-->PONE-D-25-65419-->-->Title - Assessment of Maternal Diet Inflammatory Status and Inflammatory Markers in Human Breast Milk.-->-->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: Yes

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: 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

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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 paper: “Assessment of Maternal Diet Inflammatory Status and Inflammatory Markers in Human Breast Milk by Courtney Slegers et al. is a very interesting topic and I only like to make some comments and suggestions.

Introduction:

The text is acceptable, but:

- Line 65, there is: “such as BMI and nutritional status”, but to my knowledge have the same meaning;

Materials and Methods:

- Lines 109-114, can you better explain the “automated” meaning? The self-administered recall tool is per se criticizable, but without any explanation is maybe worse;

Results:

- Lines 207-208, there is an explanation? In a western dietary condition, it is surprising;

- Lines 211-212, there is an explanation? or perhaps can it confirm that the “self admistered recall” can give not completed reliable data?

Discussion:

- Lines 295-300, it can be understandable that anti-inflammatory diets do not stimulate pro-inflammatory cytokines, but can you explain better the MCP-1 behavior? Besides what is showed in Lines 328-334?

- Lines 341-343, this is of course a limitation, but I consider much more important what I comment before of the recall tool as the authors suggest in lines 348-350;

- Lines 350-352, it is not feasible in similar conditions;

- Lines 376-378, please explain better.

Reviewer #2: The manuscript, titled 'Assessment of Maternal Diet Inflammatory Status and Inflammatory Markers in Human Breast Milk,' addresses the crucial issue of analyzing the composition of human milk and its impact on health, from the perspective of both the child and the mother. Notably, a substantial number of inflammatory markers were quantified in breast milk using flow cytometric bead arrays (13-plex panel: IL-4, IL-2, IP-10, IL-1β, TNF-α, MCP-1, IL-17A, IL-6, IL-10, IFN-γ, IL-12p70, IL-8 and free active TGF-β1). This is a unique approach.

I consider this a very valuable article. However, some issues should be corrected before final publication to improve its clarity, scientific validity and citation accuracy.

1. Considering the aim of the study: 'This study aimed to investigate whether maternal dietary inflammatory status, assessed using the DII, modulates concentrations of cytokines and chemokines in breast milk' and the fact that these relationships were confirmed only for MCP-1, and that the study indicated the need for further research, I believe the title should be modified to state that these are pilot studies. This is also confirmed by the numerous limitations of the study.

2. The introduction requires rewriting. It should be more in line with current knowledge and the stated purpose of the study. The concluding paragraph requires rewriting, and the stated purpose should be consistent throughout the manuscript—it should be concise and clearly define what the authors intend to demonstrate based on the data presented. Methodological elements are also unnecessary in this paragraph.

3. Methods:

Although the authors state that 'participants were screened against inclusion and exclusion criteria, as described in a previous study', I believe that a brief description of the study group, alongside a comparison to the general population of the country, should be included.

I am concerned about using a 24-hour dietary recall to assess dietary intake and the Dietary Inflammatory Index (DII). Was this a single interview?

If so, a single 24-hour dietary recall does not accurately reflect habitual intake patterns and is therefore not suitable for demonstrating a relationship between diet and milk concentration.

Why was a single (morning only) collection of human milk used?

Milk sampling procedures call for repeated sampling over a period of at least four hours, ideally at the following times: 06:00–12:00, 12:00–18:00, 18:00–24:00 and 24:00–06:00.

Furthermore, was this a sufficient sample size (n=101 or n=99)? Was an attempt made to calculate the minimum sample size?

Results:

Since inflammatory marker quantification was conducted on 99 participant breast milk samples (due to insufficient volume in the samples of two participants), why weren't all statistical analyses performed on n = 99? Table 1 contains data for n = 101. Why is this?

Table 2 cites 41 references (Global Daily Intake Mean (SD)[41]), but this item is not included in the bibliography (References)

Discussion

In my opinion, there is no need to recall the purpose of the study again.

The discussion should be expanded and strengthened with references to other literature — there is significantly more, and not all of them are included in the manuscript. Furthermore, since the authors state that no studies have been conducted on the link between maternal diet and inflammatory status or inflammatory markers in human breast milk, the discussion could focus on recommended diets, such as the Mediterranean diet, or the consumption of antioxidant compounds, such as carotenoids, polyphenols and antioxidant vitamins.

The manuscript lacks a conclusion or a separate summary section. Implications for further research could also be separated.

In summary, it is important to emphasise that the issues addressed in this study are highly significant in terms of infant and general public health.

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Reviewer #1: No

Reviewer #2: No

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Title - Assessment of Maternal Diet Inflammatory Status and Inflammatory Markers in Human Breast Milk.

PONE-D-25-65419R1

Dear Dr. Slegers,

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,

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

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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

**********

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

Reviewer #1: 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: 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: 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: No comment is needed because the authors have answered properly to all my questions and to all the suggestions

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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

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