Peer Review History

Original SubmissionJune 30, 2021
Decision Letter - Peter J. Hansen, Editor

PONE-D-21-21388Optimised CO2-containing medium for in vitro culture and transportation of mouse preimplantation embryos without CO2 incubatorPLOS ONE

Dear Dr. Wakayama,

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 04 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. 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,

Peter J. Hansen

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“We thank Drs S Kishigami, and Y Fujimoto, and Miss C Yamaguchi for assistance in preparing this manuscript. This work was partially funded by the Japan Society for the Promotion of Science to M.O. (17K15394), to D.I. (JP20J23364); the Naito Foundation to S.W.; Asada Science Foundation to T.W.; and the Takeda Science Foundation to T.W.”

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“This work was partially funded by the Japan Society for the Promotion of Science to M.O. (17K15394), to D.I. (JP20J23364); the Naito Foundation to S.W.; Asada Science Foundation to T.W.; and the Takeda Science Foundation to T.W.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

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. 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: No

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: - Wakayama et al. present an interesting series of studies aimed at simplifying the embryo culture process by eliminating the need for a CO2 incubator and facilitating embryo transport without cryopreservaton. Since much of the need for CO2 is due to the use of bicarbonate as the buffering agent in the medium, additional discussion of other buffering agents may be useful for the Discussion. Their sealed tube system may be even easier with a medium buffered with HEPES or MOPS instead of CO2? See Swain et al., 2009, Reproductive BioMedicine Online 18(6):799-810.

Minor comments:

- Lines 66 and 71: Would "equilibrated" be better than “aerated”?

- Line 72-73: I would suggest, “…supplied by chemical reaction via a simple handmade device provided adequate saturation of the medium with CO2.”

- Lines 110-111: I would suggest, "…increase the partial pressure of CO2 in the medium.” Or “…the amount of dissolved CO2 in the medium.”

- Line 127: Please specify if these cultures were performed in sealed tubes in/on a warming device?

- Line 183: I would suggest, “…tube to develop normally.”

- Line 184: I would suggest, “Therefore we…”

- Line 186-187: I would suggest, “…using OptC medium equilibrated in a sealed tube with CO2 produced by an Anaero pouch.”

- Line 236-238: I would suggest, “…used, the period of incubation in a thermal bottle did not affect the development rate to the blastocyst.”

- Lines 297-298: Differences between 1- and 2-cell embryos in their sensitivity top culture conditions is fairly well established in mice and relevant references should be included here.

- Line 305-307: Why couldn’t this method be adapted for domestic animals?

- Line 361-362: Some additional information on how the Anaero pouches produce CO2 would be useful. Is there any other products of the reaction released into the air?

- Lines 460-462: Chi-square and t-tests should not be used for more than 2 treatments.

Reviewer #2: In this new manuscript, the authors propose a new optimised system to culture and transport mouse embryos without a CO2 incubator. This paper is a major improvement of their previous paper as all the equipment required are commercially available. The rates and quality of blastocysts and then of pups obtained are clearly very convincing. I highly recommend publishing this manuscript.

I have only minor comments:

- I hope tat in the fial version of the tables the sentences like "Control (CO2 incubator)" and " Untreated medium" will fit in one line to make it more clear, same for number and corresponding %. On the PDF I downloaded from the website, only Table 3 was easy to read.

- I would remove the last sentence of the conclusion. If ou are doing SCNT or DNA microinjections I suspect ou would need good microinjectors and CO2 incubators in your lab anyway (at least for cell culture).

**********

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.0260645.r001
Revision 1
Author Response

Response to reviewers

Journal requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

We have followed the instruction of PLOS ONE's style completely.

2. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement.

We corrected acknowledgement and deleted our Funding Statement.

