Review Reports - Effect of Inhalation of Hydrogen Gas on Postoperative Recovery After Hepatectomy: A Randomized, Double-Blind, Placebo-Controlled Trial

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Strengths of the Study:

Rigorous Design: The study used a randomised, double-anonymised, placebo-controlled design (the gold standard for clinical trials), minimising the risk of bias.

Plausible Mechanism: The rationale is based on the antioxidant and anti-inflammatory properties of hydrogen, relevant for mitigating hepatic ischemia-reperfusion injury and the perioperative stress response.

Patient-Centred Approach: The use of QoR-40 as the primary endpoint provides a comprehensive, patient-centred measure of recovery quality, beyond traditional clinical metrics.

Weaknesses and Limitations

Sample Size and Power: The sample size was moderate (n=64 analysed), and the primary endpoint analysis was performed at alpha = 0.2 with 80% confidence intervals. This significance threshold is much less stringent than the standard alpha = 0.05 (95% CI), increasing the risk of a false-positive result (type I error).

Exploratory subdomain analysis: The key finding of improved emotions and physical independence was based on exploratory subdomain analyses that were not adjusted for multiplicity, thus reducing the statistical robustness of these findings.

Oxidative stress measurement: The assessment of oxidative stress was limited to the measurement of urinary 8-OHdG in POD3. The authors suggest that this measurement was likely post-peak and did not capture the moment of maximum oxidative damage (which could occur within the first 24 hours).

Single-centre study: The study was conducted at a single centre, which may limit the external applicability (generalizability) of the results.

Questions for the Authors

Why was alpha = 0.2 (80% CI) used for the primary endpoint instead of the standard 0.05? Is this the final result or a preliminary signal that needs confirmation?

Impact of Type I Error: Given the weak significance threshold (alpha = 0.2) and the exploratory analysis of the subdomains, how do the authors interpret the likelihood of a Type I error (false positive) for the primary finding, and what precautions should be considered when interpreting clinical significance?

With no change in systemic oxidative stress markers, could you suggest future biomarkers or sampling strategies to support the hypothesis of local action?

Psychophysical link: The key finding was improvement in the emotional and physical independence domains. The authors suggest that this may be related to the attenuation of systemic inflammation and neuroinflammation, as well as improved microcirculatory function. Is there any direct evidence or a plan to measure parameters that link H2 to the central nervous system or autonomic function (e.g., through heart rate variability, as mentioned in the Discussion)?

Regarding clinical applicability:

Dose and Duration of Therapy: The inhalation protocol was 5% H2 for at least 1 hour, three times daily, from POD1 to POD7. Was the optimal concentration, daily duration, or starting point (e.g., beginning in the operating room instead of POD1) considered? Are greater benefits expected with a different protocol?
Long-term relevance: The beneficial effect disappeared by POD7 and at discharge. What is the clinical significance of an improvement in QoR-40 limited to POD3? Does this affect long-term hospital stay or readmission rate, even though hospital stay was not significantly different between groups?

Author Response

#1 Reviewer 1

We sincerely thank the reviewer for the positive and thoughtful evaluation of the strengths of our study. We appreciate the recognition of the rigorous randomized, double-blind, placebo-controlled design, the biologically plausible mechanism of hydrogen therapy, and the patient-centred assessment using QoR-40. These points reinforce the methodological validity and clinical relevance of our work.

We thank the reviewer for the comprehensive and constructive summary of the weaknesses and limitations of our study. We fully acknowledge all of the points raised and agree that they represent important methodological considerations. Accordingly, we have revised the manuscript to explicitly address each of these limitations in the Limitations section, as detailed below.

Sample Size and Statistical Power:

We acknowledge that the sample size was moderate (n = 64), which may limit statistical power. As clarified in the revised manuscript, this study was designed as an exploratory, pilot randomized trial aimed at signal detection rather than definitive hypothesis testing.

