Peripheral mitochondrial DNA, telomere length and DNA methylation as predictors of live birth in in vitro fertilization cycles

Letizia Li Piani; Marco Reschini; Edgardo Somigliana; Stefania Ferrari; Andrea Busnelli; Paola Viganò; Chiara Favero; Benedetta Albetti; Mirjam Hoxha; Valentina Bollati

doi:10.1371/journal.pone.0261591

Peer Review History

Original SubmissionAugust 18, 2021
27 Oct 2021 Decision Letter - Gabriele Saretzki, Editor PONE-D-21-26763Peripheral mitochondrial DNA, telomere length and DNA methylation as predictors of live birth in in vitro fertilization cyclesPLOS ONE Dear Dr. Li Piani, 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 11 2021 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. 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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. Thank you for stating the following in the Competing Interests section: “I have read the journal's policy and the authors of this manuscript have the following competing interests: Dr. Somigliana reports grants from Ferring, grants and personal fees from Merck-Serono, grants and personal fees from Theramex, personal fees from Gedeon-Richter, outside the submitted work.” Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf. 3. 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 (if provided): Please address the issues of the 2 reviewers. [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: 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: 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: 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: This is an interesting study that has investigated peripheral genetic markers and corelated them prospectively with live birth rates in IVF. The results are, as the authors conclude, of no practical value in counselling patients about their potential IVF success. Nonetheless, they add to the scientific knowledge about ovarian ageing which is important for human fertility. The results of the study are not entirely novel but adds to the growing number of investigations in this field. The negative corelations are important but the results in Table 4 related to Biological age are more interesting. The mechanism of ovarian ageing related to oocyte numbers and aneuploidy should probably remain the main focus of scientific study but this paper links it to more general ageing processes. As such, the corelation is relevant. Live birth rate has been selected as the primary outcome and this is understandable as it is the most important clinical parameter for patients. To investigate the underlying biological process, it might have been more informative to consider the embryological stages e.g. fertilisation rates (what were the chronological and biological ages of the men?), embryo development rates (at what point was development compromised?) The data in Tables 1 and 2 is consistent with previously published and well accepted parameters related to IVF outcome. Of historical interest: old teachings were that “a woman with a wrinkled face would have wrinkled ovaries irrespective of age”. We are now confirming this more general association with biological markers. Specific points: It would be helpful to use the terms either DNAm age or Biological age consistently in the paper. For someone not familiar with the terms, it is confusing. The references are not numbered in the Reference section. There are several grammatical corrections needed. Table 3 – is the data for the OR for LINE-1 and Biological age correct? Reviewer #2: The topic of the work is very interesting, and the paper is well written. However, authors should make subgroups of women according to their pathology to analyse whether age biomarkers may give different results depending on each pathology. ****** 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: Yes: Alison Murdoch Reviewer #2: Yes:** María Elisa Varela Varela Sanz [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. 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Revision 1
17 Nov 2021 Author Response Response to Reviewers Reviewer #1: This is an interesting study that has investigated peripheral genetic markers and corelated them prospectively with live birth rates in IVF. The results are, as the authors conclude, of no practical value in counselling patients about their potential IVF success. Nonetheless, they add to the scientific knowledge about ovarian ageing which is important for human fertility. The results of the study are not entirely novel but adds to the growing number of investigations in this field. The negative corelations are important but the results in Table 4 related to Biological age are more interesting. The mechanism of ovarian ageing related to oocyte numbers and aneuploidy should probably remain the main focus of scientific study but this paper links it to more general ageing processes. As such, the corelation is relevant. We would like to thank you the Reviewer for the comment. Live birth rate has been selected as the primary outcome and this is understandable as it is the most important clinical parameter for patients. To investigate the underlying biological process, it might have been more informative to consider the embryological stages e.g. fertilisation rates (what were the chronological and biological ages of the men?), embryo development rates (at what point was development compromised?) We would like to thank you the Reviewer for the comment. Although the observation of the Reviewer is interesting, we deem important to be able to predict live birth rather than embryological parameters. It should be noted that, giving more attention to secondary outcomes rather than to the main clinical endopoint may lead to uncorrect conclusions with possible negative consequences. The data in Tables 1 and 2 is consistent with previously published and well accepted parameters related to IVF outcome. Of historical interest: old teachings were that “a woman with a wrinkled face would have wrinkled ovaries irrespective of age”. We are now confirming this more general association with biological markers. We would