Review Comments to the Author

Reviewer #1: - Wakayama et al. present an interesting series of studies aimed at simplifying the embryo culture process by eliminating the need for a CO2 incubator and facilitating embryo transport without cryopreservaton. Since much of the need for CO2 is due to the use of bicarbonate as the buffering agent in the medium, additional discussion of other buffering agents may be useful for the Discussion. Their sealed tube system may be even easier with a medium buffered with HEPES or MOPS instead of CO2? See Swain et al., 2009, Reproductive BioMedicine Online 18(6):799-810.

We thank you very much for your kind and valuable comments. According to your constructive comments, we added following sentence in the text.

Line 73-76.

In addition, same group also demonstrated that the HEPES or MOPS-buffered medium can support mammalian embryo development outside CO2 incubator without adverse effect[11].

Line 343-76.

To address this limitation of conventional culture, Swan et al., suggested to use HEPES or MOPS-buffered medium, which

Minor comments

- Lines 66 and 71: Would "equilibrated" be better than “aerated”?

We thank you for the kind suggestion. We changed all the words in the manuscript. Line 66, 68, 71.

- Line 72-73: I would suggest, “…supplied by chemical reaction via a simple handmade device provided adequate saturation of the medium with CO2.”

We thank you for the kind suggestion. We changed this sentence as you suggested.

Line 73-75.

To address this issue with the gas cylinder, Swain reported that the CO2 can be alternatively supplied by chemical reaction via a simple handmade device provided adequate saturation of the medium with CO2[11].

- Lines 110-111: I would suggest, "…increase the partial pressure of CO2 in the medium.” Or “…the amount of dissolved CO2 in the medium.”

We thank you for the kind suggestion. We have adopted the former sentence.

Line 115.

CO2 incubator to increase the partial pressure of CO2 in the medium.

- Line 127: Please specify if these cultures were performed in sealed tubes in/on a warming device?

We thank you for the suggestion. We described how to culture those embryos in our system.

Line 124.

Using untreated medium warmed to 37℃ placed in a thermostatic chamber,

- Line 183: I would suggest, “…tube to develop normally.”

We thank you for the kind suggestion. We changed the sentence.

Line 193.

OptC medium allowed embryos in sealed tube to develop normally.　

- Line 184: I would suggest, “Therefore we…”

We thank you for the kind suggestion. We corrected it as you suggested.

Line 194.

Therefore, we

- Line 186-187: I would suggest, “…using OptC medium equilibrated in a sealed tube with CO2 produced by an Anaero pouch.”

We thank you for the suggestion. We changed sentence because it had a slightly different meaning.

Line 195-197.

For this purpose,　OptC medium equilibrated with CO2 produced by an Anaero pouch were used for culture the embryos fertilized in vivo (not via a CO2 incubator) in a sealed tube.

- Line 236-238: I would suggest, “…used, the period of incubation in a thermal bottle did not affect the development rate to the blastocyst.”

We thank you for the kind suggestion. We changed the sentence as you suggested.

Line 248¬¬–249.

Furthermore, when in vivo-derived 2-cell embryos were used, the period of incubation in a thermal bottle did not affect the development rate to the blastocyst

- Lines 297-298: Differences between 1- and 2-cell embryos in their sensitivity top culture conditions is fairly well established in mice and relevant references should be included here.

We thank you for the kind suggestion. We corrected this sentence as follow and cited following four papers.

Line 311¬¬–312.

In this study, 1-cell embryos can be cultured for only 1 day, whereas 2-cell embryos can be cultured for 2 days by thermal bottle. However, it is well known that

Line 476 Reference

We added following 3 references:

22. Lee DR, Lee JE, Yoon HS, Roh S Il, Kim MK. Compaction in preimplantation mouse embryos is regulated by a cytoplasmic regulatory factor that alters between 1- and 2-cell stages in a concentration-dependent manner. J Exp Zool. 2001;290: 61–71. doi:10.1002/jez.1036