Exploratory Significance Threshold and Type I Error:

We recognize that the use of an exploratory alpha level (α = 0.2) is less stringent than conventional thresholds and may increase the risk of false-positive findings. This limitation is now explicitly stated, and the results are clearly framed as hypothesis-generating.

Exploratory Subdomain Analyses and Multiplicity:

We acknowledge that the QoR-40 subdomain analyses were exploratory and not adjusted for multiplicity. The associated risk of false positivity due to multiple comparisons has now been explicitly described in the revised Limitations section.

Oxidative Stress Assessment:

We agree that assessing oxidative stress using a single measurement of urinary 8-OHdG on POD3 may not capture peak perioperative oxidative injury. This methodological limitation and future directions for improved biomarker assessment are now clearly stated.

Single-Centre Design and Generalizability:

We acknowledge that the single-centre nature of this study may limit external applicability to other populations or healthcare settings. This limitation has been explicitly added to the manuscript.

Accordingly, we have revised the Limitations section of the manuscript to explicitly address these points, as described above.

Limitations

This study has several limitations. First, the sample size was moderate, reflecting the exploratory, pilot nature of this randomized trial, which may limit statistical power. The subdomain analyses were exploratory and not adjusted for multiplicity, and the primary analysis used an exploratory significance threshold (α = 0.2), which is less stringent than conventional thresholds and may increase the risk of type I error; therefore, the findings should be interpreted as hypothesis-generating rather than confirmatory. Second, because no prior evidence exists to define the optimal dose, duration, or timing of perioperative hydrogen administration, the inhalation protocol was determined primarily based on feasibility within routine postoperative care. Earlier initiation or prolonged exposure may yield greater therapeutic benefit and should be evaluated in future studies. Third, oxidative stress was assessed solely by urinary 8-OHdG measured on POD3. As oxidative stress after major surgery may fluctuate dynamically during the early postoperative phase, this single sampling point may not have captured peak perioperative changes. Future investigations should incorporate earlier or serial sampling and additional biomarkers, such as 4-HNE or F2-isoprostanes, for a more comprehensive assessment. Finally, this was a single-center study conducted in Japan, which may limit generalizability to other ethnic populations or healthcare settings. In addition, residual confounding and the possibility of response bias cannot be fully excluded, as QoR-40 is a patient-reported outcome.

From this point onward, the following comments correspond to the responses that were already provided in our previous revision.

Why was alpha = 0.2 (80% CI) used for the primary endpoint instead of the standard 0.05? Is this the final result or a preliminary signal that needs confirmation?
→
We appreciate the reviewer’s insightful question.

The use of an alpha level of 0.2 with an 80% confidence interval was determined a priori based on the exploratory, pilot nature of this randomized trial. Because this study aimed to evaluate the feasibility and detect a potential signal of benefit of perioperative hydrogen inhalation, a relaxed alpha level was selected to minimize the risk of Type II error in the context of a limited sample size. Such an approach is commonly adopted in early-phase clinical studies where avoiding false negatives is important.

Importantly, although the study was designed using this exploratory threshold, the primary endpoint (QoR-40 score on POD3) demonstrated statistical significance even under the conventional alpha level of 0.05. This reinforces the robustness of the observed effect beyond the pilot-study framework. We have clarified these points in the revised Methods and Discussion sections.

Therefore, we have added the following sentences.

Line 129-131 (Method): Given the exploratory design of this pilot study, an alpha level of 0.2 (80% CI) was prespecified to detect a potential signal of benefit while minimizing the risk of Type II error.

Line 216-218 (Discussion): Despite the exploratory alpha level of 0.2, the improvement in the QoR-40 score on POD3 still met the conventional significance threshold of p < 0.05, reducing concern about an inflated Type I error and supporting the robustness of this preliminary finding.

Impact of Type I Error: Given the weak significance threshold (alpha = 0.2) and the exploratory analysis of the subdomains, how do the authors interpret the likelihood of a Type I error (false positive) for the primary finding, and what precautions should be considered when interpreting clinical significance?

