly relevant because the primary efficacy outcomes (vaginal dryness, dyspareunia, and pruritus) were entirely subjective patient-reported measures. Such outcomes are highly susceptible to expectation effects in non-controlled studies.

The Discussion acknowledges the lack of a comparator group, but the limitation is treated somewhat briefly and does not sufficiently temper the conclusions. Statements such as “demonstrated effectiveness” and “confirmed performance” appear stronger than the study design can support.

Suggestions for the authors -substantially strengthen the limitations section and explicitly acknowledge that causality cannot be established in the absence of a control group. Conclusions should be revised to indicate that symptom improvements were observed during product use rather than definitively attributing improvements to the product itself. The authors should also discuss the magnitude of placebo responses reported in previous GSM intervention studies and place their findings within that context.

 

2. Blinding procedures are unclear - the manuscript describes the study as “single-blind,” yet it is unclear who was blinded and how blinding was maintained. In a single-arm trial where all participants receive the same intervention, participant blinding appears difficult to implement, particularly given repeated self-assessment of symptoms. Furthermore, gynecological assessments were conducted by investigators who likely knew the timing of evaluations and treatment status.

Without clarification, the statement that the study was single-blind may be misleading.

Suggestions for the authors - clearly specify who was blinded (participants, assessors, statisticians, or others), how blinding was implemented, how blinding effectiveness was maintained throughout follow-up and whether outcome assessors were independent of study conduct. If true blinding was not feasible, the term "single-blind" should be reconsidered or removed.

 

3. Insufficient information regarding sample size determination - the manuscript does not provide a sample size calculation or justification for enrolling 50 participants. Without a priori power calculation, it is difficult to assess whether the study was adequately powered for efficacy and safety endpoints, particularly microbiological and epithelial integrity outcomes.

Suggestions for the authors - include the primary endpoint used for sample size determination, assumed effect size, statistical power and significance level, expected dropout rate and state whether the sample size was determined according to regulatory requirements for medical device investigations. If no formal calculation was performed, this should be explicitly stated and discussed as a limitation.

 

4. Choice and validation of outcome measures require clarification - the efficacy assessment relies primarily on visual analogue scales for dryness, dyspareunia, and pruritus. While these are common symptom measures, the manuscript does not explain whether validated GSM-specific instruments were considered.

Furthermore, the epithelial integrity assessment appears to use an adapted scale from a 1992 publication, but details regarding validation, reproducibility, and examiner training are lacking.

Suggestions for the authors - provide additional justification for the selected outcome measures and discuss their validity in GSM research. If examiner calibration or training was performed for epithelial integrity assessment, this should be reported. The manuscript would also benefit from discussing why validated instruments such as the Vulvovaginal Symptoms Questionnaire or quality-of-life measures were not incorporated.

 

5. Clinical significance should be better distinguished from statistical significance - the manuscript reports statistically significant reductions in symptom scores. However, the clinical significance of these changes is not discussed in sufficient detail.

For example, although a 69.3% reduction in vaginal dryness appears impressive, readers would benefit from understanding whether these changes exceeded established minimal clinically important differences (MCIDs), if available.

Similarly, the manuscript reports statistical improvements beginning at 72 hours for several outcomes, yet the clinical relevance of these early changes remains uncertain.

Suggestions for the authors - discuss whether the observed improvements meet accepted thresholds for clinically meaningful benefit. If no validated MCID exists for these scales in GSM, this should be acknowledged. Inclusion of responder analyses based on clinically relevant thresholds would substantially strengthen interpretation.

 

6. Microbiological findings require more nuanced interpretation - the reported increase in Lactobacillus spp. is potentially interesting. However, the methodology used to assess the vaginal microbiota relies on conventional culture techniques targeting a limited number of organisms. The vaginal microbiome is considerably more complex than can be captured through culture-based assessment alone. Therefore, statements suggesting preservation of the vaginal ecosystem may be somewhat overstated.

Suggestions for the authors - moderate claims regarding microbiome effects and acknowledge the limitations of culture-based methods. Discussion should clarify that only selected microorganisms were assessed and that comprehensive microbiome characterization would require molecular approaches such as 16S rRNA sequencing.

 

 

7. Potential conflict of interest requires additional transparency - all authors are employees of the sponsoring company, and the study was funded entirely by the manufacturer of the investigated product. While this is transparently disclosed, it raises legitimate concerns regarding potential bias in study design, conduct, analysis, and interpretation.

The manuscript states that scientific integrity was maintained, but additional procedural safeguards are not described.

Suggestions for the authors - provide greater detail regarding independent oversight of study conduct, data management procedures, statistical analysis independence, whether external investigators or monitors were involved and whether data verification or auditing was performed. Additional transparency would enhance confidence in the findings.

 

8. Introduction – this section provides an appropriate overview of GSM but contains several grammatical and stylistic issues that affect readability.

For example, the sentence describing GSM in lines 47–49 is grammatically incomplete and should be revised. There are additional language issues throughout the section, including awkward phrasing such as “The transition period occurs between 40 to 60 years” and “management depends on the severity of it.”

Suggestions for the authors - the Introduction would benefit from professional English-language editing to improve clarity, grammar, and scientific style. Several sentences should be rewritten for precision and readability.

 

9. Eligibility criteria - restricting enrollment to sexually active women aged 50–60 years may limit generalizability. Many women affected by GSM are older than 60 years or are not sexually active.

Suggestions for the authors - justify these eligibility restrictions and discuss their implications for external validity.

 

10. Statistical reporting - several p-values are reported inconsistently, using commas rather than decimal points (e.g., p=0,002 and p=0,317). There are also instances where exact p-values are provided and others where thresholds are used.