23. Wakayama T, Maruyama Y, Imamura K, Kurohmaru M, Hayashi Y, Fukuta K. Development of early-stage embryos of the Japanese field vole, Microtus montebelli, in vivo and in vitro. J Reprod Fertil. J Reprod Fertil; 1994;101: 663–666. doi:10.1530/JRF.0.1010663

24. Chatot CL, Lewis LJ, Torres I, Ziomek CA. Development of 1-cell embryos from different strains of mice in CZB medium. Biol Reprod. 1990;42: 432–440. doi:10.1095/biolreprod42.3.432

25. Davidson A, Vermesh M, Lobo RA, Paulson RJ. Mouse embryo culture as quality control for human in vitro fertilization: the one-cell versus the two-cell model. Fertil Steril. Elsevier; 1988;49: 516–521. doi:10.1016/S0015-0282(16)59783-0

- Line 305-307: Why couldn’t this method be adapted for domestic animals?

We thank you for your kind suggestion. We delated this sentence because we also believed that our system will be useful for domestic animal too.

- Line 361-362: Some additional information on how the Anaero pouches produce CO2 would be useful. Is there any other products of the reaction released into the air?

We thank you for the kind suggestion. We have inserted a brief sentence to explaining the specification, but the detail is not released by that company. See below.

Line 376-378

Anaero pouch is a CO2 generator that produce a certain concentration of CO2 by consumes oxygen, which is designed to culture anaerobic bacteria and there is no cytotoxicity by this reaction.

- Lines 460-462: Chi-square and t-tests should not be used for more than 2 treatments.

We thank you for the suggestion. We re-calculated all tables using tukey’s test for the number of cells and tukey’s WSD test for the rate of embryo development, implantation and the birth of offspring.

Line 473-476

The number of cells were evaluated using tukey’s test. the rate of embryo development, implantation and the birth of offspring were evaluated using tukey’s WSD test. Both tests were considered to represent a statistically significant difference when P value < 0.05.

Reviewer #2: Reviewer #2: In this new manuscript, the authors propose a new optimised system to culture and transport mouse embryos without a CO2 incubator. This paper is a major improvement of their previous paper as all the equipment required are commercially available. The rates and quality of blastocysts and then of pups obtained are clearly very convincing. I highly recommend publishing this manuscript.

I have only minor comments:

- I hope tat in the fial version of the tables the sentences like "Control (CO2 incubator)" and " Untreated medium" will fit in one line to make it more clear, same for number and corresponding %. On the PDF I downloaded from the website, only Table 3 was easy to read.

We thank you for the very kind comments. We modified all tables as you suggested. See Tables.

- I would remove the last sentence of the conclusion. If ou are doing SCNT or DNA microinjections I suspect ou would need good microinjectors and CO2 incubators in your lab anyway (at least for cell culture).

We thank you for the kind suggestion. We removed that sentence. The new paragraph is follow.

Line 342-345.

In conclusion, our culture system has applications not only for embryo culture in vitro and embryo transport but also basic study for understanding the mechanisms underlying mammalian embryo development without the need to purchase and maintain an expensive CO2 incubator.

Attachments
Attachment
Submitted filename: Response to Reviewers ver.2 TW.docx
https://doi.org/10.1371/journal.pone.0260645.r002
Decision Letter - Peter J. Hansen, Editor

Optimised CO2-containing medium for in vitro culture and transportation of mouse preimplantation embryos without CO2 incubator

PONE-D-21-21388R1

Dear Dr. Wakayama,

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,

Peter J. Hansen

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

https://doi.org/10.1371/journal.pone.0260645.r003
Formally Accepted
Acceptance Letter - Peter J. Hansen, Editor

PONE-D-21-21388R1

Optimised CO2-containing medium for in vitro culture and transportation of mouse preimplantation embryos without CO2 incubator

Dear Dr. Wakayama:

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. Peter J. Hansen

Academic Editor

PLOS ONE

https://doi.org/10.1371/journal.pone.0260645.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .