Effect of Melatonin and Epigallocatechin-3-Gallate Combination on In Vitro Maturation of Mouse Oocytes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article addresses a pressing issue in reproductive biology and assisted reproductive technologies: improving oocyte quality in vitro. Oxidative stress is a known limiting factor that reduces the effectiveness of IVM. Although individual antioxidants (melatonin and EGCG) have been studied, their combined synergistic effects have been understudied. The novelty of this study lies in its shift from studying individual antioxidants to systematically testing and substantiating the effectiveness of their combination in vitro in a mouse model.
The authors evaluate not only the final outcome (blastocyst yield) but also key intermediate parameters: nuclear maturation (polar body extrusion), cytoplasmic maturation (cumulus expansion), and biochemical markers of oxidative stress (ROS and GSH levels). The results obtained have direct translational potential and can serve as a basis for optimizing IVM protocols in clinical and laboratory practice.
The article is written in clear and rigorous scientific language, adhering to all necessary standards. The material is presented logically and consistently, making it accessible to both specialists in reproductive biology and a broader range of researchers in related fields. The reference list includes 40 sources, 17 of which were published within the last five years.
Therefore, the article is of significant scientific interest and can be recommended for publication after the comments below are addressed.

Note:

• In section 2.6, on lines 155-156, the authors write, "A standard curve was constructed using 2, 5, 10, 15, and 25 µM concentrations of 155
  GSH solutions in accordance with the instructions provided in the kit," but the description of this experiment does not indicate which kit was used.
• In section 2.7, it is necessary to clarify what is meant by "Each experiment was repeated at least three times." The authors should clearly indicate whether biological replicates (independent experiments with new batches of animals/oocytes on different days) or technical replicates were used.
• The authors should clarify the statistical analysis by stating that the data distribution was tested for normality, defining the significance threshold as p < 0.05, specifying the form of data presentation (e.g., mean ± SD), and optionally noting the version of the SPSS software used.
• The scale bar value is not indicated in the caption to Figure 3.
• The caption to Table 3 is missing the note "Different letters (a, b, c) in the same column represent significant differences."

Author Response

Comments of Reviewer 1

Point 1: In section 2.6, on lines 155-156, the authors write, "A standard curve was constructed using 2, 5, 10, 15, and 25 µM concentrations of 155

GSH solutions in accordance with the instructions provided in the kit," but the description of this experiment does not indicate which kit was used.

Response 1: Thank you for this valuable comment. We have revised Section 4.6 to clearly specify the kit used for GSH measurement. The text now states that the standard curve was generated using the GSH Assay Kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions.

 

 

Point 2: In section 2.7, it is necessary to clarify what is meant by "Each experiment was repeated at least three times." The authors should clearly indicate whether biological replicates (independent experiments with new batches of animals/oocytes on different days) or technical replicates were used.

Response 2: Thank you for pointing this out. As requested, we have clarified in the revised Statistical Analysis section (lines 306-307) that all experiments were repeated at least three times using newly collected oocytes from different batches of mice, representing independent biological replicates.

 

Point 3: The authors should clarify the statistical analysis by stating that the data distribution was tested for normality, defining the significance threshold as p < 0.05, specifying the form of data presentation (e.g., mean ± SD), and optionally noting the version of the SPSS software used.

Response 3: Thank you for your constructive suggestion.  The Statistical Analysis section (lines 307-310) has been revised to state that data distribution was tested for normality, results are presented as mean ± SD, and analysis was performed using SPSS 22.0 software with a significance threshold of p < 0.05. 

 

Point 4: The scale bar value is not indicated in the caption to Figure 3.

Response 4: We thank the reviewer for pointing this out. The scale bar value (100 µm) has now been clearly indicated in the caption for Figure 3a in the revised manuscript.

 

Point 5: The caption to Table 3 is missing the note "Different letters (a, b, c) in the same column represent significant differences."

Response 5: We thank the reviewer for this helpful suggestion. As suggested, the note "Different letters (a, b, c) in the same column represent significant differences (p < 0.05)." has been added to the note of Table 3 in the revised manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper evaluated the effect of melatonin and epigallocatechin-3-gallate (EGCG) combination during IVM on oocyte quality and their subsequent developmental potential by using a mouse model. The topic is interesting; however, the manuscript requires a substantial revision.

- The title should be corrected and changed to be without any abbreviations.

- Introduction: a) The authors should emphasize the importance and the novel aspects of this work. b) Be more precise in the information and references provided.

- Animals and oocyte collection: a) Please specify the total number of mice used in the study. b) Was it not necessary to assess the oestrus cycle prior to deploying the animals for experimentation? Could the phase of the estrous cycle affect the number of oocytes available?

- Experimental design: a) Please specify the number of oocytes per experimental group. b) Why did you evaluate the NRF2 inhibitor brusatol? Justify in the introduction or discussion. c) How were the concentrations of melatonin and EGCG chosen?

- Statistical analysis: Did the authors check the data for normal distribution?