Suggestions for the authors - standardize statistical reporting throughout the manuscript according to journal style guidelines and report exact p-values whenever possible.

 

11. Adverse event reporting - the safety findings are encouraging, but adverse event reporting remains limited. It is not entirely clear whether adverse events were actively solicited or passively reported. Additionally, rates are presented as counts without detailed incidence proportions among treated participants.

Suggestions for the authors - provide a more detailed description of adverse event collection methods and include incidence rates relative to the treated population.

 

12. Figures and tables - figures generally communicate the results effectively. However, several figures would benefit from inclusion of participant numbers and confidence intervals. Table formatting also requires revision. For example, Table 1 contains terminology such as “Media pH” and “% media variation,” which appears to be a translation issue.

Suggestions for the authors - carefully revise all tables and figures for consistency, terminology, and clarity. Confidence intervals would strengthen presentation of efficacy outcomes.

 

13. Discussion - the Discussion appropriately relates findings to previous literature but occasionally overstates the implications of the study. Several passages imply that efficacy has been definitively established despite the lack of a control group.
14. Suggestions for the authors - adopt a more balanced tone throughout the Discussion and Conclusions. Statements regarding efficacy should be framed as preliminary observations requiring confirmation in randomized controlled trials. The Discussion could be strengthened by incorporating additional recent evidence on non-hormonal or combined management strategies for GSM. We sugest to the authors to consult some recent studies: https://doi.org/10.3390/clinpract15010020  or https://doi.org/10.12680/balneo.2024.757 , wich  evaluated the efficacy of combining or not of Kegel exercises with local vaginal estrogens  in therapy of women with GSM. Although the intervention differs from hyaluronic acid treatment, the study provides contemporary evidence supporting non-hormonal approaches aimed at improving GSM-related symptoms and quality of life. 

 

14. Conclusion - this study addresses a clinically important topic and provides useful preliminary data regarding the tolerability and potential symptomatic benefits of a hyaluronic acid-based vaginal moisturizer in postmenopausal women with GSM. The observed improvements in vaginal dryness, dyspareunia, and pruritus are promising and may be clinically relevant. However, the uncontrolled study design, reliance on subjective outcomes, limited follow-up duration, and sponsor involvement substantially restrict the strength of efficacy conclusions.

Author Response

Comments 1. Study design limits attribution of efficacy - the principal limitation of this study is its single-arm, before-and-after design without a placebo, comparator, or standard-of-care control group. Although symptom improvements were observed, it is impossible to determine the extent to which these changes are attributable to the investigational product rather than placebo effects, regression to the mean, increased participant attention, behavioral modifications during the study period, or natural symptom fluctuation.

This concern is particularly relevant because the primary efficacy outcomes (vaginal dryness, dyspareunia, and pruritus) were entirely subjective patient-reported measures. Such outcomes are highly susceptible to expectation effects in non-controlled studies.

The Discussion acknowledges the lack of a comparator group, but the limitation is treated somewhat briefly and does not sufficiently temper the conclusions. Statements such as “demonstrated effectiveness” and “confirmed performance” appear stronger than the study design can support.

Suggestions for the authors-substantially strengthen the limitations section and explicitly acknowledge that causality cannot be established in the absence of a control group. Conclusions should be revised to indicate that symptom improvements were observed during product use rather than definitively attributing improvements to the product itself. The authors should also discuss the magnitude of placebo responses reported in previous GSM intervention studies and place their findings within that context.

Response 1: We thank the reviewer for the rigorous and detailed evaluation of our manuscript. These comments have significantly contributed to improving the transparency and scientific balance of our work.

We agree that the absence of a control group is a significant methodological limitation. We have revised the Limitations section to explicitly state that causality cannot be established. However, we wish to clarify that this study was designed as a clinical investigation to satisfy regulatory requirements for the maintenance of a Class III medical device (ANVISA), where performance under real-use conditions is the primary focus.

We have also changed our conclusions, replacing "demonstrated effectiveness" with "observed improvements”. Lines 360-366, page 12.

Comments 2: Blinding procedures are unclear - the manuscript describes the study as “single-blind,” yet it is unclear who was blinded and how blinding was maintained. In a single-arm trial where all participants receive the same intervention, participant blinding appears difficult to implement, particularly given repeated self-assessment of symptoms. Furthermore, gynecological assessments were conducted by investigators who likely knew the timing of evaluations and treatment status.

Without clarification, the statement that the study was single-blind may be misleading.

Response 2: We appreciate this observation and have revised the manuscript to reflect that the study was open-label. The previous mention of "single-blind" referred specifically to the statistical analysis, where the statistician remained blinded to the study stages during data processing. We have removed the term "single-blind" to avoid any misleading interpretation.

 

Comments 3: Insufficient information regarding sample size determination - the manuscript does not provide a sample size calculation or justification for enrolling 50 participants. Without a priori power calculation, it is difficult to assess whether the study was adequately powered for efficacy and safety endpoints, particularly microbiological and epithelial integrity outcomes.

Suggestions for the authors - include the primary endpoint used for sample size determination, assumed effect size, statistical power and significance level, expected dropout rate and state whether the sample size was determined according to regulatory requirements for medical device investigations. If no formal calculation was performed, this should be explicitly stated and discussed as a limitation.

 

Response 3: We acknowledge that a formal statistical sample size calculation was not performed. The present study was designed as an exploratory clinical investigation intended primarily to assess the safety, tolerability, and performance of the device under real-use conditions rather than to test a specific efficacy hypothesis.

