PONE-D-24-44937Beyond antibodies: Beta-2 glycoprotein I as the unsung guardian of pregnancyPLOS ONE

Dear Dr. Nakamura,

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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: No

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: A manuscript by Nakamura and co-authors is potentially interesting. It brings a new perspective to the involvement of β2GPI in trophoblast cell function. However, the sudy has some major issues that need to be addressed.

- No details have been given for the Ethical approval for first trimester placental tissue, but only for blood samples. Please give details on who issued the approval, and the approval number for the tissue.

- Materials and Methods section needs more detail on the procedures. For example, labeling of proteins was not described well. Were these proteins from cell culture supernatants? Because the line 153 says “the same pooled serum was used as positive control”. Same to what? It is unclear.

- Only the method for cell viability was given (CCK8), and none for cell number determination, Yet, these data are presented in the Figure 1.

- Another important issue refers to the proliferation of primary trophoblast. In usual cell culture settings, cytotrophoblast does not proliferate, but rather differentiate and fuse in syncitium. Thus, it is unclear how the authors observed the proliferationin primary trophoblast cells.

- Students t-test is not apropriate for multiple compairsons. The results should be analyzed by One-Way ANOVA, followed by appropriate post-hoc test. How were the significances obtained?The authors talk about the dose -dependence (line 263). However, the statistical differences between the treatment groups is not shown, only compared to the control.

Other remarks:

In the Abstract, it is not clearly written what was investigated in the study. A sentence od two should be added to explain what was investigated and why, before the results.

Line 111 – extravillous trophoblast cell line

Line 158 and 159 – give the full names before introducing abbrevations TRX-1 and TRX-R, and briefly explain why they are added.

Line 132 – The title is not correct, no cell proliferation method is described. It is also gramatically not correct.

To what section do lines 389-394 belong? Should they be before Figure 5 lengend? This way this paragraph stands unlinked to other text.

Reviewer #2: This article in the abstract and introduction sets the scene why this research was undertaken which is easy to follow. The researchers may also wish to briefly include a few lines either in their discussion or introduction the recent online publication in Nature 11 December 2024 by Kelsey L Swingle et al Placenta-tropic VEGF mRNA lipid nanoparticles ameliorate murine pre-eclampsia, which found that "an endogenous targeting mechanism based on β2-glycoprotein I adsorption that enables LNP delivery to the placenta." This seems to further support the hypothesis of this paper that beta 2 glycoprotein I plays an important biological role in placenta biology.

The methods and ethics and statistical methodology are sound

Results figures and tables are easy to follow and have a logical sequence

My only concern is with line 281-282 (an approximately 5 fold increase.. Fig 3A). When I look at Figure 3A the increase is much less than 5 fold, going from approximately 12.5 ng/ml of total B2GPI in control to 20ng/ml of the TRXI-TRXR treated and to 16ng/ml total B2GPI for the pCMV3-APOH without TRX1-TRXR treatment.

The discussion is easy to follow and ties the results together nicely.

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Reviewer #1: No

Reviewer #2: Yes:  Bill Giannakopoulos

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<p>Beyond antibodies: Beta-2 glycoprotein I as the unsung guardian of pregnancy

PONE-D-24-44937R1

Dear Dr. Nakamura,

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,

María Teresa Llinás

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 #3: 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: (No Response)

Reviewer #3: Yes

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

Reviewer #1: (No Response)

Reviewer #3: 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: (No Response)

Reviewer #3: 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: (No Response)

Reviewer #3: 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: (No Response)

Reviewer #3: (No Response)

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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: No

Reviewer #3: No

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