Freeze all-first versus biopsy-first: A retrospective analysis of frozen blastocyst transfer cycles with preimplantation genetic testing for aneuploidy

Eun Jeong Yu; Eun-A. Park; Seung-Ah Choe; Kyung-Ah Lee; You Shin Kim

doi:10.1371/journal.pone.0267652

Peer Review History

Original SubmissionAugust 20, 2020
10 Sep 2020 Decision Letter - Stefan Schlatt, Editor PONE-D-20-23557 Freeze all-first versus biopsy-first: a retrospective analysis of frozen blastocyst transfer cycles with preimplantation genetic testing for aneuploidy PLOS ONE Dear Dr. Kim, 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. This paper performs a new analysis and is therefore an interesting contribution. However both reviewers raise serious concerns towards the rationale and the methodology which dampen the enthusiasm for publication. The authors must deal with these reservations and provide valid answers and most likely additional datasets to reach a level of validity acceptable for publication. Please ensure that your decision is justified on PLOS ONE’s publication criteria and not, for example, on novelty or perceived impact. ============================== Please submit your revised manuscript by Oct 25 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Stefan Schlatt 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 [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: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: I Don't Know ****** 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: No Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors of this manuscript have undertaken the task of analyzing retrospectively a considerable data set, in which two protocols in a single center dealing with the sequence of events in PGT-A were compared: (1) first freeze all blastocyst embryos, then thaw, then biopsy (n=211 couples, 232 blastocysts), or (2, the more common approach) first biopsy, then freeze all biopsied embryos (n=327 couples, 415 blastocysts). One or two embryos were transferred. The outcome of both protocols was analyzed up to ongoing pregnancy (12th week of gestation). Pregnancy outcome data were not provided. The authors conclude that the sequence of events, in which the embryos are first biopsied and then stored frozen is beneficial to the outcome of the treatment, as given by the ongoing pregnancy rate. Having searched the literature, this is the first report dealing with this question. Given the clear-cut beneficial outcome of first biopsy the embryo before cryostorage, it is unlikely that a randomized control trial will ever be carried out (although suggested by the authors at the end of the discussion). Both groups were not completely similar, as the basal FSH levels were higher in the first group, together with a somewhat higher mean BMI in the second group. It would certainly be appropriate to add some details on the decision processes in each of both study groups, perhaps integrated into Table 1 or in a separate Table. The patients were treated in the time interval from Januar 2016 to Dezember 2019, but the follow-up included only ongoing pregnancies until week 12 of gestation. Most pregnancies must now have gone to delivery. Isn’t it possible to add all information about the outcome of the pregnancies, including the occurrence of complications during pregnancy and the status of the newborns? The paper would benefit a lot from a thorough and careful edit by a native English speaker. The authors might consider adding one reference (Cimadomo D, Rienzi L, Romanelli V, et al. Inconclusive chromosomal assessment after blastocyst biopsy: prevalence, causative factors and outcomes after re-biopsy and re-vitrification. A multicenter experience. Hum Reprod 2018;33:1839-1846), in which embryos with inconclusive results of genetic testing were subjected to thawing and re-biopsy. They provide some information about the feasibility of the (re)-biopsy of thawed embryos and their competence to become implanted after embryo transfer, as studied in this retrospective data set. Figure 1: the authors should design the figure such, that the confidence intervals are visible. Table 1: Do the authors provide us with standard deviations, standard errors or 95% confidence intervals? Line 74: replace the word “inevitable” by “irreparable”. Line 97: all patients Line 126: zona pellucida Line 239: instead of (data not seen) please write (data not shown) Discussion, lines 238 to 248: the authors might add some information about the influence of making a hole into the zona pellucida and the risk of monozygotic twinning. Reviewer #2: Freeze all-first versus biopsy-first: a retrospective analysis of frozen blastocyst transfer cycles with preimplantation genetic testing for aneuploidy Manuscript by Jeong Yu et al., retrospectively compared the efficacy and