The sample size was determined based on regulatory considerations, feasibility, and consistency with similar clinical investigations conducted for medical devices in this therapeutic area. The selected number of participants was considered sufficient to provide an initial assessment of safety and performance and to support the intended regulatory submission.

We recognize that the absence of a priori sample size calculation may limit the ability to formally assess the statistical power for certain efficacy and microbiological outcomes. This aspect has been discussed as one limitation in the revised manuscript (lines 349-358, page 12).

Comments 4: 

Choice and validation of outcome measures require clarification - the efficacy assessment relies primarily on visual analogue scales for dryness, dyspareunia, and pruritus. While these are common symptom measures, the manuscript does not explain whether validated GSM-specific instruments were considered.

Furthermore, the epithelial integrity assessment appears to use an adapted scale from a 1992 publication, but details regarding validation, reproducibility, and examiner training are lacking.

Suggestions for the authors -provide additional justification for the selected outcome measures and discuss their validity in GSM research. If examiner calibration or training was performed for epithelial integrity assessment, this should be reported. The manuscript would also benefit from discussing why validated instruments such as the Vulvovaginal Symptoms Questionnaire or quality-of-life measures were not incorporated.

Response 4: We thank the reviewer for this important comment. The selected outcome measures were chosen because they represent the core symptoms of genitourinary syndrome of menopause (GSM), including vaginal dryness, itching, discomfort, and dyspareunia, which are among the most commonly reported symptoms and are frequently used as primary endpoints in GSM clinical studies.

We acknowledge that GSM-specific instruments like the Vulvovaginal Symptoms Questionnaire provide broader insights, Visual Analogue Scales (VAS) are widely accepted in clinical trials for quantifying the intensity of specific symptoms like dryness and dyspareunia. The selected outcome measures were chosen because they represent the core symptoms of genitourinary syndrome of menopause (GSM), including vaginal dryness, itching, discomfort, and dyspareunia, which are among the most commonly reported symptoms and are frequently used as primary endpoints in GSM clinical studies.

We add a paragraph at discussion section: lines 339-343 (page 11 and 12).

The epithelial integrity assessment was based on the epithelial integrity component described by Bachmann et al. (1992) as part of the Vaginal Health Index. In the present study, only this specific component was used as a clinical examination tool to evaluate the condition of the vaginal mucosa, using the same 1–5 scoring structure described by the authors. The scale was translated into Portuguese for operational use by the investigator; however, no modifications were made to the original scoring criteria.

The Vaginal Health Index and its individual components have been widely used in clinical studies evaluating vaginal health, vulvovaginal atrophy, and genitourinary syndrome of menopause, supporting their utility as clinical assessment tools. Nevertheless, we acknowledge that formal validation of the translated version and formal reproducibility testing were not performed in this study.

To ensure consistency, all examinations were conducted by a single trained gynecologist using standardized assessment procedures throughout the study, thereby eliminating inter-observer variability.

To avoid any misunderstanding, the manuscript has been revised to clarify that the epithelial integrity assessment was translated for study use but was not adapted or modified from the original Bachmann et al. scoring system.

Comments 5: Clinical significance should be better distinguished from statistical significance - the manuscript reports statistically significant reductions in symptom scores. However, the clinical significance of these changes is not discussed in sufficient detail.

For example, although a 69.3% reduction in vaginal dryness appears impressive, readers would benefit from understanding whether these changes exceeded established minimal clinically important differences (MCIDs), if available.

Similarly, the manuscript reports statistical improvements beginning at 72 hours for several outcomes, yet the clinical relevance of these early changes remains uncertain.

Suggestions for the authors - discuss whether the observed improvements meet accepted thresholds for clinically meaningful benefit. If no validated MCID exists for these scales in GSM, this should be acknowledged. Inclusion of responder analyses based on clinically relevant thresholds would substantially strengthen interpretation.

Response 5: The primary goal of treating GSM is to alleviate symptoms, and once other causes have been excluded, treatment is typically approached in a stepwise based on symptom severity.In this context, the goal of using a vaginal moisturizer is to reduce daily symptoms of GSM as well as to facilitate comfortable sexual activity. Although no validated minimal clinically important difference (MCID) has been established specifically for vaginal dryness, pain, or pruritus measured by VAS in women with GSM, the observed changes substantially exceeded commonly reported thresholds for clinically meaningful improvement in VAS-based symptom assessments.

At Day 22, vaginal dryness decreased from 6.16 to 1.89, corresponding to an absolute reduction of 4.27 points and a relative reduction of 69.3%. Similarly, vaginal pain decreased from 5.78 to 1.56 (4.22-point reduction; 73.1%), and vaginal pruritus decreased from 5.02 to 0.80 (4.22-point reduction; 84.1%). These reductions are considerably greater than the approximately 30% improvement, often considered clinically meaningful for VAS-based symptom measures.

In addition, responder rates were high, with symptom improvement observed in 95.6% of participants for vaginal dryness, 93.3% for vaginal pain, and 95.6% for vaginal pruritus. These findings support the clinical relevance of the observed treatment effects beyond statistical significance alone.

Thus, we acknowledge that universally validated MCID thresholds for these specific VAS scales in GSM research are not yet standardized, the magnitude of these reductions reflects a substantial relief in the patients´ lived experience. Lines 270-280 (page 10).

Comments 6: 

Comments 6: Microbiological findings require more nuanced interpretation - the reported increase in Lactobacillus spp. is potentially interesting. However, the methodology used to assess the vaginal microbiota relies on conventional culture techniques targeting a limited number of organisms. The vaginal microbiome is considerably more complex than can be captured through culture-based assessment alone. Therefore, statements suggesting preservation of the vaginal ecosystem may be somewhat overstated.

Suggestions for the authors - moderate claims regarding microbiome effects and acknowledge the limitations of culture-based methods. Discussion should clarify that only selected microorganisms were assessed and that comprehensive microbiome characterization would require molecular approaches such as 16S rRNA sequencing.

Response 6: We thank the reviewer for this valuable comment and agree that the vaginal microbiome is considerably more complex than can be fully characterized using conventional culture-based methods. We have revised the manuscript to moderate our claims regarding the microbiological findings. We now specify that the product supports the maintenance of selected beneficial microorganisms, such as Lactobacillus spp., rather than the entire ecosystem.

 We have also added a limitation statement in Discussion (lines 352-354, page 12) section that culture-based methods provide a partial assessment and that molecular approach like 16S rRNA gene sequencing would be required for a comprehensive characterization.

Comments 7: Potential conflict of interest requires additional transparency - all authors are employees of the sponsoring company, and the study was funded entirely by the manufacturer of the investigated product. While this is transparently disclosed, it raises legitimate concerns regarding potential bias in study design, conduct, analysis, and interpretation.

The manuscript states that scientific integrity was maintained, but additional procedural safeguards are not described.

Suggestions for the authors- provide greater detail regarding independent oversight of study conduct, data management procedures, statistical analysis independence, whether external investigators or monitors were involved and whether data verification or auditing was performed. Additional transparency would enhance confidence in the findings.

Response 7: We have enhanced the transparency of our disclosure. Clinical assessments were performed by investigators independent of the product’s commercial development (lines 378-381, page 12)

Comments 8: Introduction – this section provides an appropriate overview of GSM but contains several grammatical and stylistic issues that affect readability.

For example, the sentence describing GSM in lines 47–49 is grammatically incomplete and should be revised. There are additional language issues throughout the section, including awkward phrasing such as “The transition period occurs between 40 to 60 years” and “management depends on the severity of it.”

Suggestions for the authors - the Introduction would benefit from professional English-language editing to improve clarity, grammar, and scientific style. Several sentences should be rewritten for precision and readability.

Response 8: We thank the reviewer for this suggestion. The corresponding paragraph has been rewritten to improve clarity and provide a more accurate description of the menopausal transition and the underestimation of GSM prevalence. (Lines 38-46, pages 1 and 2)

Comments 9: Eligibility criteria - restricting enrollment to sexually active women aged 50–60 years may limit generalizability. Many women affected by GSM are older than 60 years or are not sexually active.

Suggestions for the authors - justify these eligibility restrictions and discuss their implications for external validity.

Response 9: We appreciate the reviewer’s comment regarding the external validity of our findings. The selection of sexually active women aged 50–60 was a deliberate methodological decision intended to maximize the internal validity of this clinical investigation. By narrowing the age range, we aimed to minimize confounding variables associated with the more diverse comorbidities and extreme physiological changes often observed in late-stage postmenopause.

While we acknowledge that this phenotypically homogeneous cohort does not represent the entire spectrum of women with GSM, it provided the necessary sensitivity to assess the rapid-onset effects of the formulation in a population with high clinical demand for non-hormonal alternatives. Lines 275-279 (Page 10).

Comments 10: Statistical reporting - several p-values are reported inconsistently, using commas rather than decimal points (e.g., p=0,002 and p=0,317). There are also instances where exact p-values are provided and others where thresholds are used.

Suggestions for the authors - standardize statistical reporting throughout the manuscript according to journal style guidelines and report exact p-values whenever possible.

Response 10: We have standardized all statistical reporting to use decimal points (e.g., p=0.002) and provided exact p-values throughout. Table 1 has been revised to correct translation issues like "Media pH" to "Mean pH".

Comments 11: Adverse event reporting - the safety findings are encouraging, but adverse event reporting remains limited. It is not entirely clear whether adverse events were actively solicited or passively reported. Additionally, rates are presented as counts without detailed incidence proportions among treated participants.

Suggestions for the authors - provide a more detailed description of adverse event collection methods and include incidence rates relative to the treated population.

Response 11: We appreciate the opportunity to clarify our safety monitoring and reporting procedures. Adverse events (AEs) were actively solicited from the start of the study. All participants were specifically instructed during the enrollment visit to report any new symptoms or health changes throughout the trial duration.

The established safety workflow required that all reported events be initially processed by the clinical research center and subsequently forwarded to the company’s pharmacovigilance department for formal documentation and analysis. Our protocol also mandated that any Serious Adverse Events (SAEs) be reported to the Ethics Committee within 24 hours of discovery; however, no SAEs were reported during this study.

For the non-serious AEs that did occur (n=6), the research center conducted a detailed medical evaluation to establish causal links. The causality assessment for these events was classified as follows:

Not clearly attributable: n=2 (4.4%), Excluded (unrelated): n=2 (4.4%), and Unlikely: n=2 (4.4%).

These events included mild cases of diarrhea, petechiae, and pruritus, as well as one moderate event (uterine pain and dark urine) which, upon investigation, was determined to be unrelated to the investigational product. We updated the manuscript (Lines 258-266, page 10)

Comments 12: Figures and tables - figures generally communicate the results effectively. However, several figures would benefit from inclusion of participant numbers and confidence intervals. Table formatting also requires revision. For example, Table 1 contains terminology such as “Media pH” and “% media variation,” which appears to be a translation issue.

Suggestions for the authors - carefully revise all tables and figures for consistency, terminology, and clarity. Confidence intervals would strengthen presentation of efficacy outcomes.

