-->PONE-D-25-63128-->-->The effect of intrauterine growth retardation on long-term lipid metabolism in the liver and nutrition regulation-->-->PLOS One

Dear Dr.  Laiyi,

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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

Reviewer #3: Yes

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: 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.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.-->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.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

Reviewer #3: Yes

Reviewer #4: No

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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

Reviewer #3: Yes

Reviewer #4: 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: This is a really interesting manuscript regarding the influence of intrauterine growth restriction on liver development and structure. There are very few minor issues that I should point out:

- In the Methods section, the word ”rats” in the last sentence of the first paragraph and in the beginning of the second paragraph should be replaced with ”offspring” for extra clarity. There is no mention whether the number of live pups in the IUGR group was different from controls.

- In the Methods section, the moment when euthanasia is performed should be more clearly described – is it performed immediately as first stated, or on designated endpoints (P1, P7...)? There seems to be a slight misunderstanding

- In the Results section, there is mention of a ”liver index”, but this should be first described in the Methods section. Is it Weight of liver/Weight of body x 100?

- In the second paragraph of the Results section, there are two instances where ”FAA” should be replaced with ”FFA”

- In the following subsection, regarding the use of folic acid, the first two sentences do not belong here, but in the Introduction section, as they depict premises for this study.

- In the first sentence in the fourth paragraph of the Discussion section, the plural should be replaced with singular (”Folic acid is commonly used as dietary supplement ...”). In the third sentence, a different word should be used insted of ”improves” to make it less confusing

- I do not think the table is relevant enough to be used in the published material

- Fig. 1 A should be either a separate figure, used in the Methods section, or it should be removed

- Is ”ns” an acronym for ”not significant”? It probably is, but there is no mention of that.

Reviewer #2: Title: The effect of intrauterine growth retardation on long-term lipid metabolism in the liver and nutrition regulation

This study provides substantial evidence, using an animal model, that intrauterine growth restriction (IUGR) leads to dysregulation of hepatic lipid metabolism in adulthood and that early postnatal folate supplementation exerts sustained beneficial effects on these metabolic disturbances. The work is well grounded in the DOHaD (Developmental Origins of Health and Disease) framework and demonstrates high clinical relevance as an early-life nutritional intervention strategy. The experimental design is sound, and the results are consistent and robust throughout the study.

� Methods

- P14 A total of 146 rat pups were included in this study; however, no sex-stratified analyses were presented. Given that responses may differ by sex, it would be important to include sex-specific analyses if such data are available.

- p14;14 Folic acid supplementation was initiated from postnatal day 21; could you please clarify the rationale for selecting this time point?

Could the authors clarify how the folic acid dosage was determined, and whether this dose corresponds to a standard physiological dose or a supraphysiological (high) dose?

- P17;1 Statistical analysis – The authors are encouraged to consider re-analyze longitudinal data using repeated-measures ANOVA or mixed-effects models to account for intra-individual correlation over time.

� Results

- The “Results” section currently includes descriptions that would be more appropriately placed in the “Methods” and/or “Discussion” sections. It would be necessary to revise this section so that it focuses exclusively on the presentation of experimental results.

Example) p18. We induced IUGR offspring models in offspring using maternal calorie and protein restriction, which has been previously validated. � This description would be more appropriate for the Methods section

P19. As reported, PPARα signals were essential for regulating TG deposition and hepatic damage for IUGR.

� Figures

- References to Figure 3J, 3K are missing from the main text.

Therefore, this manuscript represents a study with considerable merit for publication.

Thank you.

Reviewer #3: Labels G and H need to be interchanged in Figure 1 and its reference in the respective text. IUG also need to be switched to IUGR in Figure 1 legend.

Also, "We" needs to be non capital in the forts sentence of the last paragraph before discussion.

Reviewer #4: Title need to be self-explanatory; here information of folic acid seems less understandable

Methods

Need to mention how randomization was done. It is not clear how IUGR in rats was determined in method section

A flow diagram could help for better understanding

Result

Consistent with previous studies, levels of serum triglycerides (TG) and

free fatty acids (FFA) significantly increased in IUGR rats compared to

control rats during the early stages……. Please use reference

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Reviewer #1: No

Reviewer #2: Yes: Seoheui CHOISeoheui CHOISeoheui CHOISeoheui CHOI

Reviewer #3: No

Reviewer #4: Yes: Sanjoy kumer deySanjoy kumer deySanjoy kumer deySanjoy kumer dey

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Attachment

Submitted filename: PONE_D_25_63128 review.docx

Folic acid supplementation ameliorates long-term lipid metabolism following intrauterine growth restriction

PONE-D-25-63128R1

Dear Dr. Zhou Laiyi,

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.

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Kind regards,

Ewa Tomaszewska, DVM 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 #4: 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 #4: Yes

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

Reviewer #4: 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.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.-->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.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 #4: 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 #4: 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 #4: (No Response)

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Reviewer #4: Yes: Sanjoy kumer deySanjoy kumer deySanjoy kumer deySanjoy kumer dey

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