safety of the timing on the cryopreservation and trophectoderm (TE) biopsy for PGT-A program. The main observation is increased clinical and ongoing pregnancy rates in biopsy-first approach than those in freeze all-first. Authors believe that performing TE biopsy of blastocyst for PGT-A before vitrification and warming can give better clinical outcome. Major comments: My major concern with this manuscript is lack of methodological details and importantly, rationale in comparing these two groups. There is no clarity in their objective. Even after reading the manuscript several times, I did not understand when embryo transfers were done in freeze- all first group, post warming and TE biopsy. Are the results available fast enough to perform ET in the same cycle, within a few hours? Or biopsied embryos were re-frozen? The methodology is not clear on this point. If re-freezing was done, then the survival rate and embryo quality of re-frozen/thawed embryos needs to be stated, and discussion on the safety of multiple freeze/thaw would be warranted. Also, if the embryos were re-frozen, the survival rate of ~98% in both groups is very interesting. The author states, "We hypothesized that biopsy before cryopreservation may cause the embryo inevitable damage in the freezing process decreasing survival rates and implantation potential." Is it in comparison to biopsy after cryopreservation? The hypothesis has not been addressed in discussion, especially since the conclusion is contrary to the hypothesis. Was ICSI done for the freeze-all group too, when PGT was not planned at that point in time? If not, what measures were taken to avoid contamination during IVF to prevent false results in PGT. Table 1: Mean age of patients is stated as 37.0±3.9 for freeze all first and 36.7±4.1 for biopsy first, but under indications for PGT, advanced age(>40yrs) shows 125(59.2%) and 222(67.8%), respectively. There appears to be some discrepancy in the data which needs to be addressed. Minor comments: 1. Line 67 - "there is no consensus regarding the timing of TE biopsy (before versus after cryopreservation)". Please cite reference. 2. Line 76 - does the "previous data" refer to the earlier reference? It is not clear. 3. Ln 65-66 - reference given for this sentence has not been cited properly under references. 4. Ln 87 - " old age" can be rephrased as advanced maternal age and cut off can be mentioned. If any criteria were followed for the indications, please cite the references. 5. Line 127 - Blastocyst morphology ">3BC". The criteria used for grading can be stated; was 3BC the minimum grading to be eligible for biopsy? If so, it can be stated accordingly to avoid confusion. 6. Line 131 - Details could be given regarding how the authors proceeded with the biopsy material, with a focus on duration of genetic analysis. 7. Line 140 - Embryo transfer : How long after biopsy were the embryos transferred would require mentioning in the methodology section. 8. Line 149/150 - "in biopsy-first cycles, where the blastocyst had not been subjected to assist hatching in the previous cycle, assisted hatching was performed" - are the authors referring to the freeze-all group? It's not clear. 9. Table 1 : Mean survival rate - It can be stated as mean survival rate of embryos 10. Line 238 - 242 not comprehensible in discussion, could be rephrased for better clarity. 11. A further discussion on pregnancy outcomes & miscarriage rates between the two groups and also in comparing the findings with other studies would be warranted, since the study is looking into safety and efficacy of PGT timing. 12. Spelling mistakes in a few places that need correction. ****** 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.0267652.r001
Revision 1
2 Nov 2021 Author Response Reviewer #1: The authors of this manuscript have undertaken the task of analyzing retrospectively a considerable data set, in which two protocols in a single center dealing with the sequence of events in PGT-A were compared: (1) first freeze all blastocyst embryos, then thaw, then biopsy (n=211 couples, 232 blastocysts), or (2, the more common approach) first biopsy, then freeze all biopsied embryos (n=327 couples, 415 blastocysts). One or two embryos were transferred. The outcome of both protocols was analyzed up to ongoing pregnancy (12th week of gestation). Pregnancy outcome data were not provided. The authors conclude that the sequence of events, in which the embryos are first biopsied and then stored frozen is beneficial to the outcome of the treatment, as given by the ongoing pregnancy rate. Having searched the literature, this is the first report dealing with this question. Given the clear-cut beneficial outcome of first biopsy the embryo before cryostorage, it is unlikely that a randomized control trial will ever be carried