Kisspeptin improves local ovarian insulin resistance in PCOS by modulating the PI3K/AKT/GLUT4 signaling pathway

Na Sun; Pingping Sun; Lilan Sun; Ping Sun; Dandan Qi; Huagang Ma

doi:10.1371/journal.pone.0342158

Peer Review History

Original SubmissionJune 17, 2025
20 Jul 2025 Decision Letter - Sheng Wu, Editor Dear Dr. Ma, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== Please address all comments listed by two reviewers. ============================== Please submit your revised manuscript by Sep 03 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Sheng Wu, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. 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PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition). For example, authors should submit the following data: - The values behind the means, standard deviations and other measures reported; - The values used to build graphs; - The points extracted from images for analysis. Authors do not need to submit their entire data set if only a portion of the data was used in the reported study. If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access. 7. Please update your submission to use the PLOS LaTeX template. The template and more information on our requirements for LaTeX submissions can be found at http://journals.plos.org/plosone/s/latex. 8. Thank you for stating the following financial disclosure: Weifang Health Commission (Grand number: WFWSJK-2023-005) Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 9. We notice that your supplementary [tables] are included in the manuscript file. Please remove them and upload them with the file type 'Supporting Information'. Please ensure that each Supporting Information file has a legend listed in the manuscript after the references list. 10. Please include a copy of Table 2 which you refer to in your text on page 7. 11. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: No Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: The paper investigates the interaction between kisspeptin and insulin sensitivity in the ovaries, focusing on a PCOS mouse model. The study presents the concept and demonstrates that kisspeptin expression is altered in the ovaries of PCOS mice. Furthermore, external supplementation with kisspeptin enhanced GLUT4 expression, leading to improved glucose metabolism within the ovary. However the paper can be improved by doing the following: 1. Introduction: Please elaborate on how insulin sensitivity is maintained or enhanced in the ovaries despite systemic insulin resistance in PCOS. Discuss the key insulin signaling pathways involved (e.g., PI3K-Akt, MAPK) and their differential regulation in ovarian versus peripheral tissues. Additionally, the study reports downregulation of the Insulin- PI3K-Akt pathway in granulosa cells. However, some literature (e.g., PMID: 27060006) suggests insulin sensitivity in ovary under hyperandrogenic conditions with activation of insulin pathways. Please include this in the introduction. 2. Replace the term “kisspeptin antagonist” with “KISS1 receptor antagonist” or “KISS1R antagonist” throughout the manuscript for scientific accuracy. 3. In the Methods section, provide complete details of the in vivo model: o Duration of DHEA treatment for follicle collection. References supporting the DHEA model. What was the rationale for using the DHEA model vs DHT? o Time points for testosterone measurement following DHEA treatment. o Was insulin supplemented to the culture media for the primary granulosa cells. Were experiments done with and without above. o Please specify in methods and legends if kisspeptin and kisspeptin antagonist was added to PCOS-IR cells ? o Catalog numbers for all reagents and kits used (e.g., testosterone and insulin kits, PMSG, goat serum) 4. In Figure 1A: o Label the antral follicles and cysts clearly. o Add data on the body weight of mice at or near the time of sacrifice. 5. In Figure 2, clarify whether the study rationale is based on local kisspeptin production in the ovary or on circulating levels. Since kisspeptin was added to the culture media, this likely simulates serum levels-please clarify in discussion. If Kisspeptin Receptor expression was measured by any means, please include it in the paper. 6. For Figure 3, specify the number of biological and technical replicates used. Include this information in the Methods section and figure legend. Also, though mentioned in the legend, it may be helpful to call the control group PCOS-IR, since Control is vehicle+ HFD group in figure 2. Add Data from Control-IR animals in supplementary if available, to see the independent effect of kisspeptin. 7. (Same as point 7) Ensure that replicate numbers are clearly indicated for all experiments in Figure 4. 8. In the Discussion, please comment on the observation that the rise in testosterone in the model group compared to controls is relatively modest, yet the difference in glucose tolerance test (GTT) results and HOMA-IR is quite significant. This may suggest aggravated insulin resistance in the presence of slightly increased testosterone. Also would be interesting to add some details on if insulin resistance preceeds morphological changes in the ovary. Reviewer #2: The authors conclude that there is decreased kisspeptin production and that treating with kisspeptin improves insulin sensitivity. Did the authors measure serum kisspeptin levels? Ideally, serum kisspeptin levels would be reported at baseline and after HCG treatment for ovarian induction. Levels were measured after 24h of primary culture, so it is possible that expression could have changed during culture or in response to HCG stimulation. The conclusions do to make it clear that all granulosa cell studies evaluating insulin resistance are in vitro studies. This needs to be clarified in the discussion and conclusions, particularly in the abstract. "Kisspeptin improves insulin resistance" in vitro in granulosa cells... Page 18: misspelled "Previous" ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: reviewer comments ffinal.docx https://doi.org/10.1371/journal.pone.0342158.r001
