The effects of L-carnitine and fructose in improved Ham’s F10 on sperm culture in idiopathic severe asthenospermia within 24h

Dehui Chang; Fudong Li; Yindong Kang; Yixin Yan; Feiyan Kong; Wei Jiang; Dongxing Wang; Zhigang Cao; Liuting Xu; Chuang Huang; Yafen Kang; Xuejun Shang; Bin Zhang

doi:10.1371/journal.pone.0306235

Peer Review History

Original SubmissionJune 13, 2024
9 Aug 2024 Decision Letter - Xuejiang Guo, Editor PONE-D-24-23439The effects of L-carnitine and fructose in improved Ham's F10 on sperm culture in idiopathic severe asthenospermia within 24hPLOS ONE Dear Dr. Zhang, 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. Especially the points from Reviewer #2 that the author should introduce the definition, occurrence, ART outcome, ways to improve ART outcome in men with idiopathic severe asthenospermia, as well as how to recruit these patients. Please submit your revised manuscript by Sep 23 2024 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. 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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. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. 3.Thank you for stating the following financial disclosure: "Supported by the Fundamental Research Funds for the Central Universities and the grant numbers is 31920240072. Supported by the the Health Special Project fund of PLA and the grant numbers is 21BJZ43. Supported by the Special Scientific Research Projects for Family Planning, Prenatal and Postnatal Care of PLA and the grant numbers is 21JSZ13. Supported by the Medical and Health Project fund of the 940th Hospital and the grant numbers is 2021yxky017, 2021. Supported by the Medical and Health Project fund of the 940th Hospital and the grant numbers is 2023YXKY006 and 2023YXKY024, 2023. Supported by the Natural Science Foundation of Gansu Province and the grant numbers is 22JR5RA001, 2022. Supported by the Natural Science Foundation of Gansu Province and the grant numbers is 23JRRA531, 2023. Supported by the Lanzhou science and technology project Bureau and the grant numbers is 2023-2-63, 2023." Please state what role the funders took in the study. 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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. [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 Reviewer #2: Yes ******** 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: Yes 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: 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: In this study, the semen of patients with severe asthenospermia was collected and studied. Based on Ham’s F10 medium, different concentrations of L-carnitine and fructose were added into, and different culture temperatures were used to study the effects of the above three factors on the sperm motility, survival rate, deformity rate and DNA fragmentation index of sperm cultured in vitro for 24 hours. The meanings of this study are to prolong the survival time of sperm in vitro, improve the quality and motility of sperm, and improve the success rate of clinical assisted reproductive technology. This study has certain clinical significance. Through orthogonal test, the results of this study found that when 8mg/mL L-carnitine, 0.375mg/mL fructose were added into Ham 's F10 basic medium at the temperature of 36.5 °C, the semen quality of patients with severe asthenospermia could be effectively improved, which was better than that treat with Ham’s F10 basic medium. However, there are several defects that need to be fixed before the article can be accepted: 1. The writing is not standardized, such as the mixed use of mg/mL and mg/ml. 2.When the table crosses the page, the table needs to be adjusted to one page or the continuation table is used. 3.In the study, there were many groups but the abbreviations of the group names were not direct enough and could not be understood at a glance. Such as the saline group could be abbreviated as NS, the Ham’s F10 basic medium group could be abbreviated as HF, the different concentrations of L-carnitine group could be abbreviated as L+ numbers, and the different concentrations of fructose group could be abbreviated as F+ numbers. 4.There are writing errors in the text that need to be corrected. For example, in 3.4.5, there was no significant difference, but marked with p<0.05. Reviewer #2: Idiopathic severe asthenospermia is a common condition for male infertility, and with relatively bad result even with ART, thus the research on improving ART outcome on these patients is desired. This research found L-carnitine and fructose can improve sperm motility, which may be used to improve ART outcome. 1. The authors should focus on the idiopathic severe azoospermia. In the introduction and discussion, as well as in the method part, the author should introduce the definition, occurrence, ART outcome, ways to improve ART outcome in men with idiopathic severe azoospermia, as well as how to recruit these patients. 2. Some figures should put into the supplementary data, only keeping important results in the main document. 3. Revision of language is suggested. ****** 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 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. https://doi.org/10.1371/journal.pone.0306235.r001
Revision 1
5 Sep 2024 Author Response Author’s Response to Reviewers‘ Comments Ms. No.: PONE-D-24-23439 Title: The effects of L-carnitine and fructose in improved Ham's F10 on sperm culture in idiopathic severe asthenospermia within 24h Journal: PLOS ONE Editor and Reviewer comments: Especially the points from Reviewer #2 that the author should introduce the definition, occurrence, ART outcome, ways to improve ART outcome in men with idiopathic severe asthenospermia, as well as how to recruit these patients. Reply: According to WHO laboratory manual for the examination and processing of human semen (5th Edition), asthenospermia is defined as progressive motility < 32% (abstinence for 2-7 days). Idiopathic asthenospermia is understood as a form of asthenospermia with unexplained and unknown etiology. However, the definition of severe asthenozoospermia is different in different race. Severe asthenozoospermia refers to patient with 1 ~ 10% (China) or < 20% (Spain) progressive motility. Therefore, idiopathic severe asthenospermia refers to Chinese patient with 1 ~ 10% progressive motility with undiscovered etiology in this study. Although conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) can lead to successful pregnancy in couples with only male infertility, progressive motility has a certain influence on the outcome of ART. Therefore, using sperm culture medium to improve sperm motility as much as possible is helpful to improve ART outcome. The above detailed explanations are supported by updated literature. Ethics approval by the Human Ethics Committee of the 940 Hospital (IRB approval No. 2016KYLL086). And the use of this experimental semen specimens and the experimental protocol were agreed. The clinical trial number: 2016CLT-U002.We