out (although suggested by the authors at the end of the discussion). Both groups were not completely similar, as the basal FSH levels were higher in the first group, together with a somewhat higher mean BMI in the second group. It would certainly be appropriate to add some details on the decision processes in each of both study groups, perhaps integrated into Table 1 or in a separate Table. The patients were treated in the time interval from Januar 2016 to Dezember 2019, but the follow-up included only ongoing pregnancies until week 12 of gestation. Most pregnancies must now have gone to delivery. Isn’t it possible to add all information about the outcome of the pregnancies, including the occurrence of complications during pregnancy and the status of the newborns? -> We added your point to the table 1 and result section The paper would benefit a lot from a thorough and careful edit by a native English speaker. ->We received English correction. The authors might consider adding one reference (Cimadomo D, Rienzi L, Romanelli V, et al. Inconclusive chromosomal assessment after blastocyst biopsy: prevalence, causative factors and outcomes after re-biopsy and re-vitrification. A multicenter experience. Hum Reprod 2018;33:1839-1846), in which embryos with inconclusive results of genetic testing were subjected to thawing and re-biopsy. They provide some information about the feasibility of the (re)-biopsy of thawed embryos and their competence to become implanted after embryo transfer, as studied in this retrospective data set. ->Thanks for your suggestion. We added this article on result and reference section. Figure 1: the authors should design the figure such, that the confidence intervals are visible. ->We added the confidence intervals on figure. Table 1: Do the authors provide us with standard deviations, standard errors or 95% confidence intervals? ->We have corrected your point. Line 74: replace the word “inevitable” by “irreparable”. ->We have corrected your point. Line 97: all patients ->We have corrected your point. Line 126: zona pellucida ->We have corrected your point. Line 239: instead of (data not seen) please write (data not shown) ->We have corrected your point. Discussion, lines 238 to 248: the authors might add some information about the influence of making a hole into the zona pellucida and the risk of monozygotic twinning. -> we corrected what was pointed out “Also, this change has been related with increased risk of monozygotic twin because of blstocyst herniation and embryo splitting through non-natural gap”(Line 279-281) Reviewer #2: Freeze all-first versus biopsy-first: a retrospective analysis of frozen blastocyst transfer cycles with preimplantation genetic testing for aneuploidy Manuscript by Jeong Yu et al., retrospectively compared the efficacy and safety of the timing on the cryopreservation and trophectoderm (TE) biopsy for PGT-A program. The main observation is increased clinical and ongoing pregnancy rates in biopsy-first approach than those in freeze all-first. Authors believe that performing TE biopsy of blastocyst for PGT-A before vitrification and warming can give better clinical outcome. Major comments: My major concern with this manuscript is lack of methodological details and importantly, rationale in comparing these two groups. There is no clarity in their objective. Even after reading the manuscript several times, I did not understand when embryo transfers were done in freeze- all first group, post warming and TE biopsy. Are the results available fast enough to perform ET in the same cycle, within a few hours? Or biopsied embryos were re-frozen? The methodology is not clear on this point. If re-freezing was done, then the survival rate and embryo quality of re-frozen/thawed embryos needs to be stated, and discussion on the safety of multiple freeze/thaw would be warranted. Also, if the embryos were re-frozen, the survival rate of ~98% in both groups is very interesting. ->Thanks for your comment. We tried to explain about this point on material and method part. In the freeze-all first cycles, thawing blastocysts were prioritized based on the best quality before biopsy. Embryos were warmed in the early morning on the day before transfer, then cultured for 2-3h and assessed for TE biopsy. Biopsy results confirmed on the morning of scheduled transfer and transferred at least one euploid embryo. In biopsy-first cycles, once biopsy results confirmed at least one euploid embryo, patients were scheduled for a frozen ET cycle.