→

We thank the reviewer for raising this important point. We acknowledge that using an exploratory alpha level of 0.2 increases the possibility of a Type I error (false positive). This threshold was selected intentionally to avoid missing a potential signal of benefit in this pilot, early-phase study with a limited sample size. Therefore, the findings should be interpreted as preliminary and hypothesis-generating rather than confirmatory. However, it is noteworthy that the primary endpoint—the difference in total QoR-40 score on POD3—also reached statistical significance under the conventional alpha level of 0.05. This reduces concern regarding an inflated Type I error and suggests that the observed effect is unlikely to be solely due to chance. Nevertheless, clinical interpretation should remain cautious, and validation in larger, adequately powered trials is required before drawing definitive conclusions.

To address this point clearly, we have added the following clarification to the Discussion section.

With no change in systemic oxidative stress markers, could you suggest future biomarkers or sampling strategies to support the hypothesis of local action?

→

We sincerely appreciate the reviewer’s insightful comment. In this study, urinary 8-OHdG was measured only on POD3, corresponding to the timing of QoR-40 assessment. Because oxidative stress after major surgery may fluctuate dynamically during the early postoperative phase, a single sampling point may not have fully captured the relevant perioperative changes. Future investigations should incorporate earlier or serial measurements—particularly within the first postoperative 24 hours—to better characterize the temporal course of oxidative stress. Furthermore, while 8-OHdG is a representative marker of oxidative DNA damage, additional biomarkers such as 4-HNE or F2-isoprostanes may complement its assessment and allow a more comprehensive evaluation of hydrogen’s potential biological effects. Such multimodal assessment may also help distinguish systemic responses from possible liver-localized effects. 　

Accordingly, we have revised the Limitations section to clarify these points.

Line 272-277 (Limitation): The evaluation of oxidative stress relied solely on urinary 8-OHdG measured on POD3. Because oxidative stress after major surgery may fluctuate dynamically in the early postoperative phase, the single sampling point may not have captured the most relevant perioperative changes. Future studies should incorporate earlier or serial sampling and consider additional biomarkers such as 4-HNE or F2-isoprostanes to achieve a more comprehensive assessment.

Psychophysical link: The key finding was improvement in the emotional and physical independence domains. The authors suggest that this may be related to the attenuation of systemic inflammation and neuroinflammation, as well as improved microcirculatory function. Is there any direct evidence or a plan to measure parameters that link H2 to the central nervous system or autonomic function (e.g., through heart rate variability, as mentioned in the Discussion)?

→

We thank the reviewer for this important question. In this study, heart rate variability (HRV) was measured as an exploratory indicator of autonomic function; however, no significant differences were observed between the hydrogen and control groups. HRV is highly sensitive to postoperative physiological factors—such as fluid balance, hemodynamic fluctuations, pain, and medications—and this variability makes it challenging to detect stable between-group differences in the early postoperative period. A larger sample size or more standardized measurement conditions may therefore be required to evaluate autonomic effects with sufficient precision.

While the improvements in the emotional and physical independence domains raise the possibility of a psychophysiological impact of hydrogen, the present findings do not provide direct evidence of autonomic or central nervous system modulation. Future studies employing more frequent or continuous HRV monitoring, together with complementary neurophysiological or autonomic markers, will be essential to clarify these potential mechanisms.

To address this point, we have added a clarifying sentence to the Discussion section.

Line 237-245 (Discussion): In the present study, HRV was assessed as an exploratory autonomic marker; however, no significant between-group differences were observed. Because HRV is highly sensitive to postoperative influences such as fluid balance, hemodynamic fluctuations, pain, and medications, detecting stable differences in the early postoperative period may require a larger sample size or more standardized monitoring conditions. Thus, while improvements in emotional and physical independence domains raise the possibility of psychophysiological effects, the current findings do not provide direct evidence of autonomic modulation by hydrogen. Future studies incorporating more intensive HRV monitoring and complementary autonomic biomarkers will be needed to clarify these mechanisms.