Response 12: We appreciate the reviewer's suggestion to enhance the clarity of our visual data. In response, we have implemented the following changes:

Inclusion of Participant Numbers: All figure and table captions have been updated to explicitly include the number of participants (n) involved in each specific analysis.

Improved Fluidity and Terminology: Captions and table headers were rewritten to ensure a more professional and fluid scientific style. For example, in Table 1, the term "Media pH" was corrected to "Mean pH," and "% media variation" was adjusted to "% mean variation" to resolve translation inconsistencies.

Participant Flowchart: As suggested to improve transparency regarding study eligibility and enrollment, we have inserted a Study Flowchart (Figure 2). This figure clearly illustrates the screening process, reasons for exclusion, and the final number of participants who completed the study (n=45), providing a clearer overview of the study population as described in the Results section. (Lines 180-181; page 5).

Comments 13: Discussion - the Discussion appropriately relates findings to previous literature but occasionally overstates the implications of the study. Several passages imply that efficacy has been definitively established despite the lack of a control group.

Suggestions for the authors- adopt a more balanced tone throughout the Discussion and Conclusions. Statements regarding efficacy should be framed as preliminary observations requiring confirmation in randomized controlled trials. The Discussion could be strengthened by incorporating additional recent evidence on non-hormonal or combined management strategies for GSM. We sugest to the authors to consult some recent studies: https://doi.org/10.3390/clinpract15010020 or https://doi.org/10.12680/balneo.2024.757 , wich evaluated the efficacy of combining or not of Kegel exercises with local vaginal estrogens in therapy of women with GSM. Although the intervention differs from hyaluronic acid treatment, the study provides contemporary evidence supporting non-hormonal approaches aimed at improving GSM-related symptoms and quality of life.

Response 13: We thank the reviewer for this suggestion and carefully examined the cited studies. The publications provide relevant contemporary evidence regarding the management of GSM, particularly in the context of the potential additive effects of pelvic floor rehabilitation (Kegel exercises) in estrogen-based therapies.

However, after consideration, we decided not to incorporate these studies into the Discussion because their interventions differ substantially from the therapeutic approach evaluated in our study. As a result, direct comparison with our findings would be limited and could potentially lead to inappropriate extrapolation regarding the mechanisms and effectiveness of the interventions.

Nevertheless, in response to the reviewer's comment, we have revised the Discussion and Conclusions to adopt a more balanced interpretation of the findings, emphasizing the preliminary nature of the efficacy observations.

Comments 14: 

Conclusion - this study addresses a clinically important topic and provides useful preliminary data regarding the tolerability and potential symptomatic benefits of a hyaluronic acid-based vaginal moisturizer in postmenopausal women with GSM. The observed improvements in vaginal dryness, dyspareunia, and pruritus are promising and may be clinically relevant. However, the uncontrolled study design, reliance on subjective outcomes, limited follow-up duration, and sponsor involvement substantially restrict the strength of efficacy conclusions.

Reviewer 3 Report

Comments and Suggestions for Authors

Although the study findings is clinically relevant, the novelty of the present study is not clear. The beneficial effects of vaginal hyaluronic acid on symptoms of vulvovaginal atrophy/genitourinary syndrome of menopause, including vaginal dryness, pruritus, vaginal pH, and overall symptom improvement, have already been reported in recent studies. The manuscript does not clearly explain how the current study differs from or advances the findings of the previously published literature. The authors should explicitly address the novelty of their work and clarify what new scientific or clinical information is provided beyond the existing evidence. Without a clear distinction from the existing literature, the scientific contribution and originality of the manuscript remain limited. (1. Jafarzade et al. A comparison of hyaluronic acid and estradiol treatment in vulvovaginal atrophy. Eur Rev Med Pharmacol Sci. 2024 Jan;28(2):571-576. doi: 10.26355/eurrev_202401_35054: 2. Agrawal et al. A randomized, pilot trial comparing vaginal hyaluronic acid to vaginal estrogen for the treatment of genitourinary syndrome of menopause. Menopause. 2024 Sep 1;31(9):750-755. doi: 10.1097/GME.0000000000002390). Please address following querries: whether any new outcome measures or mechanistic insights were evaluated. How the present findings contribute additional evidence beyond the studies already published.

 

 

Author Response

Comments 1: Although the study findings is clinically relevant, the novelty of the present study is not clear. The beneficial effects of vaginal hyaluronic acid on symptoms of vulvovaginal atrophy/genitourinary syndrome of menopause, including vaginal dryness, pruritus, vaginal pH, and overall symptom improvement, have already been reported in recent studies. The manuscript does not clearly explain how the current study differs from or advances the findings of the previously published literature. The authors should explicitly address the novelty of their work and clarify what new scientific or clinical information is provided beyond the existing evidence. Without a clear distinction from the existing literature, the scientific contribution and originality of the manuscript remain limited. (1. Jafarzade et al. A comparison of hyaluronic acid and estradiol treatment in vulvovaginal atrophy. Eur Rev Med Pharmacol Sci. 2024 Jan;28(2):571-576. doi: 10.26355/eurrev_202401_35054: 2. Agrawal et al. A randomized, pilot trial comparing vaginal hyaluronic acid to vaginal estrogen for the treatment of genitourinary syndrome of menopause. Menopause. 2024 Sep 1;31(9):750-755. doi: 10.1097/GME.0000000000002390). Please address following querries: whether any new outcome measures or mechanistic insights were evaluated. How the present findings contribute additional evidence beyond the studies already published.