(Line 169-174) The author states, "We hypothesized that biopsy before cryopreservation may cause the embryo inevitable damage in the freezing process decreasing survival rates and implantation potential." Is it in comparison to biopsy after cryopreservation? The hypothesis has not been addressed in discussion, especially since the conclusion is contrary to the hypothesis. ->Thanks for your comment. We are sorry for confusion. We changed the paragraph from “biopsy before cryopreservation” to “biopsy after cryopreservation-thawing”(Line 75) Was ICSI done for the freeze-all group too, when PGT was not planned at that point in time? If not, what measures were taken to avoid contamination during IVF to prevent false results in PGT. ->We used ICSI in all cases. ICSI is commonly used in pre-implantation genetic testing (PGT) cases to eliminate any risk of sperm DNA contamination. Table 1: Mean age of patients is stated as 37.0±3.9 for freeze all first and 36.7±4.1 for biopsy first, but under indications for PGT, advanced age(>40yrs) shows 125(59.2%) and 222(67.8%), respectively. There appears to be some discrepancy in the data which needs to be addressed. ->Freeze all first group included more young patient with recurrent spontaneous abortion than biopsy first group. Minor comments: 1. Line 67 - "there is no consensus regarding the timing of TE biopsy (before versus after cryopreservation)". Please cite reference. -> We have corrected your point. Reference 8 : Aoyama N, Kato K. Trophectoderm biopsy for preimplantation genetic test and technical tips: A review. Reprod Med Biol. 2020;19(3):222-31. 2. Line 76 - does the "previous data" refer to the earlier reference? It is not clear. ->We changed the word from “data” to “study”. This refers to an article published prior to our study. 3. Ln 65-66 - reference given for this sentence has not been cited properly under references. ->We found new reference and change the reference Greco E, Litwicka K, Minasi MG, Cursio E, Greco PF, Barillari P. Preimplantation Genetic Testing: Where We Are Today. Int J Mol Sci. 2020;21(12). 3. Ln 87 - " old age" can be rephrased as advanced maternal age and cut off can be mentioned. If any criteria were followed for the indications, please cite the references. -> We have corrected your point and added the references. (line 89) 4. Line 127 - Blastocyst morphology ">3BC". The criteria used for grading can be stated; was 3BC the minimum grading to be eligible for biopsy? If so, it can be stated accordingly to avoid confusion. -> We newly described about this point at the material and method part (line 128) 6. Line 131 - Details could be given regarding how the authors proceeded with the biopsy material, with a focus on duration of genetic analysis. -> We newly described about this point at the material and method part (line 128-140) 7. Line 140 - Embryo transfer : How long after biopsy were the embryos transferred would require mentioning in the methodology section. -> We newly described about this point at the material and method part (line 169-174) 8. Line 149/150 - "in biopsy-first cycles, where the blastocyst had not been subjected to assist hatching in the previous cycle, assisted hatching was performed" - are the authors referring to the freeze-all group? It's not clear. -> This sentence corresponds to biopsy-first group. 9. Table 1 : Mean survival rate - It can be stated as mean survival rate of embryos -> Thanks for your comment. We have corrected your point. 10. Line 238 - 242 not comprehensible in discussion, could be rephrased for better clarity. -> Thanks for your comment. We have corrected your point. “Hatching status could make significant different in clinical outcomes between biopsy-first and freeze all frist groups.” 11. A further discussion on pregnancy outcomes & miscarriage rates between the two groups and also in comparing the findings with other studies would be warranted, since the study is looking into safety and efficacy of PGT timing. ->Thanks for your comment. We have corrected your point. 12. Spelling mistakes in a few places that need correction. -> Thanks for your comment. We have corrected your point. Attachments Attachment Submitted filename: response to reviewer.docx https://doi.org/10.1371/journal.pone.0267652.r002
26 Nov 2021 Decision Letter - Stefan Schlatt, Editor PONE-D-20-23557R1Freeze all-first versus biopsy-first: a retrospective analysis of frozen blastocyst transfer cycles with preimplantation genetic testing for aneuploidyPLOS ONE Dear Dr. Kim, 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. The authors have addressed the concerns of the reviewers in a not fully adequate way. There are still doubts on the validity of the conclusions. The major concerns is that patient selection is responsible for some of the observed effects. Before a final decision can be made the authors have to deal once more with the critical concerns and add additional evidence (and supportive data) that their conclusions are truly valid. Please submit your revised manuscript by Jan 10 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, Stefan