Dose and Duration of Therapy: The inhalation protocol was 5% H2 for at least 1 hour, three times daily, from POD1 to POD7. Was the optimal concentration, daily duration, or starting point (e.g., beginning in the operating room instead of POD1) considered? Are greater benefits expected with a different protocol?

→

We thank the reviewer for this important question. Because this study represents an early-phase pilot investigation conducted in an area with no prior evidence regarding the optimal hydrogen dose, concentration, or timing, the inhalation protocol was determined primarily based on feasibility in the postoperative clinical environment. After major hepatectomy, patients undergo scheduled rehabilitation, meals, and routine nursing care throughout the day; therefore, we selected a regimen that could be implemented consistently without disrupting standard postoperative management. In this sense, the protocol reflects a clinically practical exposure schedule in the absence of any established evidence defining the optimal regimen.

Hydrogen inhalation could begin only on POD1 because many patients remain intubated and on mechanical ventilation immediately after surgery, during which hydrogen administration is not technically feasible with currently available devices. It is possible that earlier initiation—such as intraoperatively or immediately post-extubation—or longer daily exposure might yield greater therapeutic benefit. These possibilities should be evaluated in future studies designed specifically to optimize dosing strategies.

Accordingly, we have revised the Limitations section to clarify these points.
Line 267-271 (Limitation): Because no prior evidence exists to define the optimal dose, duration, or timing of perioperative hydrogen administration, the inhalation schedule used in this study was determined primarily based on feasibility within routine postoperative care. Earlier initiation or prolonged exposure may potentially enhance therapeutic effects and should be evaluated in future investigations.

Long-term relevance: The beneficial effect disappeared by POD7 and at discharge. What is the clinical significance of an improvement in QoR-40 limited to POD3? Does this affect long-term hospital stay or readmission rate, even though hospital stay was not significantly different between groups?

→

We appreciate the reviewer’s thoughtful question. It is correct that the between-group difference diminished by POD7 and at discharge. However, POD3 corresponds to the peak period of postoperative discomfort, functional limitation, and stress response after hepatectomy. Therefore, an improvement in QoR-40 at this early critical window is clinically meaningful, as it may reflect accelerated psychophysical recovery. Importantly, QoR-40 scores naturally approach an upper limit as patients recover, and by POD7 most individuals have returned close to their baseline functional status. This ceiling effect reduces the ability to detect between-group differences at later time points. Thus, the disappearance of statistical differences by POD7 likely reflects earlier recovery in the hydrogen group rather than the absence of a biological effect.

Although no significant difference was observed in hospital stay, these outcomes are strongly influenced by standardized clinical pathways and institutional discharge criteria, which may mask modest improvements in early well-being. Hydrogen-related enhancement of early recovery may still contribute to improved patient experience, even if not directly reflected in length-of-stay metrics.

We have added a statement to the Discussion to clarify this point.

Line 211-215 (Discussion): Although the between-group difference did not persist to POD7 or discharge, this may reflect accelerated early recovery rather than the absence of a biological effect. POD3 represents the period of greatest postoperative discomfort, and improvement at this time point is clinically meaningful. By POD7, most patients approach their preoperative functional status, producing a ceiling effect that narrows detectable differences.

Reviewer 2 Report

Comments and Suggestions for Authors

There are a few comments.

It would be helpful to clearly specify the diagnostic categories included under “Others.”
Please provide a detailed breakdown or definition of this group.

A comparison of the pathological characteristics and clinical stages of HCC and ICC between the hydrogen and placebo cohorts would strengthen the study. Including this analysis may enhance the interpretability of the clinical outcomes.

Please ensure that the first word in each table entry is capitalized.
For example, in Table 3, “parameters” should be revised to “Parameters.”