informed each participant the details of the study: We need to collect some semen amples from you, and only use the remaining semen samples from the routine semen test. The sample is only used in this study and will not affect routine treatment or diagnosis. We obtained written informed consent forms from each participants included in this study. We recruited 60 patients with idiopathic severe asthenozoospermia (the criteria was that forward motility of sperm is 1 ~ 10% and with unknown etiology), aged 24-35 (26.5±4.31) years, participated in this study at the Urology Center of the 940th Hospital, from September 2017 to August 2018. We informed each participant the details of the study: We need to collect some semen samples from you, and only use the remaining semen samples from the routine semen test. The sample is only used in this study and will not affect routine treatment or diagnosis. Informed consent forms were obtained from the patients included in this study. Reply to Journal Requirements: 1. We update the manuscript to meet PLOS ONE's style requirements, including those for file naming. 2. We provide additional details regarding participant consent, which is stated in the ethics statement in the Methods and online submission information. Informed consent forms were obtained from the patients included in this study. We upload the Informed consent for clinical participant. 3. Dehui Chang and Bin Zhang are the funders of above funds. Dehui Chang contributed to the whole project development. Bin Zhang contributed to carrying out the literature review, the language translation, grammar checking, and manuscript writing. 4. We confirm at this time the submission contains all raw data required to replicate the results of this study. We submit and upload 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. - Some minor data errors have been corrected, and the icons has been corrected.. 5. By using PACE digital diagnostic tool, We transformed and uploaded the figure files. Reply to Reviewer #1: 1. Thank you for your review. We standarded the writing and corrected mg/mL to mg/ml. 2. When the table spreads, we have adjusted the table to one page or used a continuation table. 3. According to your suggestion, we rewrote the abbreviations of the group names as follows: A1: NS, B1: L4, C1: L8, D1: L12, E1: L16, F1:HF. A2: NS, B2: F0.125, C2: F0.250, D2: F0.375, E2: F0.500, F2:HF. A3:NS (NS--30min, NS3-1h, NS3-12h, and NS3-24h), B3:HF (HF3-30min, HF3-1h, HF3-12h, and HF3-24h), C3: m-HF (m-HF -30min, m-HF -1h, m-HF -12h, and m-HF -24h). 4. We corrected all writing errors in the text. Reply to Reviewer #2: 1. In the introduction, we have introduced the definition, occurrence, ART outcome and methods to improve ART outcome in patients with idiopathic azoospermia and idiopathic severe asthenospermia in detail. In the discussion, we emphasized the significance of this study for improving sperm motility in patients with idiopathic asthenospermia in vitro. 2. Because the contents of table 1 and table 2 are duplicated, we deleted table 1 and kept table 2 and 3. We put some figures into the supplementary data, and kept important results in the main document. 3. This paper has been significantly revises. Please refer to a website to help modify the language of the article. The editor’s native language is English. Thank you! Thank you for your help and support! Your sincerely yours. Bin Zhang 2024-09-05 Attachments Attachment Submitted filename: renamed_70cc5.docx https://doi.org/10.1371/journal.pone.0306235.r002
25 Oct 2024 Decision Letter - Xuejiang Guo, Editor PONE-D-24-23439R1The effects of L-carnitine and fructose in improved Ham's F10 on sperm culture in idiopathic severe asthenospermia within 24hPLOS ONE Dear Dr. Zhang, 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 09 2024 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, Xuejiang Guo, Ph.D. Academic Editor PLOS ONE Journal Requirements: 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 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 #2: 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 #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: 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 #2: 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 #2: Yes ****** 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 #2: The authors have addressed all my comments. However, they added a paragraph in the INTRODUCTION part for the azoospermia. Deletion of this paragraph is suggested since this work is not related to the azoospermia at all. ****** 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 #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. https://doi.org/10.1371/journal.pone.0306235.r003
Revision 2
16 Nov 2024 Author Response Reply to Journal Requirements: 1. At present, we have no laboratory protocols, and we will strengthen our work in this area. Thank you for your proposal. 2. The references have been revised and adjusted. The DOI and PMID code is supplemented in the reference [6], [19], [20], [21], and [22]. The original reference [18] was adjusted to reference [7], and references [7-17] was adjusted to reference [8-18]. 3. We reviewed the reference list and ensure that it is complete and correct, in which all cited papers were searched on Pubmed and have not been retracted. Reply to Reviewer #2 about “6. Review Comments to the Author”: We deleted the paragraph in the INTRODUCTION part for the azoospermia since this work is not related to the azoospermia at all, according to the suggestion. The reference for this paragraph is the original reference [18]. The original reference [18] was about the study between male infertility and precision medicine and systems proteomics, so it was adjusted to the paragraph: “Male infertility is mainly manifested as azoospermia” in the INTRODUCTION part (line 75-76). For this reason, we adjusted the original reference [18] to reference [7], and references [7-17] to reference [8-18]. Thank you for your help and support! Attachments Attachment Submitted filename: renamed_52410.docx https://doi.org/10.1371/journal.pone.0306235.r004
26 Nov 2024 Decision Letter - Xuejiang Guo, Editor The effects of L-carnitine and fructose in improved Ham's F10 on sperm culture in idiopathic severe asthenospermia within 24h PONE-D-24-23439R2 Dear Dr. Zhang, 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. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. 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, Xuejiang Guo, Ph.D. Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0306235.r005
Formally Accepted
5 Dec 2024 Acceptance Letter - Xuejiang Guo, Editor PONE-D-24-23439R2 PLOS ONE Dear Dr. Zhang, 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 If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks 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. 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. Xuejiang Guo Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0306235.r006

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