PONE-D-24-37315Cytokines and appetite-regulating hormones in human milk and associations with infant growth across four sites in a longitudinal cohort: The Mothers, Infants and Lactation Quality StudyPLOS 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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

Reviewer #5: 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: Actually it's fantastic work, but you have long abstract if it short will be great papnd my comment

Clarity and Structure: Simplify complex sentences and provide a concise summary of the methodology.

Scientific Rigor: Clarify the rationale for marker selection and indicate if multiple comparison adjustments were made.

Results Presentation: Use tables or figures to enhance accessibility and specify adjustments for comparisons.

Findings Interpretation: Expand on mechanisms linking HM composition to growth, especially in The Gambia, while addressing potential confounders.

Ethical Considerations: Explicitly state ethical approval and informed consent procedures.

Language and Style: Revise grammatical issues and lengthy sentences for improved readability.

Public Health Implications: Highlight how findings could inform interventions in resource-poor settings.

Limitations: Mention limitations like selection bias or uncontrolled confounders.

Reviewer #2: Review Comments to the Author

General Comments:

The study titled "Cytokines and appetite-regulating hormones in human milk and associations with infant growth across four sites in a longitudinal cohort" investigates important and novel aspects of maternal and infant health. The multi-site design and extensive data collection add significant value to the field. However, there are areas where the manuscript can be improved to enhance clarity, interpretability, and scientific rigor.

Strengths:

Relevance and Novelty: The focus on cytokines and appetite-regulating hormones in human milk across diverse populations is novel and addresses a significant research gap.

Design and Scope: The multi-country longitudinal design adds robustness to the findings and allows for meaningful comparisons.

Detailed Analysis: The use of ANCOVA and linear regression models provides a comprehensive assessment of associations between milk markers and infant growth.

Major Comments:

Clarity in Methodology:

While the manuscript describes sample collection and analytical methods in detail, additional clarification on how non-detectable values were handled across all sites is needed. For example, were ND values imputed consistently across all markers?

The description of adjustments made for batch variability and inter-assay variability could be expanded to include specific steps to ensure reproducibility.

Statistical Analysis:

The use of log-transformed data for normality and subsequent back-transformation for reporting results is appropriate. However, it would help if the authors justified why specific covariates (e.g., maternal BMI) were chosen over others in the ANCOVA models.

Sensitivity analyses stratified by SCM status were mentioned but not extensively discussed in the results. Including a supplementary table summarizing key findings from this stratification could improve the manuscript.

Discussion and Interpretation:

The interpretation of cytokine levels differing across countries could be expanded to include potential cultural, dietary, and genetic influences.

The associations between human milk markers and infant growth in The Gambia, but not in other sites, are interesting but require a more in-depth discussion regarding possible underlying mechanisms.

Data Availability and Ethics:

The authors state that data are restricted due to confidentiality. Consider specifying how researchers can request access and what criteria will be used to evaluate such requests.

The manuscript adheres to ethical standards, but more detail on how local differences in ethical approvals were harmonized across sites could be provided.

Minor Comments:

Abstract:

The conclusion in the abstract mentions “atopic diseases” but does not clearly connect this to the study findings. Consider rephrasing to highlight only findings directly supported by the data.

Figures and Tables:

Figure legends could benefit from additional details to ensure standalone interpretability.

Table 3 presents adjusted means but does not specify confidence intervals consistently.

Language and Grammar:

Minor grammatical errors are present, such as “The results likely reflect, that different environmental exposures...” which could be rephrased for clarity.

Conclusion:

The manuscript is a valuable contribution to understanding the interplay between human milk composition and infant growth in diverse settings. However, addressing the above concerns will strengthen the rigor and clarity of the paper.

Reviewer #3: The manuscript provides a well-conducted piece of scientific research, with clear and reliable data that supports the conclusions. The experiments are carried out with careful attention to detail, ensuring that proper controls, replication, and sample sizes are in place. The conclusions was been writing based on the data in a thoughtful and logical way, accurately reflecting the findings of the research.

The statistical analysis appears to have been performed appropriately and rigorously R soft wears. However, it might be beneficial to consider increasing the cut of point of 1.5 or 2. This adjustment could help reduce the exclusion of significance certain variables, potentially leading to a more balanced and interpretable model.

Its good data in the manuscript or included as supporting information, or alternatively. In addition to summary, because it is correct statistics raw data points underlying the means, medians, and variance measures are available. This allows for greater transparency and enables other researchers to verify and build upon the findings.

Reviewer #4: Well-written and interesting paper.

The authors collected information on socio-economic status ob 3 vists by the mothers. However, it is not presented in any table or figure. Please include this in the manuscript, if this information was collected. As socioeconomic conditions were mentioned as possible reasons for the significant findings from Gambia, analysis of this variable may be important. The authors to conisder this.

Reviewer #5: Major Comments

1. Study Design and Methodology

Clarity of Objectives: The objectives of the study are clearly stated, focusing on cytokine and appetite-regulating hormone concentrations in human milk and their association with infant growth. However, further elaboration on how these objectives align with existing literature would strengthen the introduction.

Sample Size Justification: While the manuscript mentions a sample size of 825 mother-infant dyads, it lacks a detailed justification for this size in terms of statistical power. Including a power analysis would enhance the credibility of the findings.

2. Data Collection and Analysis

Cytokine Measurement Techniques: The methods used for measuring cytokines and appetite-regulating hormones should be described in greater detail, including any standardization processes or validation methods that were employed.

Statistical Analysis: The use of analysis of covariance (ANCOVA) is appropriate; however, the manuscript should specify which covariates were included in the model. Additionally, it would be beneficial to discuss how potential confounding factors were controlled.

3. Results Interpretation

Geometric Means Reporting: The results section reports geometric means for cytokines and hormones, but it could benefit from including confidence intervals or standard deviations to provide a clearer picture of variability.

Contextualization of Findings: The differences observed among study sites are significant; however, the discussion should better contextualize these findings within existing research on environmental factors affecting human milk composition.

4. Infant Growth Associations

Z-score Calculations: The method for calculating Z-scores for infant growth should be explicitly detailed to ensure reproducibility. It would also be helpful to clarify how these scores were interpreted in relation to growth standards.

Causal Inferences: While associations between HM markers and infant growth are noted, caution should be exercised in making causal inferences without longitudinal data supporting these claims.

5. Ethical Considerations

Ethics Statement Clarity: The ethics statement is comprehensive but could benefit from a more concise summary of ethical approvals obtained across different sites to enhance readability.

6. Funding and Competing Interests

Transparency in Funding Sources: The funding disclosure is provided; however, it should explicitly state whether funders had any role in study design or data interpretation to maintain transparency.

7. Conclusion Strengthening

Implications of Findings: The conclusion summarizes the results effectively but could further emphasize the implications for public health or future research directions based on the findings regarding human milk composition and infant growth.

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Reviewer #1: Yes:  Hussein Mussa Muafa

Reviewer #2: Yes:  Mahdy Ali Ahmad Osman

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

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Cytokines and appetite-regulating hormones in human milk and associations with infant growth across four sites in a longitudinal cohort: The Mothers, Infants and Lactation Quality Study

PONE-D-24-37315R1

Dear Dr. Sophie Hilario Christensen,

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Kind regards,

Mehran Rahimlou, PhD

Academic Editor

PLOS ONE

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