like to thank you the Reviewer for the comment. Specific point: It would be helpful to use the terms either DNAm age or Biological age consistently in the paper. For someone not familiar with the terms, it is confusing. The text has been corrected accordingly. The references are not numbered in the Reference section. The text has been corrected accordingly. There are several grammatical corrections needed. The text has been corrected by an English speaker. Table 3 – is the data for the OR for LINE-1 and Biological age correct? We would like to thank the Reviewer for the comment. Table 3 shows data as mean values while Table 4 shows OR. Therefore, lower panel of Table 3 has been cancelled. This work aims to analyse the potential of different age-indicators (telomere length, mitochondrial DNA and DNA methylation pattern) as possible markers of success after IVF treatments. The topic is of interest for the reproductive field. The manuscript is well-written, and authors provide key references to the whole comprehension of the work. We would like to thank you the Reviewer for the comment. However, there are several points that could be improved: Major points: -The authors recruited a total of 181 patients for IVF or ICSI. These people had different pathologies: endometriosis, anovulatory problems, tubal factor, even the male factor could be affecting the results. It is possible that the imprint of aging changes in a different manner in different pathologies. In addition, it does not seem compatible to find a common pattern between women with a specific reproductive pathology and women undergoing IVF because of male factor. Making subgroups of patients, according to each pathology would help to understand whether the analysed biomarkers (telomere length, mitochondrial DNA and DNA methylation pattern) do work for any given pathology, rather than for all of them together. It would be better to compare the results of age markers of each subgroup/pathology with healthy controls of similar age. We thank the Reviewer for the comment. Following Reviewer’s advice, we performed a subgroup analysis based on the leading cause of infertility. We found out that only two groups (endometriosis and mixed factor) were characterized by a statistically significant difference of biological age and age acceleration between women who delivered and who did not. While we cannot draw any conclusions in case of mixed factor both for the heterogeneity of the population and the reduced sample size, the result related to endometriosis is intriguing. Besides, this data was confirmed even comparing endometriosis to unexplained infertile women, that could be considered the control group. It could be hypothesized that endometriosis may accelerate ovarian aging more than expected. The data and the relative text in the Results and Discussion has been included in the manuscript (Supplementary table). -Line 232 and 233: “The contrast of our findings with those reported by Busnelli et al. is more difficult to reconcile”. Could you please try to give a possible explanation for these differences, for example referring to the limitations of either your or their study? We thank the Reviewer for the comment, the sentence was better clarified introducing some explanations for these differences. -Please explain the meaning of your results to facilitate the reading of the manuscript. For example: Odds ratio of 0.9 (in the case of mDNA) would indicate that the probability of having a live newborn is lower each time your DNA methylation fingerprint corresponds to a year older. We thank the Reviewer for the comment. This aspect has now been more thoroughly discussed in the text. References have been added. -Table 2. The number of oocytes retrieved, and the number of cleavage stage embryos is repeated twice with different values. Please explain better in the text or below the table what is the difference between both results. It is really not easy to understand. We thank the Reviewer for the comment. In regard to oocytes, “ N. of oocytes retrieved” refers to the average value of oocytes after the retrieval, while “No oocytes retrieved” refers to the number of women without oocytes retrieved. We have added this explanation below the table, as suggested, to make this difference clearer. In regard to “number of cleavage stage embryos” we wrongly expressed the same value in mean and median. We have corrected this mistake. Minor points: -Line 65 and Line 72: “Aging” and “ageing” should be unified. Corrected -Line 122: “sample” should be replaced by “samples”. Corrected -Line 245: “Leucocytes” should be replaced by “Leukocytes”. Corrected -Line 243, 247 and 249: “telomere length” should be replaced by “TL”. Corrected -Line 202 and onwards: The type of statistical analysis performed should be explained in materials and methods, and only a brief reference could be done in results if necessary. We thank the Reviewer for the comment, the sentence was modified as requested. -Since the manuscript body refers to the DNAm age when speaking of “Biological age (years)”, please explain write underneath the table that equivalence because it is confusing. The text has been corrected accordingly. Reviewer #2: The topic of the work is very interesting, and the paper is well written. However, authors should make subgroups of women according to their pathology to analyse whether age biomarkers may give different results depending on each pathology. We thank the Reviewer for the comment. Following Reviewer’s advice, we performed a subgroup analysis based on the leading cause of infertility. We found out that only two groups (endometriosis and mixed factor) were characterized by a statistically significant difference of biological age and age acceleration between women who delivered and who did not. While we cannot draw any conclusions in case of mixed factor both for the heterogeneity of the population and the reduced sample size, the result related to endometriosis is intriguing. Besides, this data was confirmed even comparing endometriosis to unexplained infertile women, that could be considered the control group. It could be hypothesized that endometriosis may accelerate ovarian aging more than expected. The data and the relative text in the Results and Discussion has been included in the manuscript (Supplementary table). Attachments Attachment Submitted filename: Response_to_Reviewers.docx https://doi.org/10.1371/journal.pone.0261591.r002