Response 1: We thank the reviewer for highlighting these recent studies and agree that the beneficial effects of HA in vulvovaginal atrophy/genitourinary syndrome of menopause have been previously reported. We have revised the manuscript to better clarify the specific contribution of the present study in relation to the existing literature, and we add the first reference in the introduction.

Although the general therapeutic potential of HA is already recognized, the novelty of our study lies in the evaluation of this specific medical-device formulation under real-use conditions, with a detailed short-term temporal assessment of symptom evolution and local tolerability. In particular, our study evaluated clinical outcomes at early time points, including 24 hours, 72 hours, 6 days, and 22 days after treatment initiation. This design allowed us to characterize not only the overall symptomatic improvement, but also the onset and progression of symptom relief over time.

This is clinically relevant because patients with genitourinary syndrome of menopause often seek rapid relief from symptoms such as vaginal dryness, dyspareunia, and pruritus, which can substantially affect daily comfort, sexual activity, and quality of life. While previous studies have mainly focused on medium- or longer-term outcomes, our findings suggest that symptomatic improvement may begin as early as 72 hours after product use. This provides additional practical information for clinicians regarding the expected time course of response to this specific non-hormonal vaginal moisturizer.

In addition, the present study included assessments beyond subjective symptom scores, including epithelial integrity, vaginal pH, and selected culture-based microbiological parameters. Although culture-based methods do not provide a comprehensive characterization of the vaginal microbiome, they allowed us to evaluate selected microorganisms, including Lactobacillus spp., during product use. Therefore, our findings add preliminary evidence regarding the local safety, tolerability, epithelial effects, and selected microbiological profile of this formulation.

We have updated the Introduction and Discussion sections to clarify that the contribution of this study is not the demonstration that HA is a new therapeutic class, but rather the characterization of the short-term performance, safety profile, and early symptom-relief pattern of this specific HA-based medical device.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed the concerns raised through methodological clarifications, interpretative improvements, and editorial corrections. These revisions have substantially enhanced the manuscript's clarity, transparency, and interpretability. Therefore, I consider the manuscript suitable for acceptance in its current form

Comments on the Quality of English Language

N/A

Author Response

Comments: The authors have satisfactorily addressed the concerns raised through methodological clarifications, interpretative improvements, and editorial corrections. These revisions have substantially enhanced the manuscript's clarity, transparency, and interpretability. Therefore, I consider the manuscript suitable for acceptance in its current form

Response: We are deeply grateful for the reviewer’s final assessment and for the recommendation for acceptance. We sincerely appreciate the rigorous and constructive feedback provided throughout this revision process.

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript is considerably stronger than the previous version and is approaching publication quality. However, several issues remain that should be addressed before acceptance:

1. Study design limitations are acknowledged but efficacy claims remain somewhat overstated - the authors have appropriately expanded the limitations section and now explicitly acknowledge that the absence of a comparator group limits causal inference. This is an important improvement. Nevertheless, several statements throughout the Discussion and Conclusions continue to imply treatment efficacy more strongly than can be supported by an uncontrolled, open-label study. For example, phrases suggesting that the product "demonstrated" efficacy or "supports its use" may still be interpreted as causal conclusions rather than observations from a single-arm investigation.

Please further revise the Discussion and Conclusion to consistently emphasize that symptom improvements were observed during product use, causality cannot be established, placebo effects and regression to the mean cannot be excluded and confirmation in randomized controlled trials remains necessary.

2. Sample size justification remains insufficient - the authors now state that sample size was determined according to regulatory requirements rather than an a priori power calculation, which improves transparency. However, the manuscript still does not provide sufficient detail regarding the regulatory rationale, target performance outcomes, assumptions used for enrollment targets and expected dropout rates. Please include a brief explanation of the regulatory framework used to determine sample size and clarify whether the study was intended primarily as a safety/performance investigation rather than an efficacy trial.
3. Conflict of interest and sponsor involvement require additional transparency - the manuscript continues to indicate that all authors are employees of the sponsoring company. While this is disclosed, the revised version does not provide meaningful additional information regarding safeguards implemented to minimize bias. Important questions remain unanswered such as: was data monitoring performed independently? was statistical analysis conducted by an external party? was source-data verification performed? were investigators independent from sponsor management?

Expand the conflict-of-interest and study oversight sections to provide greater transparency regarding data integrity procedures and quality-control measures.

4. Clinical significance could be discussed more rigorously - the addition of absolute symptom reductions is helpful and strengthens interpretation of the findings. However, the manuscript still does not discuss whether validated minimal clinically important differences (MCIDs) exist for these outcomes and if the observed changes exceed accepted thresholds for meaningful clinical benefit. Either discuss available MCID literature or explicitly state that validated thresholds are unavailable for these measures in GSM research.
5. Outcome assessment - the description of epithelial integrity assessment has improved substantially and now includes examiner training and use of the Vaginal Health Index framework. However, the manuscript would benefit from a brief explanation regarding why validated GSM-specific symptom instruments or quality-of-life questionnaires were not incorporated.
6. Adverse event reporting - the adverse event section is improved and now includes severity classification and causality assessment. Nevertheless, the manuscript should clarify whether adverse events were actively solicited at each visit and whether participants maintained symptom diaries.
7. Statistical reporting - most statistical reporting inconsistencies have been corrected. However, the authors should ensure that exact p-values are reported whenever possible and formatting follows journal guidelines consistently throughout all tables and figures.
8. Figures and tables - figures and tables are generally clear and improved compared with the previous version. The correction of terminology such as "mean pH" and "mean variation" is appreciated. Consider adding confidence intervals and participant numbers directly within figure legends, to facilitate interpretation.
9. We suggest that you discuss more about complementary therapies like Kegel exercises, Kegel exercises combined or not with estrogens. As for reference material, we would like to suggest a few interesting study titles: Șerbănescu, L.; Rotar, V.; Brezeanu, D.; Mirea, S.; Ionescu, E. V.; Ionescu, P. Evaluating the Efficacy of Combined Intravaginal Estriol Therapy and Kegel Exercises in Managing Menopausal Atrophic Vulvovaginitis. Clin Pract, 2025, 15(1), 20.                                                                                             Șerbănescu, L.; Mirea, S.; Ionescu, P.; Petrica, L.A.; Iorga, I.C.; Surdu, M.; Surdu, T.V.; Rotar, V. Involuntary Urine Loss in Menopause—A Narrative Review.  Clin. Med.2025, 14(21), 7664. https://doi.org/10.3390/jcm14217664