Revision 1
23 Sep 2025 Author Response 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Answer: Thank you for your valuable suggestion. We will strictly follow the file naming convention you pointed out to name the files. Please review the files we have submitted. 2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. Answer: We sincerely thank you for your valuable suggestion. As per your guidance, we have provided the original images for all Western blots. Please refer to the newly submitted files. 3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Answer: Thank you for your reminder. In accordance with your guidance, we have registered and verified our ORCID iD. 4. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on methods of anesthesia and/or analgesia. Answer: Thank you very much for your valuable suggestion. We have incorporated your instructions and added the method of euthanasia for mice in the Methods section of the manuscript. Please review the latest version we have provided. 5.Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section. Answer: Thank you for your reminder. We have carefully checked the manuscript and ensured that the ethical statement is only presented in the Methods section. Any repeated ethical statements outside the Methods section have been removed accordingly. 6.We note that your Data Availability Statement is currently as follows: If the data are all contained within the manuscript and/or Supporting Information files, enter the following: All relevant data are within the manuscript and its Supporting Information files. Answer: Thank you for your reminder. We have made the revisions as per your instructions. Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition). For example, authors should submit the following data: - The values behind the means, standard deviations and other measures reported; - The values used to build graphs; - The points extracted from images for analysis. Authors do not need to submit their entire data set if only a portion of the data was used in the reported study. If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access. Answer: Thank you for your reminder. Please refer to the latest supplementary information file we have provided. 7. Please update your submission to use the PLOS LaTeX template. The template and more information on our requirements for LaTeX submissions can be found at http://journals.plos.org/plosone/s/latex. Answer: Thank you for your suggestion. We have submitted all the files as per your instructions. 8. Thank you for stating the following financial disclosure: Weifang Health Commission (Grand number: WFWSJK-2023-005) Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. Answer: We sincerely thank you once again for your valuable suggestion. In accordance with your guidance, we have incorporated the supplementary explanation into the cover letter. Kindly review our latest revised manuscript. 9.We notice that your supplementary [tables] are included in the manuscript file. Please remove them and upload them with the file type 'Supporting Information'. Please ensure that each Supporting Information file has a legend listed in the manuscript after the references list. Answer: We sincerely thank you once again for your valuable suggestion. In accordance with your guidance, the relevant content has been removed from the manuscript. 10. Please include a copy of Table 2 which you refer to in your text on page 7. Answer: We sincerely thank you once again for your valuable suggestion. In accordance with your guidance, we have carefully reviewed the manuscript and corrected the content as Supplementary Table 2. 11. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. Answer: We sincerely thank you once again for your suggestion. We will review the references in accordance with your guidance. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Partly 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No Reviewer #2: Yes 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The paper investigates the interaction between kisspeptin and insulin sensitivity in the ovaries, focusing on a PCOS mouse model. The study presents the concept and demonstrates that kisspeptin expression is altered in the ovaries of PCOS mice. Furthermore, external supplementation with kisspeptin enhanced GLUT4 expression, leading to improved glucose metabolism within the ovary. However the paper can be improved by doing the following: 1. Introduction: Please elaborate on how insulin sensitivity is maintained or enhanced in the ovaries despite systemic insulin resistance in PCOS. Discuss the key insulin signaling pathways involved (e.g., PI3K-Akt, MAPK) and their differential regulation in ovarian versus peripheral tissues. Answer: We sincerely thank you for your valuable suggestion. In accordance with your guidance, we have added the relevant information to the Introduction section of the manuscript. We kindly invite you to review the latest version of the manuscript. Additionally, the study reports downregulation of the Insulin- PI3K-Akt pathway in granulosa cells. However, some literature (e.g., PMID: 27060006) suggests insulin sensitivity in ovary under hyperandrogenic conditions with activation of insulin pathways. Please include this in the introduction. Answer: Thank you for your reminder. In accordance with your guidance, we have added the relevant references to the Introduction section of the manuscript. We kindly invite you to review the latest version of the manuscript. 