Stress and DNA Methylation of Blood Leukocytes among Pregnant Latina Women

Round 1

Reviewer 1 Report

The authors tried to examine a group of genes some of which were reported to be differentially methylated in stress. There are several issues in the paper which are confusing for the readers specially those who are interested in biology or clinical interpretation &/or utility of the study

Secondary analysis was done from EPIC array data which had >850000 loci. They selected 409 CpG covering their gene of interest. Therefore the multiple testing correction was done on those 409 loci. None of the locus passed even this level of multiple correction.

Authors have not mentioned anywhere about the magnitude of differential methylation.  Interpretation of such methylation data based on p-value and unnecessary statistical correction is simply confusing.

Authors may aware of the fact that methylation is very much tissue specific and there is significant degree of person-to-person variation in methylation data. They have very correctly mentioned the need of pre-pregnancy blood sample to be used as control in the experimental design.

All the participants in this study were pregnant women. So there could be no confusion regarding the sex at all. Yet what was the rationale for "removal of outliers of median methylation values and wrong sex prediction samples" as mentioned in the methylation analysis section? Do you really need sex prediction in such individuals who were pregnant? Overuse of this type of statistical correction in biological/health research field is perhaps not needed.

In the method section, the authors mention that they extracted T-lymphocytes. So did the methylation from DNA from these DNA samples really needed further cell compositional analysis to complicate. If DNA was extracted from whole blood containing neutrophil, lymphocytes (T- and B- lymphocytes), then perhaps such compositional analysis would make some sense. 

With the prior knowledge of the "phenotype of interest - stress", the authors could have used hormone level (measured from the collected blood samples for the trial to make some biologically relevant data analysis.

What was the rationale for Inclusion of African American female participants along with “Latina"?

The authors are definitely aware of the fact that methylation of CpG in promoter or enhancers may not have similar effect as methylation of gene body, or intergenic regions. They have not mentioned anything anywhere about the loci they have examined that could have some biological meaningful insight.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

The authors of the manuscript 'Stress and DNA methylation of blood leukocytes among pregnant Latina women' present an important topic that is  methodologically and scientifically important. The manuscript is well structured and well written. However, the methods could use some more details to make it more reproducible. Please add in catalog numbers of the reagents used and version numbers for the various packages use in analysis.

English quality is good. Does not require editing. 

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

Dear Authors,

The manuscript entitled "Stress and DNA methylation of blood leukocytes among pregnant Latina women" represents a valuable work in the field.

Below you will find my comments.

1) In the introduction section, i think it would be better for the readers to add more details regarding the action of the studied genes (NR3C1, BDNF, FKBP5, HSD11B2, SLC6A4, CRHR1, CRHR2, and 123 NR3C2)

2) How the authors concluded to study only these genes.

3) Maybe it is a good idea to be added as suplementary the association of specific genes with other function. For this purpose the database https://string-db.org/ can assist in this.

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Revised version has not improved the paper. Health related research work is supposed to put some emphasis on health &/or biology and potential impact on practical application.

Author Response

This paper presents findings of a pilot project that relates to human health very directly, by studying an important potential mechanism leading to poor birth outcomes for a high risk group- Latina women. Though we had null findings, this study adds to the very limited literature on this vulnerable group.