Author Response

We thank the reviewer for the rigorous and detailed evaluation of our manuscript. These comments have significantly contributed to improving the transparency and scientific balance of our work. We appreciate the opportunity to further clarify our methodology and findings.

Comments 1: The revised manuscript is considerably stronger than the previous version and is approaching publication quality. However, several issues remain that should be addressed before acceptance:

1. Study design limitations are acknowledged but efficacy claims remain somewhat overstated - the authors have appropriately expanded the limitations section and now explicitly acknowledge that the absence of a comparator group limits causal inference. This is an important improvement. Nevertheless, several statements throughout the Discussion and Conclusions continue to imply treatment efficacy more strongly than can be supported by an uncontrolled, open-label study. For example, phrases suggesting that the product "demonstrated" efficacy or "supports its use" may still be interpreted as causal conclusions rather than observations from a single-arm investigation. Please further revise the Discussion and Conclusion to consistently emphasize that symptom improvements were observed during product use, causality cannot be established, placebo effects and regression to the mean cannot be excluded and confirmation in randomized controlled trials remains necessary.

Response 1: We recognize that in an uncontrolled, open-label study, the use of causal verbs can be misleading. Accordingly, we have conducted a comprehensive revision of the Discussion (line 373) and Conclusion (lines 390 and 391, page 12) sections to ensure the language reflects the observational nature of our findings. This adjustment ensures that the results are framed as performance data within our specific study population, without overstating the ability to infer direct causality.

 

Comments 2:  Sample size justification remains insufficient - the authors now state that sample size was determined according to regulatory requirements rather than an a priori power calculation, which improves transparency. However, the manuscript still does not provide sufficient detail regarding the regulatory rationale, target performance outcomes, assumptions used for enrollment targets and expected dropout rates. Please include a brief explanation of the regulatory framework used to determine sample size and clarify whether the study was intended primarily as a safety/performance investigation rather than an efficacy trial.

We thank the reviewer for the opportunity to further clarify the methodological framework of our study. We would like to provide additional context regarding the regulatory history of the product and the rationale for the study design:

The investigational product, Provagin®, was previously registered and marketed as a cosmetic. Following a change in national legislation (ANVISA, Brazil), products of this nature were reclassified as Class III medical devices. This transition necessitated a formal clinical investigation to maintain its registration under the new regulatory framework.

As the product was already established on the market, the study was designed primarily as a clinical performance and safety investigation rather than a primary efficacy trial for a novel drug. The objective was to confirm the safe and appropriate use of the existing formulation specifically for women with GSM under the more rigorous medical device standards.

The sample size of 50 participants was designed to be sufficient for observing target performance outcomes (specifically, a clinically significant reduction in GSM symptoms and maintenance of physiological vaginal parameters) and to detect potential adverse effects in real-use conditions.

It is important to note that the regulatory dossier submitted to the health authority includes not only the data from this clinical study but also long-term market safety data continuously monitored by our corporate pharmacovigilance department.

Comments 3: Conflict of interest and sponsor involvement require additional transparency - the manuscript continues to indicate that all authors are employees of the sponsoring company. While this is disclosed, the revised version does not provide meaningful additional information regarding safeguards implemented to minimize bias. Important questions remain unanswered such as: was data monitoring performed independently? was statistical analysis conducted by an external party? was source-data verification performed? were investigators independent from sponsor management?

Expand the conflict-of-interest and study oversight sections to provide greater transparency regarding data integrity procedures and quality-control measures

Response 3: We have updated the conflicts of interest section (lines 411-415, page 13) to provide the following clarifications:

The entire execution of the study, including the development of the operational protocol, participant recruitment, and the full clinical follow-up from the signing of the Informed Consent Form to the final participant visit, was performed by an independent clinical investigation center. The center is an autonomous entity contracted by the sponsor; importantly, no employees of the sponsoring company work at the clinical site or are involved in the direct management of participants.

All investigators were clinically independent and not subject to the sponsor's management or product development goals. The sponsor’s role was limited to GCP (Good Clinical Practice) monitoring. This involved periodic source-data verification (reviewing documents to ensure data accuracy and participant safety) to ensure the study remained in strict compliance with the protocol and ethical standards. This monitoring was strictly an oversight function and did not involve any interference with the clinical conduct or decision-making during the study.

The statistical analysis of the data was conducted by the independent research center.

Comments 4: Clinical significance could be discussed more rigorously - the addition of absolute symptom reductions is helpful and strengthens interpretation of the findings. However, the manuscript still does not discuss whether validated minimal clinically important differences (MCIDs) exist for these outcomes and if the observed changes exceed accepted thresholds for meaningful clinical benefit. Either discuss available MCID literature or explicitly state that validated thresholds are unavailable for these measures in GSM research

Response 4: We have updated the discussion (lines 350-358, page 12) section to explicitly state that, to date, universally validated MCID thresholds for Visual Analogue Scale (VAS) scores regarding vaginal dryness, dyspareunia, and pruritus have not been established specifically for GSM research.