2. Replace the term “kisspeptin antagonist” with “KISS1 receptor antagonist” or “KISS1R antagonist” throughout the manuscript for scientific accuracy. Answer: We sincerely thank you for your valuable suggestion. In accordance with your guidance, we have made the necessary corrections in the manuscript. We kindly invite you to review the latest version. 3. In the Methods section, provide complete details of the in vivo model: o Duration of DHEA treatment for follicle collection. References supporting the DHEA model. What was the rationale for using the DHEA model vs DHT? Answer: We sincerely thank you for your valuable suggestion. In accordance with your guidance, we have added the relevant references regarding model establishment in the manuscript. We kindly invite you to review the latest version. o Time points for testosterone measurement following DHEA treatment. Answer: We sincerely thank you for your valuable suggestion. In accordance with your guidance, we have added clarification regarding the measurement time points in the manuscript. o Was insulin supplemented to the culture media for the primary granulosa cells. Were experiments done with and without above. Answer: We sincerely thank you for your valuable suggestion. In accordance with your guidance, we have corrected and clarified the culture medium for granulosa cells in the manuscript. We kindly invite you to review the latest version. o Please specify in methods and legends if kisspeptin and kisspeptin antagonist was added to PCOS-IR cells ? Answer: We sincerely thank you for your valuable suggestion. In accordance with your guidance, we have added the supplementary explanation in the manuscript. We kindly invite you to review the latest version. o Catalog numbers for all reagents and kits used (e.g., testosterone and insulin kits, PMSG, goat serum) Answer: We sincerely thank you once again for your valuable suggestion. In accordance with your guidance, we have added the supplementary information in the manuscript. We kindly invite you to review the latest version. 4. In Figure 1A: o Label the antral follicles and cysts clearly. Answer: We sincerely thank you once again for your suggestion. In accordance with your guidance, we have added the markings in Figure 1. We kindly invite you to review the latest version of the manuscript. o Add data on the body weight of mice at or near the time of sacrifice. Answer: We sincerely thank you once again for your valuable suggestion. We have added the time of mouse euthanasia in the manuscript. We kindly invite you to review the latest version. 5. In Figure 2, clarify whether the study rationale is based on local kisspeptin production in the ovary or on circulating levels. Since kisspeptin was added to the culture media, this likely simulates serum levels-please clarify in discussion. If Kisspeptin Receptor expression was measured by any means, please include it in the paper. Answer: We thank the reviewer for this insightful comment. In response, we have clarified in the Discussion section that the rationale for our study is based on local ovarian production of kisspeptin, rather than circulating serum levels. Although kisspeptin was added to the culture media in vitro to mimic its physiological effects, the in vivo relevance is primarily due to its local synthesis within the ovary. Furthermore, we have now included information regarding kisspeptin receptor (KISS1R) expression in granulosa cells, which was measured and confirmed, in the revised manuscript. We kindly invite the reviewer to refer to the updated Discussion and Methods sections for these clarifications. 6. For Figure 3, specify the number of biological and technical replicates used. Include this information in the Methods section and figure legend. Also, though mentioned in the legend, it may be helpful to call the control group PCOS-IR, since Control is vehicle+ HFD group in figure 2. Add Data from Control-IR animals in supplementary if available, to see the independent effect of kisspeptin. Answer: We sincerely thank you once again for your valuable suggestion. In accordance with your guidance, we have renamed the groups in the manuscript. We kindly invite you to review the latest version. 7. (Same as point 7) Ensure that replicate numbers are clearly indicated for all experiments in Figure 4. Answer: We sincerely thank you once again for your valuable suggestion. We have clarified the number of experimental replicates in the Methods section and figure legends, and have corrected the group names in the manuscript. We kindly invite you to review the latest version. 8. In the Discussion, please comment on the observation that the rise in testosterone in the model group compared to controls is relatively modest, yet the difference in glucose tolerance test (GTT) results and HOMA-IR is quite significant. This may suggest a Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0342158.r002
8 Oct 2025 Decision Letter - Sheng Wu, Editor Dear Dr. Ma, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Nov 22 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Sheng Wu, Ph.D. Academic Editor PLOS ONE Journal Requirements: 1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes ****** Reviewer #1: (No Response) ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org Attachments Attachment Submitted filename: 2 nd review.docx https://doi.org/10.1371/journal.pone.0342158.r003
Revision 2