Author Response

#1 Reviewer 2
1. It would be helpful to clearly specify the diagnostic categories included under “Others.”

Please provide a detailed breakdown or definition of this group.
→

We appreciate the reviewer’s helpful comment. We have now clarified the diagnostic categories included under “Others” in Table 1. Specifically, “Others” comprises benign hepatobiliary diseases, including intrahepatic lithiasis (n = 2), hepatic hemangioma (n = 3), and multiple hepatic cysts (n = 2).

This information has been added to the table 1 legend for improved clarity: Others includes benign hepatobiliary diseases such as intrahepatic lithiasis (n = 2), hepatic hemangioma (n = 3), and multiple hepatic cysts (n = 2).

A comparison of the pathological characteristics and clinical stages of HCC and ICC between the hydrogen and placebo cohorts would strengthen the study. Including this analysis may enhance the interpretability of the clinical outcomes.

→

We sincerely appreciate the reviewer’s valuable suggestion. This pilot study enrolled a heterogeneous population that included not only HCC and ICC but also colorectal liver metastases and several benign hepatobiliary conditions, making it difficult to apply a uniform staging system across all participants. Nonetheless, in response to the reviewer’s concern, we reviewed the disease stage in patients with malignant tumors only. This assessment showed no appreciable imbalance in stage distribution between the hydrogen and placebo groups, suggesting broadly comparable oncologic backgrounds among these patients.

To increase clarity for readers, we have added the following note to the Table 1 legend: Among patients with malignant tumors, no meaningful imbalance in disease stage was observed between groups.

Please ensure that the first word in each table entry is capitalized.

For example, in Table 3, “parameters” should be revised to “Parameters.”

→

Thank you for this helpful comment. We carefully reviewed all table entries and ensured that each item begins with a capital letter. Minor inconsistencies were corrected accordingly.

Reviewer 3 Report

Comments and Suggestions for Authors

Review Report for Manuscript ID hydrogen-4019180

Journal Hydrogen (ISSN 2673-4141)

Title: Effect of Inhalation of Hydrogen Gas on Postoperative Recovery After Hepatectomy: A Randomized, Double-Blind, Placebo-Controlled Trial

The paper presents the original results from a randomized, double-blinded placebo-controlled trial among patients after hepatectomy which compares the effects of inhaled hydrogen in comparison to placebo on the postoperative recovery. The manuscript is well-written, organized and structured in accordance to the scientific form of writing papers. Some points should be addressed.

Comments:

The Introduction part is too short and does not convince the reader about the importance of considering molecular hydrogen gas in medical treatment. There is lack of rationale behind the idea of hydrogen as reduction agent and practically insoluble compound (very limited aqueous solubility) in medical treatment. So the Introduction should present the rationale behind the idea as well as possible plausible mechanisms of actions of molecular hydrogen in the pathophysiological pathways of patients with damaged liver that undergo hepatectomy.
Although he inclusion and exclusion criteria are already reported there is necessity of summarizing the most important points. For example the smoking status, the use of some medicines, the alcohol consumption etc. that may interfere with the obtained outcomes should be emphasized. Clear inclusion and exclusion criteria need to added in section 2.1 with the emphasize on medications, smoking status and alcohol consumption which per se may affect the reported endpoints.
In Table 1 smoking status and consumption of alcohol if available should also be indicated. BMI if available should also be added.
There should be explanation in the Discussion why there were significant changes in QoR on day three and not on day 7 or at the discharge.
In the limitations it should be emphasized that the obtained results can not be extrapolated easily to different ethnicity or even different geographical area. Due to limited number of patients there is possibility for false positivity” present in the “multiplicity”of significance tests carried out on the same sample, since each comparison is not carried out “with all other variables being equal”, due to the failure to neutralize all known and unknown confounding factors. As in every survey, bias is possible, as is the underreporting of undesirable outcome or overreporting of desirable behaviour.

