Single sperm karyotyping of testicular sperm in non-obstructive and obstructive azoospermia using next generation sequencing

Sumiko Sueyoshi; Akifumi Ijuin; Hiroe Ueno; Ai Miyakoshi; Haru Hamada; Misaki Toda; Naoki Tsuchiya; Miki Tanoshima; Mayuko Kurumizaka; Marina Saito; Yuki Oike; Kazumi Takeshima; Teppei Takeshima; Shinnosuke Kuroda; Yasushi Yumura; Shin Saito; Ryoko Asano; Taichi Mizushima; Etsuko Miyagi; Hideya Sakakibara; Hiroki Kurahashi; Akira Yanagihara; Mariko Murase; Tomonari Hayama

doi:10.1371/journal.pone.0338222

Peer Review History

Original SubmissionJuly 7, 2025
7 Jul 2025 Author Response Attachments Attachment Submitted filename: Response_to_Reviewers_letter.docx https://doi.org/10.1371/journal.pone.0338222.r001
3 Sep 2025 Decision Letter - Joël Drevet, Editor Dear Dr. Hayama , 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, Joël R Drevet, 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. 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. Additional Editor Comments: The submission was evaluated by a cytogenetics expert who provided a detailed assessment, which you will find below. Given the significant weaknesses in the presentation of the data and their excessive interpretation in some cases, a “major revision” is recommended, while taking into account that the technology developed may be of some interest. Please carefully follow the reviewer's comments. Some sections could be condensed to avoid unnecessary repetition. In addition, the discussion should focus on your own data. [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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: No ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: No ****** Reviewer #1: I had the opportunity to review the paper entitled “Single sperm karyotyping of testicular sperm of non-obstructive and obstructive azoospermia using next generation sequencing” by Tomonari Hayama et al. The study aims to evaluate the incidence of chromosomal abnormalities in sperm from patients with non-obstructive azoospermia, considering previous studies that reported an increased incidence of such abnormalities. Although the results are not entirely novel, the methodology proposed here—based on approaches commonly used for PGT-A analysis—is of interest. However, the paper requires substantial revision before it can be considered for publication in PLOS ONE, particularly the discussion section, as explained below. In addition, there are numerous typographical errors, inappropriate sentences, and cytogenetic inaccuracies that must be corrected. Major remarks: 1- The discussion section, in its current form, is not suitable and needs to be revised and considerably reduced. - the authors should provide a clear explanation of the reported 20% failure rate, and discuss the protocol in more detail, particularly its feasibility and reproducibility for other teams attempting to replicate this strategy. - the results should be discussed separately for each group in light of previous studies, with particular attention to the NOA group, where high variability has been observed. This discussion should also be connected to the high aneuploidy rate reported in oocytes. - it is unnecessary to repeat large portions of the introduction at the beginning of the discussion. This section should instead focus on the interpretation of the results. - please clarify why it could be relevant to propose PGT-A for couples similar to patient 15. In this specific case, was a sperm FISH analysis performed for chromosome 7? - Finally, the discussion should focus more closely on the study results, avoiding excessive speculation. In several places, sentences extend beyond the scope of the findings and should be revised. 2- There are several cytogenetic mistakes and misinterpretations that significantly compromise the accuracy of the results. As a cytogeneticist, it is evident that the manuscript has not been critically reviewed by a specialist in this field. Below, I summarize the most important issues; however, I strongly recommend that the authors consult with an expert in cytogenetics to ensure accuracy and consistency throughout the paper. Line 89: The authors refer to “an antibody,” but this is incorrect. It should be described as a specific DNA probe labeled with a fluorochrome. Line 231: The karyotypes are not reported according to ISCN nomenclature (e.g., “46XY” should be “46,XY”). In addition, the complete karyotype formula should be provided for the BT group, particularly for patients with reciprocal translocations, as this is essential for proper interpretation of the results. When describing the absence or duplication of a whole chromosome, the appropriate terms are nullisomy and disomy, respectively, and should be used consistently throughout the manuscript. The section Sperm karyotyping of the BT group requires a thorough revision according to both translocation segregation patterns and ISCN nomenclature. For example, in patient 6, sample 3, the karyotype should be written as 23,X,+der(19)t(19;22),-19. This clarification is necessary for accurate interpretation. For the same patient, the reported karyotype 23,X,del(22) is questionable. It may in fact correspond to 23,X,+der(22)t(19;22),-22. The authors should carefully review this point. The manuscript contains numerous typographical errors and poorly constructed sentences. A thorough language revision is strongly recommended. Line 369: Please clarify the mention of “chr 23.” This terminology is incorrect and should be revised. Line 387: The phrase “zygote stage of meiosis” is inappropriate and scientifically incorrect. Please rephrase this sentence to accurately reflect the intended meaning. Best, ****** 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 . Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0338222.r002
Revision 1
