PREreview of Sex-dependent effects of a gestational ketogenic diet on offspring birth and lifespan

Summary:

This study investigates the impact of a gestational ketogenic diet (KD) in mice on offspring development and long-term health. The researchers aimed to determine the sex-dependent effects of a KD administered during late gestation (gestational days G8-G18) on various offspring outcomes, including metabolic profile, emotional behavior, circadian rhythms, reproductive success, and lifespan. Pregnant female mice were subjected to either a KD (84% fat, 11% protein, 5% carbohydrate) or a control diet (13% fat, 20% protein, 67% carbohydrate), and offspring were monitored throughout their lives. The study found that gestational KD significantly reduced litter size and litter mass, altered the sex ratio at birth, and reduced the lifespan of male offspring. Additionally, male offspring in the KD group exhibited increased body mass starting at 2.2 years of age. We found the study relevant because previous studies have shown mixed results or focused on KDs applied throughout the entire gestation. However, the effects of exposing developing offspring to a KD during pregnancy particularly the long - term health outcomes are not well understood. We found the study interesting and relevant, and after reading it, we provide here a series of suggestions and comments that in our opinion would improve the paper.

Major Comments:

One of the main limitations of this study may be the differences in litter sizes between the experimental groups, which may have a profound effect on the phenotype of the offspring, independently of the diet that the dam had during pregnancy. This aspect should be addressed in this study. Authors could set the same number of pups between the experimental groups and then analyze possible differences in phenotype. The authors should address the potential limitations of a small sample size, particularly concerning the number of female animals. A power analysis could be included to justify the sample size used. Additionally, in this type of studies, it is important to clarify whether litter size or individual pups were considered the experimental unit, and discuss how this choice might influence the interpretation of results. See for example: PMID: 16391426 DOI: 10.1093/ilar.47.1.5.

While the experiment is elegantly conducted, it does not allow to disentangle the effect of the KD and an abrupt switch in macronutrient availability. Therefore, the study conclusions would be further supported/disputed if other control groups (e.g., KD during all pregnancy, progressively increasing fat content until reaching the KD to avoid a sharp change in nutrient availability) had been included. Likewise, adding another group with control diet supplemented with ketones could help elucidate whether the effects are attributable to these metabolites vs other factors implicated in the KD.

The authors report an effect of the gestational KD on male body weight (figure6). However, this data might be biased by the body weight of the deceased mice. Differences in body weight became apparent only after day 750. By this time, large differences in the survival rate were present between groups. The fact that these differences are detected then but not before suggest that there might be differences in the body weight of the deceased/surviving mice. This could be addressed by conducting sensitivity analyses with data of mice living longer than X days. Is the weight of mice living longer than 750 days really increasing sharply, or is it that once the lighter mice are dead, the average weight is driven by the heavier surviving mice?

The authors should consider analyzing a separate cohort of animals for in-depth organ-specific studies that may provide information on mechanisms of action behind the observed phenotype. This would allow for a more comprehensive investigation of potential organ dysfunction that may not be apparent from post-mortem analyses. Specifically, consider adding histological or functional assessments of key organs (e.g., brain, liver, reproductive organs) at multiple time points throughout the lifespan of the offspring, not just at the endpoint. This could reveal earlier changes or subtle pathologies that contribute to the observed lifespan reduction and other phenotypes.

The authors should revise the title of the figure related to circadian activity rhythms to reflect the actual findings. Since the text indicates no effects on activity rhythms were found, but the authors acknowledge that this doesn't exclude changes in other aspects of circadian rhythms, a more accurate title would be, for example, 'Effect of gestational KD on circadian activity rhythms' or 'Gestational KD does not alter offspring circadian activity rhythms'. This will prevent potential misinterpretation of the data.

Minor Comments:

We found that Figure 2F and 6B were a bit difficult to read. We suggest to improve the clarity of these figures. The use of dotted lines to distinguish between experimental groups makes it difficult to visually differentiate the data. We suggest using distinct colors for different experimental groups. Additionally, consider using a combination of color and line style (e.g., one color for males, another for females, and solid vs. dashed lines to further differentiate groups) to enhance readability and prevent potential confusion. Please ensure that figure legends clearly explain the color and line style conventions.

While the statistical methods seem generally appropriate, a more detailed description of the specific tests used for each comparison (e.g., which post-hoc test was used following ANOVA) would improve the rigor of the reporting.

The authors should expand the discussion of the study's limitations. In addition to the points already raised, consider discussing potential limitations related to the animal model, the specific KD formulation used, and the generalizability of the findings to human populations.

The data in figure 5, panel A, CD show a bimodal distribution. It might worth exploring why the animals seems to cluster into 2 subgroups

Competing interests

The authors declare that they have no competing interests.