-->PONE-D-25-68905-->-->Quality assessment of endotoxin contamination in consumables used for assisted reproductive technology-->-->PLOS One

Dear Dr. Haishima,

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Dear respected Authors, -->--> Based on the evaluation from our reviewers, I have decided that your manuscript requires a major revision. Please follow the reviewers' recommendations point by point and submit your revised version to PLOS One. -->--> -->-->Best regards,

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Academic Editor

PLOS One

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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: Partly

Reviewer #2: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

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-->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

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-->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

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-->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 study suggests that contaminated containers “may reduce ART success rates,” but no clinical outcome data are provided to support this claim. Extrapolating from laboratory results (embryo growth and sperm motility) of potential biological effects to actual clinical outcomes of ART is not conclusive. Therefore, conclusions about reduced ART success rates should be interpreted in the discussion section appropriately, with a review of previous research, or supported by clinical outcome data

In this study, the observed concentrations of hydrogen peroxide were compared with the reported toxicity threshold for human mesenchymal stem cells. However, mesenchymal stem cells are not biologically equivalent to human gametes or embryos, which are generally known to be more sensitive to oxidative stress. Therefore, using MSC data as a reference for reproductive cell safety may not be appropriate. This section is discussed in detail in the Discussion.

The human cell-based pyrogen test (HCPT) plays a key role in the conclusions of this study; more details on the validation and robustness of this method would be helpful.

Were spike and recovery tests performed for the specific substances tested?

Was inter-donor variability assessed?

How many independent blood donors were included in each test condition?

Reviewer #2: This work is relevant and sould be published. Small linguistic issues, however: Headers: "Effect on mice and human embryo development" should be "Effect on mouse and human embryo development" ; "Removal performance of residual hydrogen peroxide by drying treatment" should be "Removal of residual hydrogen peroxide" .

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-->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.

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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: Ashraf Kariminik

Reviewer #2: No

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NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Quality assessment of endotoxin contamination in consumables used for assisted reproductive technology

PONE-D-25-68905R1

Dear Dr. Haishima,

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.

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Kind regards,

Mohammad Faezi Ghasemi, Ph.D

Academic Editor

PLOS One

Additional Editor Comments (optional):

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

Reviewer #2: All comments have been addressed

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-->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

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-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

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-->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

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-->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

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-->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 have addressed all of my previous comments thoroughly. The methods, results, discussion, and conclusion sections have been appropriately revised based on the feedback provided. I have no further major concerns regarding the scientific content or presentation of this manuscript. The authors have responded professionally and the manuscript appears to be in compliance with standard ethical guidelines.

Reviewer #2: Appropriate changes have been made. The authors have successfully met all concerns raised by the reviewers.

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-->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: Ashraf Kariminik

Reviewer #2: No

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