22 Nov 2021 Decision Letter - Gabriele Saretzki, Editor PONE-D-21-26763R1Peripheral mitochondrial DNA, telomere length and DNA methylation as predictors of live birth in in vitro fertilization cyclesPLOS ONE Dear Dr. Li Piani, 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: One of the previous reviewers still requires some minor revision.============================== Please submit your revised manuscript by Jan 06 2022 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, Gabriele Saretzki, PhD 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. Additional Editor Comments: One of the previous reviewers still requires some minor revision. [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: (No Response) 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 #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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 #1: Yes 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 #1: Yes 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 #1: The short title of the paper overemphasises the results of the paper. The authors state that the association is “insufficient to claim a clinical use”. The paper is an interesting indicator for further research to understand the genetic basis of oocyte aging rather than clinical testing for treatment. Line 339: this final clause also still implies the predictive potential for DNAm. Knowing how the IVF sector responds to such comments (another expensive unhelpful test for the patient) it would be ethically more acceptable to be cautious. In practice, the predictive power would need to be many times more powerful to alter patient selection. Abstract/ Objective Line 27. The brackets around (biological age…rate) are confusing implying that these are the factors that are part of DNAm calculation. Line 142 equates Biological age and DNAm age so why not use just one term? The paper is written assuming that the main readers will be those in the field of Reproductive medicine/science so it is better to write to their understanding. Reviewer #2: Reviewer 2 is satisfied with the response of the authors. Some of the points raised by reviewer 2 were written under “reviewer 1”. However, reviewer 2 has checked all the comments that were raised by Reviewer 2 from the beginning. ****** 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: Yes:** Alison Murdoch 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.0261591.r003
Revision 2
26 Nov 2021 Author Response Response to Reviewer #1 The short title of the paper overemphasises the results of the paper. The authors state that the association is “insufficient to claim a clinical use”. The paper is an interesting indicator for further research to understand the genetic basis of oocyte aging rather than clinical testing for treatment. Line 339: this final clause also still implies the predictive potential for DNAm. Knowing how the IVF sector responds to such comments (another expensive unhelpful test for the patient) it would be ethically more acceptable to be cautious. In practice, the predictive power would need to be many times more powerful to alter patient selection. We thank the Reviewer for the comment. We completely agree that DNAm age should be much more powerful to be considered useful for a clinical purpose. However, in our conclusion we wanted only to emphasize the urgency of exploring ovarian DNAm aging mechanisms, not inviting to adopt it clinically. As short title could be misleading, we modified it. Similarly, we added a sentence to clarify the message before line 339. Abstract/ Objective Line 27.The brackets around (biological age…rate) are confusing implying that these are the factors that are part of DNAm calculation. We thank the Reviewer for the comment, the sentence was modified as requested. Line 142 equates Biological age and DNAm age so why not use just one term? The paper is written assuming that the main readers will be those in the field of Reproductive medicine/science so it is better to write to their understanding. Corrected. Reviewer #2: Reviewer 2 is satisfied with the response of the authors. Some of the points raised by reviewer 2 were written under “reviewer 1”. However, reviewer 2 has checked all the comments that were raised by Reviewer 2 from the beginning. We would like to thank the Reviewer for the comment. Attachments Attachment Submitted filename: Response to Reviewer.docx https://doi.org/10.1371/journal.pone.0261591.r004
6 Dec 2021 Decision Letter - Gabriele Saretzki, Editor Peripheral mitochondrial DNA, telomere length and DNA methylation as predictors of live birth in in vitro fertilization cycles PONE-D-21-26763R2 Dear Dr. Li Piani, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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, Gabriele Saretzki, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): All outstanding issues have been addressed now. 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: 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 #1: Thanks for agreeing the changes and good luck with further basic science studies. There is a lot to learn in this area. ****** 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: Yes:** Professor Alison Murdoch https://doi.org/10.1371/journal.pone.0261591.r005
Formally Accepted
14 Jan 2022 Acceptance Letter - Gabriele Saretzki, Editor PONE-D-21-26763R2 Peripheral mitochondrial DNA, telomere length and DNA methylation as predictors of live birth in in vitro fertilization cycles Dear Dr. Li Piani: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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. Gabriele Saretzki Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0261591.r006

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