23 Oct 2025 Author Response PONE-D-25-35308 Single sperm karyotyping of testicular sperm of non-obstructive and obstructive azoospermia using next generation sequencing PLOS ONE Dear Dr. Hayama , 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 deeply revised version of the manuscript that addresses the points raised during the review process (see below). Please submit your revised manuscript by Oct 18 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. Please include the following items when submitting your revised manuscript: 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, Joël R Drevet, 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. 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. Additional Editor Comments: The submission was evaluated by a cytogenetics expert who provided a detailed assessment, which you will find below. Given the significant weaknesses in the presentation of the data and their excessive interpretation in some cases, a “major revision” is recommended, while taking into account that the technology developed may be of some interest. Please carefully follow the reviewer's comments. Some sections could be condensed to avoid unnecessary repetition. In addition, the discussion should focus on your own data. [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: Yes 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 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: No 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: I had the opportunity to review the paper entitled “Single sperm karyotyping of testicular sperm of non-obstructive and obstructive azoospermia using next generation sequencing” by Tomonari Hayama et al. The study aims to evaluate the incidence of chromosomal abnormalities in sperm from patients with non-obstructive azoospermia, considering previous studies that reported an increased incidence of such abnormalities. Although the results are not entirely novel, the methodology proposed here—based on approaches commonly used for PGT-A analysis—is of interest. However, the paper requires substantial revision before it can be considered for publication in PLOS ONE, particularly the discussion section, as explained below. In addition, there are numerous typographical errors, inappropriate sentences, and cytogenetic inaccuracies that must be corrected. Major remarks: 1- The discussion section, in its current form, is not suitable and needs to be revised and considerably reduced. - the authors should provide a clear explanation of the reported 20% failure rate, and discuss the protocol in more detail, particularly its feasibility and reproducibility for other teams attempting to replicate this strategy. - the results should be discussed separately for each group in light of previous studies, with particular attention to the NOA group, where high variability has been observed. This discussion should also be connected to the high aneuploidy rate reported in oocytes. - it is unnecessary to repeat large portions of the introduction at the beginning of the discussion. This section should instead focus on the interpretation of the results. - please clarify why it could be relevant to propose PGT-A for couples similar to patient 15. In this specific case, was a sperm FISH analysis performed for chromosome 7? - Finally, the discussion should focus more closely on the study results, avoiding excessive speculation. In several places, sentences extend beyond the scope of the findings and should be revised. 2- There are several cytogenetic mistakes and misinterpretations that significantly compromise the accuracy of the results. As a cytogeneticist, it is evident that the manuscript has not been critically reviewed by a specialist in this field. Below, I summarize the most important issues; however, I strongly recommend that the authors consult with an expert in cytogenetics to ensure accuracy and consistency throughout the paper. Line 89: The authors refer to “an antibody,” but this is incorrect. It should be described as a specific DNA probe labeled with a fluorochrome. Line 231: The karyotypes are not reported according to ISCN nomenclature (e.g., “46XY” should be “46,XY”). In addition, the complete karyotype formula should be provided for the BT group, particularly for patients with reciprocal translocations, as this is essential for proper interpretation of the results. When describing the absence or duplication of a whole chromosome, the appropriate terms are nullisomy and disomy, respectively, and should be used consistently throughout the manuscript. The section Sperm karyotyping of the BT group requires a thorough revision according to both translocation segregation patterns and ISCN nomenclature. For example, in patient 6, sample 3, the karyotype should be written as 23,X,+der(19)t(19;22),-19. This clarification is necessary for accurate interpretation. For the same patient, the reported karyotype 23,X,del(22) is questionable. It may in fact correspond to 23,X,+der(22)t(19;22),-22. The authors should carefully review this point. The manuscript contains numerous typographical errors and poorly constructed sentences. A thorough language revision is strongly recommended. Line 369: Please clarify the mention of “chr 23.” This terminology is incorrect and should be revised. Line 387: The phrase “zygote stage of meiosis” is inappropriate and scientifically incorrect. Please rephrase this sentence to accurately reflect the intended meaning. Best, 6. PLOS authors have the option to publish the peer review history of their article (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. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0338222.r003
16 Nov 2025 Decision Letter - Joël Drevet, Editor Dear Dr. Hayama, 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 Dec 31 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, Prof. Joël R Drevet, Ph.D. Academic Editor PLOS ONE Journal Requirements: 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. 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. Additional Editor Comments: After this first round of revisions, the manuscript has been significantly improved. However, there are still a few additional points that could be addressed to make it even better. I am therefore returning it to you so that you make a few more minor changes. Please note that this will be the final revision phase offered by the editorial office, so try to cover all the points raised by the reviewer. