Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Below is a detailed review of the manuscript:

 “Lowered maternal and paternal plasma concentrations of choline are associated with the severity of congenital heart defects in the offspring” (Nutrients, peer-reviewed version).

General assessment

This manuscript examines the associations between parental plasma choline concentrations and the severity of congenital heart disease (CHD) in offspring at an average age of 3 years, using a trio family model. This topic is timely and relevant, particularly given the growing interest in one-carbon metabolism and the influence of parental factors, including diet and their metabolism, on offspring health.

The obtained results may have potential practical implications for guiding dietary and supplementation recommendations. However, some methodological and interpretative issues require clarification before publication.

The work is structured in accordance with the journal's requirements.

Abstract – in accordance with the journal's requirementsIntroduction

Introduction

It provides basic information on the metabolic effects of paternal factors before conception, and this aspect should be expanded upon to justify the research.

 

Methods

There are several points that require clarification in this section.

The main limitation of the study stems from the timing of biomarker measurement, which raises problems with the interpretation of the results. Biomarkers were measured on average 3.1 years after the child's birth, a time distant from the periconceptional period. This raises the question of whether current choline concentrations truly reflect periconceptional status and whether the observed association may be due to dietary patterns rather than a direct effect on embryogenesis. The methodology lacks information regarding the diets of parents and children – were such questions asked in the questionnaire? Furthermore, the issue of vitamin supplementation during pregnancy requires clarification – whether it was administered and, if so, with which preparations. Another concern is the study's inclusion criteria. The authors acknowledge that the study included children with CHD with and without associated syndromes. It would be helpful to specify which associated syndromes are being addressed, as, for example, genetic syndromes can affect one-carbon metabolism. It is also not fully clear whether BMI, socioeconomic status, supplementation, dietary choline intake, or genetic factors (e.g., MTHFR, BHMT) were controlled for. Considering these variables when studying one-carbon metabolism is particularly important.

Another issue is that the threshold of < 10 µmol/L for choline is not a universally recognized clinical cut-off for deficiency - it is rather an interpretative value and this aspect needs to be emphasized in the paper.

The number of fathers with available data was only 43–44 in the biomarker analyses, which may be important for the obtained results and requires the possibility of a Type I error discussion. There is no information as to why samples were missing. There is no information on whether collinearity in the models was checked and whether interactions were tested, which could increase the value of the work.

 

Discussion

The discussion should be more balanced regarding the obtained results, and the paragraph on Limitations should emphasize more the issues related to the number of studies, the time of analysis and the potential influence of lifestyle and diet on the results.

 

References

References: please ensure full and properly formatted references with DOI. The completeness of the references requires checking and correction.

The paper [12] from 2025 in Nutrients may be "ahead of print" or potentially an article from the same group - it requires checking whether it was actually published (risk of self-plagiarism extension of the concept). Citations 38 need to be verified – the full data in the references is missing.

 

Comments on the Quality of English Language

There is a mistake in the abbreviations section: "methylenetetrahydrofolate reeducate" instead of "reductase"

In the text: Prevalence of Congenital Heart Failure in Newborns. This is an error – PAN refers to: Prevalence of Congenital Heart Defects in Newborns. Reference [1] is correct.

Author Response

Please see separate document

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This new manuscript tests for an association between the severity of congenital heart defects against one carbon metabolite levels in the plasma and urine of the child patients (mean age 3yr) and their parents. The cohort is from a German cardiac defects registry. Cardiac defects were classified as “mild” or “severe” based on definitions from EUROCAT or PAN (both were done). Plasma and urine from the index case and parents were assayed for choline, betaine, and folate metabolites. Results find that plasma choline values in the mother and father, but not in the child, were associated with CHD severity. <10 umol/L choline ad greater odds of severe CHD aOR 3.7; 95% CI 1.1 – 12.2 in mother; similar values for father.  Multiple studies implicate one-carbon metabolism in healthy cardiac development in preclinical models and associations in surveillance studies, so the hypothesis is biologically plausible.

The study is well designed, analyzed, and discussed. Minor concerns would improve the manuscript further and can be readily addressed.

 

1. Alcohol is a known cardiac teratogen and alcohol consumption was obtained from the parents. Rates of self-reported alcohol exposure during pregnancy in Germany can be as high as 40%. Was the data analysis stratified by alcohol intake? If not, this needs to be stated as a limitation of the study. This is of especial concern given growing evidence of alcohol-choline interactions during pregnancy.

 

2. CHDs were classified as ‘mild’, ‘moderate’ or ‘severe’ based on the EUROCAT and PAN classifications, which appear to be designed to facilitate treatment rather than inform underlying mechanisms. Unfortunately, this classification ignores that CHDs have multiple mechanisms of causation and emerge at different times of development. Thus, lumping CHDs by severity buries important information that could otherwise inform the association with choline and the actual risk. For example, deficits in chamberization and looping occur in the early first trimester, whereas ASDs and VSDs can originate during 1^st trimester or from perinatal insults; that is, ‘severe’ defects generally originate during 1^st trimester and the mild deficits often reflect failures to close around birth. As they are mechanistically distinct, choline may play very different roles on CHD risk and severity. This needs to be acknowledged as a limitation of the study.

 

3. Table 3 is unclear. What are the two lines of data for most measurements? Is the upper line mean and the lower line median? This should be stated explicitly rather than relying on the different brackets. Perhaps in the Title?

 

4. That these CHDs emerge mostly during the first trimester, and parental / child choline values are not made until 3yr after birth is a significant limitation that needs to be acknowledged.

Typos:

                Line 323 – mating not matting

                Line 323 – what is meant by 1/8 choline?

 

Author Response

Please see separate document

Author Response File: Author Response.pdf