Schlatt Academic Editor PLOS ONE [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: No Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A 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: No 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: No 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 authors of this manuscript evaluate the effect of a freeze-all first strategy versus a biopsy-first strategy on the outcome of PGT-A in a cohort of more than 500 treatment cycles. The analysis was retrospective, but the strength of this study is that no other study has dealt with this topic so far. The paper has already undergone a major revision based up two extensive and high quality reviews. The quality of the manuscript has much improved, but a number of issues still remain. As this is a retrospective analysis, it is important to explain why both strategies were used (freeze-all first versus biopsy-first). Between lines 87 and 93 some information is given, but not quantified. Especially the statement that poor ovarian reserve requiring repeated stimulations as a reason for freeze-all first is worrying, as this subpopulation is characterized by lower likelihood of pregnancy, thereby skewing the outcome of the study. This would also explain the slightly higher (but statistically significant) basal FSH-serum levels in the one study group. The authors should clearly state and quantify the reasons for selection into the one or into the other study group, either in the Material and Method section and/or in the Results section of their manuscript, as already requested by Reviewer 2. One reviewer requested the authors to add live birth numbers to their results section. This was indeed partially done, but the authors should also implement Table 1 with these additional data. They should add the live birth numbers to the clinical pregnancy rate per ongoing ET, to ongoing pregnancy rate per ET and to miscarriage. The first reviewer also asked for data on incident complications during pregnancy and data on the newborn babies, but these data were not given (although stated to be given). Please, amend this valuable information, that will greatly benefit the quality of this study. The authors should also clarify in Table 1, whether the mean values are accompanied by standard error of the mean or by standard deviations. The authors have transferred either one or two euploid embryos. Twin pregnancies may have resulted. In addition, the hole inserted into the zona pellucida of the blastocyst embryos has had an effect on the timing of the hatching, as stated in the discussion. For that reason, the authors should also include information about the multiple pregnancies (including whether these were monozygotic or dizygotic twin pregnancies). Table 2 should also be implemented with an estimate of the miscarriage rate, as PGT-A may lower the incidence of miscarriages. It is of interest, whether the miscarriage rate is indeed different in one of both strategies. Figure 1 should also have some indication of the statistical analysis (such as p-values). As the reviewers requested: confidence intervals were given. One statement in the Introduction is misleading to me: why should the timing of cryopreservation and biopsy in the blastocyst stage interfere with genome activation and epigenetic imprinting? I would transfer lines 64 to 67 to line 57, after the first sentence. This sequence of statements is more logical. Although languagewise the manuscript has been improved, the first few sentences of the discussion must be improved. For example: Line 29: Abstract: the sentence should be: “Potential use of preimplantation genetic testing for aneuploidy (PGT-A) is increasing.” Line 126: zona pellucida instead of zona pellucid. Line 207: lower pregancy rates Line 239: data not shown instead of data not seen Line 241: frequently often: remove one of both words Reviewer #2: Authors have addressed my comments in the revised version and I do not have any other issues with the manuscript. ****** 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 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.0267652.r003
Revision 2