27 Nov 2025 Author Response 1.Fig 1A- There are no arrow marks. Please add them. Answer: Thank you very much for your valuable suggestion. We sincerely apologize for the confusion caused by our oversight. In accordance with your instructions, we have replaced Figure 1, and we kindly invite you to review the updated version now provided in the revised manuscript. 2.Please indicate changes using coloring in the text. Answer: Thank you for your feedback. We have highlighted all revised sections in the manuscript as instructed. Please review the latest version we have provided. 3.Reviewer question: Please indicate the line numbers for the addition regarding kisspeptin receptor expression in granulosa cells. I can’t find them. “Answer: We thank the reviewer for this insightful comment. In response, we have clarified in the Discussion section that the rationale for our study is based on local ovarian production of kisspeptin, rather than circulating serum levels. Although kisspeptin was added to the culture media in vitro to mimic its physiological effects, the in vivo relevance is primarily due to its local synthesis within the ovary. “Furthermore, we have now included information regarding kisspeptin receptor (KISS1R) expression in granulosa cells, which was measured and confirmed, in the revised manuscript”. Answer: We sincerely thank the reviewer for carefully checking our previous revision. We apologize for the oversight—although we stated in the last response that information regarding KISS1R expression in granulosa cells had been added, the corresponding text was not clearly incorporated in the revised manuscript, which caused confusion. We appreciate the reviewer’s patience in pointing this out. In the current revision, we have corrected this issue and have now explicitly added the relevant description based on published evidence. The revised manuscript now includes the following statement in the Discussion section: “In addition, previous studies have shown that kisspeptin is specifically expressed in granulosa cells, whereas its receptor KISS1R exhibits specific expression in oocytes.” This information clarifies the cell-specific distribution of kisspeptin and its receptor in the ovary, thereby strengthening the mechanistic rationale of our study. We appreciate the reviewer’s valuable comments, which have helped us improve the accuracy and clarity of the manuscript. Attachments Attachment Submitted filename: Response_to_Reviewers_auresp_2.docx https://doi.org/10.1371/journal.pone.0342158.r004
23 Dec 2025 Decision Letter - Sheng Wu, Editor Dear Dr. Ma, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ===================== Request from the Editorial Office: We noted that the following related study was recently published by some of your coauthors: Kisspeptin regulates the proliferation and apoptosis of ovary granulosa cells in polycystic ovary syndrome by modulating the PI3K/AKT/ERK signalling pathway (https://doi.org/10.1186/s12905-022-02154-6) In order to comply with our Publication Criteria (http://journals.plos.org/plosone/s/criteria-for-publication#loc-2), please discuss this study in your Introduction and provide a sound scientific rationale for the submitted work. In your Discussion section, please discuss your findings in light of this existing paper. Please contact plosone@plos.org with any questions about this request. Thank you. ===================== Please submit your revised manuscript by Feb 05 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Sarah Jose, Ph.D. Staff Editor PLOS One Journal Requirements: 1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. https://doi.org/10.1371/journal.pone.0342158.r005
Revision 3
30 Dec 2025 Author Response Journal Requirements: 1.If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. Response: Thank you very much for your reminder. 2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Response: Thank you very much for your reminder. Attachments Attachment Submitted filename: Response_to_Reviewers_auresp_3.docx https://doi.org/10.1371/journal.pone.0342158.r006
7 Jan 2026 Decision Letter - Sheng Wu, Editor Dear Dr. Ma, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== ACADEMIC EDITOR: Please submit your revised manuscript by Feb 21 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Sheng Wu, Ph.D. Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0342158.r007
Revision 4
14 Jan 2026 Author Response Please specify the major differences between your article and the newly published study [Kisspeptin regulates the proliferation and apoptosis of ovary granulosa cells in polycystic ovary syndrome by modulating the PI3K/AKT/ERK signalling pathway (https://doi.org/10.1186/s12905-022-02154-6)]. The discussion is currently very general and superficial. Answer: We sincerely thank you for your valuable suggestions. In accordance with your comments, we have comprehensively and systematically reorganized the Discussion section, and further clarified the similarities and differences between our study and the newly published literature. Please refer to the revised manuscript we have submitted. Attachments Attachment Submitted filename: Response_to_Reviewers_auresp_4.docx https://doi.org/10.1371/journal.pone.0342158.r008
19 Jan 2026 Decision Letter - Sheng Wu, Editor Kisspeptin improves local ovarian insulin resistance in PCOS by modulating the PI3K/AKT/GLUT4 signaling pathway PONE-D-25-32818R4 Dear Dr. Ma, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support . If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Sheng Wu, Ph.D. Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0342158.r009
Formally Accepted
Acceptance Letter - Sheng Wu, Editor PONE-D-25-32818R4 PLOS One Dear Dr. Ma, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Sheng Wu Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0342158.r010

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