- Results: a) The authors should also check the content as there are methods on the results section. b) “Conjectures/Suggestions” could be separate from the results part; please describe only results.

- In results text, the data description and statistical comparisons must be accurate. Revise the text for better organization and clarity.

- There are no statistical indications in figure legends 2 and 3 and Table 3 (meaning of asterisk and letters).

- The Discussion section is very concise than usual. Most of the discussion is mentioning some results and citing some other studies. The physiological concept integrating the obtained functional interrelationships between the measured parameters is desirable. Moreover, it is suggested to add some description of the practical significance of the findings to the discussion.

- Abbreviations should be defined at first mention and used consistently thereafter.

Author Response

Point 1: The title should be corrected and changed to be without any abbreviations.

Response 1: We thank the reviewer for this correction. The title has been revised to remove all abbreviations and is now presented in full to improve clarity and readability.

 

Point 2: Introduction: a) The authors should emphasize the importance and the novel aspects of this work. b) Be more precise in the information and references provided.

Response 2: Thank you for your constructive suggestion. The Introduction has been carefully revised (Lines 31–96) to better emphasize the importance and novelty of the present study. In addition, the information has been made more precise and the references have been revised and appropriately cited to support the updated statements.

 

Point 3: Animals and oocyte collection: a) Please specify the total number of mice used in the study. b) Was it not necessary to assess the oestrus cycle prior to deploying the animals for experimentation? Could the phase of the estrous cycle affect the number of oocytes available?

Response 3: Thank you for your constructive suggestion.(a) Female mice were used for oocyte collection, and oocytes obtained from superovulated mice were pooled across independent experimental replicates. Therefore, experimental outcomes were analyzed and reported based on the number of oocytes or embryos, as indicated in the Results section.

(b) This point has been clarified in the revised Methods section (Lines 232–235), where superovulation with pregnant mare serum gonadotropin is described as a means to minimize variability associated with the estrous cycle.

Point 4: Experimental design: a) Please specify the number of oocytes per experimental group. b) Why did you evaluate the NRF2 inhibitor brusatol? Justify in the introduction or discussion. c) How were the concentrations of melatonin and EGCG chosen?

Response 4: Thank you for bringing this to our attention. (a) The number of oocytes used in each experimental group has now been clearly specified in the Results section.

(b) Brusatol was included as a separate treatment during in vitro maturation to evaluate the role of endogenous NRF2 signaling in regulating oxidative stress and oocyte maturation. This rationale has now been clarified in the Introduction (Lines 91-94), where we explain that brusatol was used as a pharmacological inhibitor of NRF2 to explore the involvement of this pathway in oocyte maturation under IVM conditions.

(c)The concentrations of melatonin and EGCG used in this study were selected based on preliminary experiments conducted prior to the formal study, in which different concentrations were evaluated to identify relatively optimal doses for supporting oocyte in vitro maturation. This clarification has now been added to the Methods section (Lines 242–243).

 

Point 5: Statistical analysis: Did the authors check the data for normal distribution?

Response 5: Thank you for your constructive suggestion. The data were tested for normal distribution prior to statistical analysis and this information has been explicitly added in Lines 307–310.

 

Point 6:  Results: a) The authors should also check the content as there are methods on the results section. b) “Conjectures/Suggestions” could be separate from the results part; please describe only results.

Response 6: Thank you for this helpful comment. We have carefully revised the Results section to ensure that it contains only experimental results. Methodological descriptions and speculative statements have been removed from the Results and relocated to the Materials and Methods or Discussion sections as appropriate. 

 

Point 7: In results text, the data description and statistical comparisons must be accurate. Revise the text for better organization and clarity.

Response 7: Thank you for your comment. We thank the reviewer for the suggestion. The Results section has been revised for clarity, and all data descriptions and statistical comparisons have been carefully checked and accurately presented.

 

Point 8: There are no statistical indications in figure legends 2 and 3 and Table 3 (meaning of asterisk and letters).

Response 8: Thank you for pointing this out. The statistical indications have now been added to the legends of Figures 2 and 3 and Table 3, including explanations of the meaning of asterisks and different letters.

 

Point 9: The Discussion section is very concise than usual. Most of the discussion is mentioning some results and citing some other studies. The physiological concept integrating the obtained functional interrelationships between the measured parameters is desirable. Moreover, it is suggested to add some description of the practical significance of the findings to the discussion.

Response 9: Thank you for your comment. The Discussion section has been substantially revised to integrate the functional relationships among redox regulation, oocyte maturation, and embryonic development, and to highlight the practical implications of a combined antioxidant strategy for optimizing in vitro maturation systems.

 

Point 10: Abbreviations should be defined at first mention and used consistently thereafter.

Response 10: Thank you for this helpful comment. All abbreviations have now been defined at their first occurrence in the manuscript and are used consistently throughout the text.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors responded satisfactorily to the comments and made changes that improved the manuscript. This reviewer has no further comment.