31 Mar 2022 Author Response Reviewer #1: The authors of this manuscript evaluate the effect of a freeze-all first strategy versus a biopsy-first strategy on the outcome of PGT-A in a cohort of more than 500 treatment cycles. The analysis was retrospective, but the strength of this study is that no other study has dealt with this topic so far. The paper has already undergone a major revision based up two extensive and high quality reviews. The quality of the manuscript has much improved, but a number of issues still remain. As this is a retrospective analysis, it is important to explain why both strategies were used (freeze-all first versus biopsy-first). Between lines 87 and 93 some information is given, but not quantified. Especially the statement that poor ovarian reserve requiring repeated stimulations as a reason for freeze-all first is worrying, as this subpopulation is characterized by lower likelihood of pregnancy, thereby skewing the outcome of the study. This would also explain the slightly higher (but statistically significant) basal FSH-serum levels in the one study group. The authors should clearly state and quantify the reasons for selection into the one or into the other study group, either in the Material and Method section and/or in the Results section of their manuscript, as already requested by Reviewer 2. � We added your point to the material and methods (line 89-94). As a result of reviewing the raw data again, there was no statistically significant difference between FSH and BMI. One reviewer requested the authors to add live birth numbers to their results section. This was indeed partially done, but the authors should also implement Table 1 with these additional data. They should add the live birth numbers to the clinical pregnancy rate per ongoing ET, to ongoing pregnancy rate per ET and to miscarriage. The first reviewer also asked for data on incident complications during pregnancy and data on the newborn babies, but these data were not given (although stated to be given). Please, amend this valuable information, that will greatly benefit the quality of this study. � We added your point to the table 1 and result section The authors should also clarify in Table 1, whether the mean values are accompanied by standard error of the mean or by standard deviations. � We added your point to the table 1 and result section The authors have transferred either one or two euploid embryos. Twin pregnancies may have resulted. In addition, the hole inserted into the zona pellucida of the blastocyst embryos has had an effect on the timing of the hatching, as stated in the discussion. For that reason, the authors should also include information about the multiple pregnancies (including whether these were monozygotic or dizygotic twin pregnancies). � We added your point to the result section Table 2 should also be implemented with an estimate of the miscarriage rate, as PGT-A may lower the incidence of miscarriages. It is of interest, whether the miscarriage rate is indeed different in one of both strategies. � We changed the term from ‘spontaneous abortion’ to ‘miscarriage’ in table 2 Figure 1 should also have some indication of the statistical analysis (such as p-values). As the reviewers requested: confidence intervals were given. � We have corrected your point One statement in the Introduction is misleading to me: why should the timing of cryopreservation and biopsy in the blastocyst stage interfere with genome activation and epigenetic imprinting? I would transfer lines 64 to 67 to line 57, after the first sentence. This sequence of statements is more logical. � We have corrected your point Although languagewise the manuscript has been improved, the first few sentences of the discussion must be improved. For example: Line 29: Abstract: the sentence should be: “Potential use of preimplantation genetic testing for aneuploidy (PGT-A) is increasing.” Line 126: zona pellucida instead of zona pellucid. Line 207: lower pregancy rates Line 239: data not shown instead of data not seen Line 241: frequently often: remove one of both words � We have corrected your point. Reviewer #2: Authors have addressed my comments in the revised version and I do not have any other issues with the manuscript. Attachments Attachment Submitted filename: response to reviewers_032322.docx https://doi.org/10.1371/journal.pone.0267652.r004
13 Apr 2022 Decision Letter - Stefan Schlatt, Editor Freeze all-first versus biopsy-first: a retrospective analysis of frozen blastocyst transfer cycles with preimplantation genetic testing for aneuploidy PONE-D-20-23557R2 Dear Dr. Kim, 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, Stefan Schlatt Academic Editor PLOS ONE Additional Editor Comments (optional): The authors responded appropriately to the reviewers concerns and suggestions. The paper has much improved, contains valid and original datasets which will be important for the field. Reviewers' comments: https://doi.org/10.1371/journal.pone.0267652.r005
Formally Accepted
18 Sep 2022 Acceptance Letter - Stefan Schlatt, Editor PONE-D-20-23557R2 Freeze all-first versus biopsy-first: a retrospective analysis of frozen blastocyst transfer cycles with preimplantation genetic testing for aneuploidy Dear Dr. Kim: 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. Stefan Schlatt Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0267652.r006

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