Author Response

#1 Reviewer 3
1. The Introduction part is too short and does not convince the reader about the importance of considering molecular hydrogen gas in medical treatment. There is lack of rationale behind the idea of hydrogen as reduction agent and practically insoluble compound (very limited aqueous solubility) in medical treatment. So the Introduction should present the rationale behind the idea as well as possible plausible mechanisms of actions of molecular hydrogen in the pathophysiological pathways of patients with damaged liver that undergo hepatectomy.

→

We sincerely appreciate the reviewer’s valuable feedback. We agree that the original Introduction required a more detailed rationale for the clinical relevance of molecular hydrogen and its potential mechanisms of action in patients undergoing hepatectomy. In response, we have substantially expanded the Introduction to better explain:

(1) the role of reactive oxygen species (ROS) in hepatic ischemia–reperfusion injury,

(2) experimental and translational evidence supporting hydrogen's selective antioxidant and cytoprotective effects, and

(3) the clinical need for interventions that improve early postoperative recovery.

These additions provide a clearer scientific foundation for evaluating hydrogen inhalation as a perioperative adjunct therapy.

The revised text has been inserted into the Introduction as shown below, with all changes marked in red.

Line 49-63, 69-71:

Hepatectomy inevitably induces oxidative stress due to hepatic ischemia–reperfusion (I/R) injury, which contributes to postoperative inflammation, hepatocellular damage, and de-layed functional recovery [1–3]. Excess reactive oxygen species (ROS) generated during I/R play a central role in this process. Molecular hydrogen has attracted growing interest as a potential medical gas because it selectively scavenges the highly cytotoxic hydroxyl radical and peroxynitrite, thereby attenuating oxidative stress at the molecular level [4]. Beyond its antioxidant properties, hydrogen has been shown to modulate inflammatory cytokine production, stabilize mitochondrial function, and improve microvascular perfusion in various organ systems. These cytoprotective mechanisms are supported by extensive preclinical work demonstrating that inhaled hydrogen reduces infarct size, mitigates I/R injury, and preserves organ function in rodent and porcine models [4–6]. Furthermore, early-phase human studies in post–cardiac arrest syndrome have shown that hydrogen inhalation is feasible, safe, and capable of reducing biochemical markers of oxidative stress [7,8]. Together, these findings indicate that hydrogen—despite its limited aqueous solubility—rapidly diffuses into tissues and exerts biologically meaningful antioxidant and anti-inflammatory effects.

These mechanisms provide a biologically plausible rationale for investigating hydrogen therapy in hepatectomy, where oxidative stress and transient hepatocellular injury are unavoidable. Although hydrogen has limited aqueous solubility, previous studies have confirmed that inhaled hydrogen rapidly diffuses into tissues and exerts organ-protective effects in both animal models and early human studies [4–8].

Despite improvements in perioperative care, many patients—particularly older indi-viduals—experience substantial fatigue and delayed recovery following liver resection. Previous studies have shown that postoperative quality of recovery (QoR) often remains impaired during the early postoperative period, highlighting the need for interventions that enhance patient-centered recovery trajectories [9–12].

Given the mechanistic promise of hydrogen and the ongoing clinical need to enhance early recovery, hydrogen inhalation represents a rational and potentially feasible perioperative intervention [4–8]. We therefore conducted a randomized, double-blind, placebo-controlled trial to evaluate whether perioperative hydrogen inhalation initiated in the immediate postoperative period could attenuate excessive oxidative stress and improve early postoperative recovery. The primary endpoint was the total Quality of Recovery score on postoperative day 3 (POD3) using the validated QoR-40 instrument [13–16]. Secondary endpoints included safety and postoperative clinical outcomes, and exploratory analyses assessed oxidative stress using urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) and related biomarkers.

