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PONE-D-21-23121Trimester-specific phthalate exposures in pregnancy are associated with circulating metabolites in childrenPLOS ONE

Dear Dr. Goodrich,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: Summary

The authors attempt to perturb biochemical pathways associated with exposure to phthalates during gestation, which influence the development of cardiometabolic risk? The goal is unclear, but I believe that the authors are looking to provide proof of concept that global metabolomics could identify new biomarkers in human subjects to elucidate the effects of exposure in later life, as mentioned later on in the manuscript.

Basic summary: Phthalates were measured during each trimester and correlated with the metabolic profiles, of the children, of the participants in later life (the author does a good job of justifying and identifying studies in animals that show similar findings to theirs).

However, overall, it is not clear what their goal(s) are- leaving many unanswered questions.

Importance

The paper lacks focus on the importance of the study. Pregnant women typically know that they should avoid smoking, alcohol, and raw fish, etc., but is everyone aware that they should probably avoid lipstick, perfumes, etc., probably not. However, if we were able to discover a biomarker related to the mechanism of phthalate exposure, how would we intervene given that the ‘damage’ has already been done? Or is the goal to search for biomarkers of cardiometabolic syndrome? I think the former, please clarify why such biomarkers are needed/how they will be helpful for clinical decision making/intervention.

Major Comments

1. Clarify the goal of the study- Is your goal to identify biomarkers for cardiometabolic exposure or to elucidate the mechanism of phthalate exposure to predict cardiometabolic disease? Initially I thought the former but as I read the manuscript the later seemed more fitting. What are the clinical markers of cardiometabolic risk?

2. Cardiometabolic disorders represent a cluster of interrelated risk factors, primarily hypertension, elevated fasting blood sugar, dyslipidemia, abdominal obesity, and elevated triglycerides. How will new biomarkers be useful/what aspect of the diagnostic decision making is hindered? Assuming we find biomarkers for associated with risk how will they help with intervention?

3. Did you have a control group? How do you know that the statistical differences were due to phthalate exposure during gestation, and not sex, age, diet, etc.? Admittedly challenging to control for in human studies!

4. Looks like a lot of negative ion FA’s being identified in the male (boy) cohort- have you looked at %CV variability of each given that smaller molecules do not always ionize well in negative mode?

5. What is the distribution of the female (girl) cohort in terms of age? Testosterone is noted as being statistically significant but may also be due to age. The clinical lab applies different age-based reference intervals for total- and free-testosterone. Might be useful to report tanner staging results by sex in the supplemental in addition to age distribution of females (girls).

6. How did you normalize the data and adjust for batch effects? More detail needed in the method section overall. What mass spectrometer was used- Thermo Tribrid IQX MS, for example. Liquid chromatography- mobile phase, gradient, column temperature, etc. What software was used for data analysis? Should break this section down into metabolomics and lipidomics as details will vary between the two.

7. What extraction was carried-out per-application, metabolomics vs lipidomics?

8. Not clear what the comparisons are. When you say ‘girls only comparison? Is this between trimesters? Clarify overall comparisons that were carried-out.

Reviewer #2: This manuscript reports associations between 9 maternal urinary phthalate metabolites and untargeted metabolomics profiles in serum samples collected from children aged 8-14 years. The results present a large number of phthalate associations across trimester with serum metabolites among the entire cohort of children as well as within gender/sex. The primary weakness of the paper include:

- the introduction could consolidated to focus on the underlying biology of maternal phthalate exposure and pediatric outcomes. As written, the introduction is very long and provides information that is not specific to the research objective. It seems as though some of this information could be moved to the discussion.

- methods are completely lacking information on how the metabolites were measured using mass spectrometry. Additionally, there is inadequate information on data processing software, code and techniques.

- it is unclear why the analysis was stratified by gender/sex rather than a formal interaction test.

- the discussion suffers from the same issue as the introduction. It is rambling and hard to discern what is important. I would recommend consolidating the discussion into discreet paragraphs related to: overall cohort results, sex specific results, rationale biological explanation that links underlying biology to the results.

- results/tables would benefit from including standardized metabolite ID's (HMDB, chemspider) that will help with replicating these results in future studies.

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Reviewer #1: No

Reviewer #2: No

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Trimester-specific phthalate exposures in pregnancy are associated with circulating metabolites in children

PONE-D-21-23121R1

Dear Dr. Goodrich,

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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Timothy J Garrett, PhD

Academic Editor

PLOS ONE

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