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed ******** 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: No ****** Reviewer #1: I have had the opportunity to review for the second time the manuscript entitled “Single sperm karyotyping of testicular sperm in non-obstructive and obstructive azoospermia using next generation sequencing” submitted to “Plos One”. The paper has been clearly improved and seems to be suitable for publication after few improvements, especially concerning the redundancy. One point is worth discussing. You clearly confirm a high rate of chromosomal abnormalities in the sperm of patients with NOA. However, before proposing single sperm sequencing prior to PGT-A, you need to consider several points: - The cost: on average, 5 to 7 blastocysts can be expected per ICSI. Compared to the analysis of 10 spermatozoa, PGT-A therefore seems less expensive. - The effectiveness of PGT-A in detecting oocyte aneuploidy, which is impossible to achieve with a sperm analysis. - The risk associated with biopsy and embryo freezing/thawing. This risk appears to be very low today. Therefore, in my opinion, you are proposing a very interesting new technique, but one that is difficult to implement in medical practice because it is less relevant than PGT-A analysis and non-invasive PGT-A using culture media. Regarding the structure of the text, here are a few comments: The introduction could be shortened, and some elements could be deleted. There are errors, redundancies, and repetitions; see some examples according to lines number in the highlighted version. - Lines 198-200 and 203-204: repetitions - Lines 231-234: number of patients already mentioned previously - paragraphs 246-254: data from the table, and please note that it is not necessary to point out the difference between OA and NOA. The OA and NOA diagnosis has already been made previously, so please avoid lines 246-248. - paragraphs 257-267: as the details for each group in Table 3, please summarize as follows: “no difference in all groups with a total success rate of...”. - lines 278-282, in the introduction - line 347, , Not “surprisingly”, “as expected” - lines 388-391 and 397-398 unnecessary Your best ****** 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.] 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.0338222.r004
Revision 2
18 Nov 2025 Author Response PONE-D-25-35308R1 Single sperm karyotyping of testicular sperm in non-obstructive and obstructive azoospermia using next generation sequencing PLOS ONE Dear Dr. Hayama, 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 Dec 31 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. Please include the following items when submitting your revised manuscript: 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, Prof. Joël R Drevet, Ph.D. Academic Editor PLOS ONE Journal Requirements: 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. 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. Additional Editor Comments: After this first round of revisions, the manuscript has been significantly improved. However, there are still a few additional points that could be addressed to make it even better. I am therefore returning it to you so that you make a few more minor changes. Please note that this will be the final revision phase offered by the editorial office, so try to cover all the points raised by the reviewer. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed 2. 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: 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 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: Yes 5. 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: No 6. 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: I have had the opportunity to review for the second time the manuscript entitled “Single sperm karyotyping of testicular sperm in non-obstructive and obstructive azoospermia using next generation sequencing” submitted to “Plos One”. The paper has been clearly improved and seems to be suitable for publication after few improvements, especially concerning the redundancy. One point is worth discussing. You clearly confirm a high rate of chromosomal abnormalities in the sperm of patients with NOA. However, before proposing single sperm sequencing prior to PGT-A, you need to consider several points: - The cost: on average, 5 to 7 blastocysts can be expected per ICSI. Compared to the analysis of 10 spermatozoa, PGT-A therefore seems less expensive. - The effectiveness of PGT-A in detecting oocyte aneuploidy, which is impossible to achieve with a sperm analysis. - The risk associated with biopsy and embryo freezing/thawing. This risk appears to be very low today. Therefore, in my opinion, you are proposing a very interesting new technique, but one that is difficult to implement in medical practice because it is less relevant than PGT-A analysis and non-invasive PGT-A using culture media. Regarding the structure of the text, here are a few comments: The introduction could be shortened, and some elements could be deleted. There are errors, redundancies, and repetitions; see some examples according to lines number in the highlighted version. - Lines 198-200 and 203-204: repetitions - Lines 231-234: number of patients already mentioned previously - paragraphs 246-254: data from the table, and please note that it is not necessary to point out the difference between OA and NOA. The OA and NOA diagnosis has already been made previously, so please avoid lines 246-248. - paragraphs 257-267: as the details for each group in Table 3, please summarize as follows: “no difference in all groups with a total success rate of...”. - lines 278-282, in the introduction - line 347, , Not “surprisingly”, “as expected” - lines 388-391 and 397-398 unnecessary Your best 7. PLOS authors have the option to publish the peer review history of their article (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.] 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. Attachments Attachment Submitted filename: Nov18_Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0338222.r005
19 Nov 2025 Decision Letter - Joël Drevet, Editor Single sperm karyotyping of testicular sperm in non-obstructive and obstructive azoospermia using next generation sequencing PONE-D-25-35308R2 Dear Dr. Hayama, 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, Prof. Joël R Drevet, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Thanks for having followed the suggestions and remarks. Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed ******** 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: I have had the opportunity to review for the thrid time the manuscript entitled “Single sperm karyotyping of testicular sperm in non-obstructive and obstructive azoospermia using next generation sequencing” submitted to “Plos One”. The paper has been clearly improved and seems to be suitable for publication as it. Authors answered to all my queries. Your best ****** 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 ******** https://doi.org/10.1371/journal.pone.0338222.r006
Formally Accepted
Acceptance Letter - Joël Drevet, Editor PONE-D-25-35308R2 PLOS ONE Dear Dr. Hayama, 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 Prof. Joël R Drevet Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0338222.r007

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