Although he inclusion and exclusion criteria are already reported there is necessity of summarizing the most important points. For example the smoking status, the use of some medicines, the alcohol consumption etc. that may interfere with the obtained outcomes should be emphasized. Clear inclusion and exclusion criteria need to added in section 2.1 with the emphasize on medications, smoking status and alcohol consumption which per se may affect the reported endpoints.
3. In Table 1 smoking status and consumption of alcohol if available should also be indicated. BMI if available should also be added.

→

We sincerely appreciate the reviewer’s valuable comment. We agree that smoking status, alcohol consumption, and BMI may influence postoperative recovery and should therefore be clearly reported. In response, these variables have been added to Table 1 to provide a more complete description of baseline characteristics. In addition, a clarifying statement has been added to Section 2.1 to explain how lifestyle and medication-related factors were considered during eligibility assessment.

Line 86-89: Smoking history, alcohol consumption, and BMI were recorded as part of the baseline characteristics because these factors may influence postoperative recovery; however, they were not applied as exclusion criteria unless associated with severe organ dysfunction or unstable medical conditions.

There should be explanation in the Discussion why there were significant changes in QoR on day three and not on day 7 or at the discharge.

→

We appreciate the reviewer’s insightful comment. The temporal pattern of QoR-40 changes—significant improvement on POD3 but not on POD7 or at discharge—is indeed an important point. The physiological rationale for the POD3 difference has already been discussed in our original manuscript, where we noted that hydrogen inhalation may preferentially preserve psychophysical components of early recovery, such as emotional stability and functional independence, during the period of greatest postoperative stress. To address the reviewer’s concern more explicitly, we have additionally clarified in the Discussion why the between-group difference did not persist beyond POD3. Specifically, by POD7 most patients regain substantial functional capacity, resulting in a ceiling effect that narrows detectable differences despite early improvements.

The following text has been added to the Discussion section:
Line 211-215: Although the between-group difference did not persist to POD7 or discharge, this may reflect accelerated early recovery rather than the absence of a biological effect. POD3 represents the period of greatest postoperative discomfort, and improvement at this time point is clinically meaningful. By POD7, most patients approach their preoperative functional status, producing a ceiling effect that narrows detectable differences.

In the limitations it should be emphasized that the obtained results can not be extrapolated easily to different ethnicity or even different geographical area. Due to limited number of patients there is possibility for false positivity” present in the “multiplicity”of significance tests carried out on the same sample, since each comparison is not carried out “with all other variables being equal”, due to the failure to neutralize all known and unknown confounding factors. As in every survey, bias is possible, as is the underreporting of undesirable outcome or overreporting of desirable behaviour.

→

We sincerely appreciate this important comment. We fully agree that the generalizability of our findings is limited. Because this pilot randomized trial was conducted at a single center in Japan, the results should not be extrapolated directly to different ethnic populations or geographical regions. Additionally, the modest sample size inherently increases the risk of false-positive findings, particularly in the presence of multiple comparisons and residual confounding from unmeasured variables that cannot be fully controlled in an early-phase study. Furthermore, as the QoR-40 is a patient-reported outcome measure, the possibility of response bias—including underreporting of undesirable symptoms or overreporting of well-being—cannot be completely excluded.

To address these points, we have expanded the Limitations section to explicitly acknowledge these issues

Line 278-284: Because this pilot trial was conducted at a single center in Japan, the findings may not be readily generalizable to other ethnic populations or geographical regions. The modest sample size also increases the risk of false-positive findings, especially given the multiplicity of comparisons and the inability to fully adjust for all known and unknown confounding factors. Furthermore, because QoR-40 is a patient-reported outcome, the possibility of response bias—such as underreporting of undesirable symptoms or overreporting of perceived well-being—cannot be entirely excluded.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has been well revised.

Author Response

We sincerely thank the reviewer for the positive evaluation and for acknowledging that the manuscript has been well revised. We greatly appreciate the reviewer’s time and constructive feedback, which have helped us improve the clarity and quality of our work.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have made the changes that I have required in the previous report. I have no further comments.

Author Response