However, we have contextualized our findings based on broader clinical literature. In many clinical domains, a 30% reduction or an absolute change of 1.5 to 2 points on a 10-point VAS is widely accepted as a threshold for a clinically meaningful benefit. Our results demonstrated absolute reductions of over 4 points and relative reductions exceeding 69% for all primary symptoms. In addition, the high responder rates (over 93% for all symptoms) reinforce that the observed changes represent a profound and meaningful improvement in the patients’ lived experience

 

Comments 5: Outcome assessment - the description of epithelial integrity assessment has improved substantially and now includes examiner training and use of the Vaginal Health Index framework. However, the manuscript would benefit from a brief explanation regarding why validated GSM-specific symptom instruments or quality-of-life questionnaires were not incorporated

Response 5: We appreciate the reviewer's insight regarding the use of multidimensional assessment tools. The decision to prioritize Visual Analogue Scales (VAS) for core symptoms (vaginal dryness, dyspareunia, and pruritus) was based on the specific nature of this investigation. As clarified in previous responses, this study was a clinical performance and safety investigation required for the regulatory maintenance of an existing product reclassified as a Class III medical device. In this context, the primary objective was to confirm the device's ability to alleviate the hallmark symptoms of GSM and maintain local safety parameters under real-use conditions.

VAS is a validated, sensitive, and widely accepted standard in GSM clinical research for quantifying symptom intensity and was considered sufficient to demonstrate the device's performance for regulatory purposes. While quality-of-life (QoL) questionnaires provide valuable broader perspectives, they were not included as primary endpoints to maintain the study's focus on objective clinical performance and safety. We have added a brief justification in the Methods (lines 154-157, page 4).

Comments 6: Adverse event reporting - the adverse event section is improved and now includes severity classification and causality assessment. Nevertheless, the manuscript should clarify whether adverse events were actively solicited at each visit and whether participants maintained symptom diaries.

Response 6: To ensure comprehensive safety monitoring, all participants were explicitly instructed at the beginning of the study to contact the research center immediately should any signs or symptoms arise during the study period. For this purpose, the investigational product label was designed to include not only the product information but also the research center’s contact telephone number, ensuring that participants had immediate access to the clinical team for any eventuality or to report potential adverse events.

Comments 7: Statistical reporting - most statistical reporting inconsistencies have been corrected. However, the authors should ensure that exact p-values are reported whenever possible and formatting follows journal guidelines consistently throughout all tables and figures.

Response 7: We appreciate the reviewer's attention to the precision of our statistical reporting. We have conducted a final, thorough review of all tables and figures to ensure full consistency with the journal's guidelines. Specifically, we have standardized all p-values to use decimal points instead of commas (e.g., p = 0.002 instead of p = 0,002) as previously suggested. Regarding the request for exact p-values, we have provided the precise calculated values for all results where p ≥ 0.001. For results showing high statistical significance where the software outputs p < 0.001, we have maintained this standard notation, as it represents the limit of precision for the statistical tests performed (Friedman and post-hoc analyses). We believe this follows the standard scientific convention for reporting highly significant results.

Comments 8: Figures and tables - figures and tables are generally clear and improved compared with the previous version. The correction of terminology such as "mean pH" and "mean variation" is appreciated. Consider adding confidence intervals and participant numbers directly within figure legends, to facilitate interpretation.

Response 8: As an extra measure of clarity and in response to point 8, the number of participants (n) has been explicitly included in all tables and figure captions to ensure transparency in the reported data.

Comments 9: We suggest that you discuss more about complementary therapies like Kegel exercises, Kegel exercises combined or not with estrogens. As for reference material, we would like to suggest a few interesting study titles: Șerbănescu, L.; Rotar, V.; Brezeanu, D.; Mirea, S.; Ionescu, E. V.; Ionescu, P. Evaluating the Efficacy of Combined Intravaginal Estriol Therapy and Kegel Exercises in Managing Menopausal Atrophic Vulvovaginitis. Clin Pract, 2025, 15(1), 20.  Șerbănescu, L.; Mirea, S.; Ionescu, P.; Petrica, L.A.; Iorga, I.C.; Surdu, M.; Surdu, T.V.; Rotar, V. Involuntary Urine Loss in Menopause—A Narrative Review.  Clin. Med.2025,14(21), 7664. https://doi.org/10.3390/jcm14217664

Response 9: We have now incorporated a paragraph in the Discussion section (lines 364-369, page 12) that discusses contemporary evidence regarding complementary non-hormonal strategies, specifically pelvic floor rehabilitation (Kegel exercises). We cited the suggested studies to highlight how these physical therapies contribute to improving the quality of life and symptomatic relief in women with GSM, complementing the hydration-focused benefits provided by hyaluronic acid moisturizers.

 

Reviewer 3 Report

Comments and Suggestions for Authors

I thank the authors for their careful revision of the manuscript and for providing detailed responses to the reviewers' comments. The authors have adequately addressed the concerns raised during the previous review round and have incorporated the requested revisions and clarifications.

Author Response

Comments 1: I thank the authors for their careful revision of the manuscript and for providing detailed responses to the reviewers' comments. The authors have adequately addressed the concerns raised during the previous review round and have incorporated the requested revisions and clarifications.

Response 1: We would like to thank the reviewer for their final recommendation for acceptance and for the constructive guidance provided during the previous rounds of review.