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

Abstract

Low-carbohydrate, high-fat ketogenic diets (KDs) are used for treating drug-resistant epilepsy, and other potential benefits, such as treating neurological disorders, metabolic syndrome, and cancer are being explored. In addition to these and other medical applications, KDs have also become popular for rapid weight-loss and enhancing athletic performance. However, the potential negative effects of exposing developing offspring to KDs during pregnancy (gestational KD) are poorly understood, and especially the long-term health consequences. In this study, we investigated the effects of a partial gestational KD administered during the second half of pregnancy and assessed the consequences on the offspring over their entire lifespan. We found that, compared to controls, a gestational KD significantly reduced dams' litter size and litter mass and altered the litter sex ratio, reducing the proportion of female offspring, which also had lower body mass early in their life. In contrast, male offspring exposed to a gestational KD suffered a significantly reduced lifespan and a late-onset increase in body mass. We found no evidence that a KD diet influenced some adult offspring behaviors (locomotion, anxiety, depression, circadian rhythms, food and water consumption) or reproductive success. Our results with laboratory mice may not translate to human health, but nevertheless, they should raise concerns that even a partial maternal KD during pregnancy may have detrimental effects on offspring health and longevity.

Fig 1. Experimental timeline.

In the upper portion, timeline and manipulation of maternal diet until weaning of the offspring. Sample sizes depict the offspring number in the two cohorts. In the lower portion, the husbandry conditions illustrated per cage (squares around the mice) and offspring age during the behavioral assays. Green color: French cohort; Blue color: Austrian cohort. Abbreviations: CD = control diet, KD = ketogenic diet, BHB = β-hydroxybutyric acid, EPM = elevated plus maze, FST = forced swim test, SPF = specific pathogen free.

Fig 2. Effects of a partial KD in dams’ glucose and ketone levels and body mass during gestation.

(A-C) Box plots showing the dynamics of (A) blood glucose, (B) BHB, and (C) body mass in dams fed either a control diet or ketogenic diet (**p < 0.01, ***p < 0.001). Box plots include all values with outliers, median, individual values, first and last quartiles, whiskers drawn within the 1.5 interquartile range value.

Fig 3. A partial gestational KD impacted female, but not male, pups.

(A) Litter size by gestational diet. (B) Number of female (left) and male (right) pups per litter by diet. (C) Offspring sex ratio by diet. (D) Maternal body mass difference between G18.5 and post-delivery (G18.5-G19.5) by diet (left) and estimated mean pup mass calculated by dividing the maternal mass difference by the litter size (right). (E) Pup body mass at P9 by diet and sex. (F) Dynamics of pup body mass during the first month by diet and sex (mean±SEM). Box plots include all values with outliers, median, individual values, first and last quartiles, whiskers drawn within the 1.5 interquartile range value. Statistics A, B, D, and E: Student’s T-test; C: χ² test, F: repeated measures ANOVA. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4. A partial gestational KD did not affect metabolism, body mass, nor food and water intake.

(A) Blood glucose (right) and blood BHB levels (left) in adult offspring by diet and sex. (B) Body mass dynamics across time by diet and sex. (C) Total food (left) and water consumed over 78 h. Box plots include all values with outliers, median, individual values, first and last quartiles, whiskers drawn within the 1.5 interquartile range value. Statistics A-C: ANOVA, p > 0.05.

Fig 5. A partial gestational KD did not affect adult offspring spontaneous locomotion, susceptibility to anxiety and depression, nor circadian rhythms.

(A) Total distance traveled during 1 hour in the open field test (left) and time spent in the center (right) by diet and sex. (B) Time spent in the closed arm (left) and the open arm (right) during a 5-minute elevated plus maze assay. (C) Time spent immobile (left) or actively swimming (right) during a 5-minute forced swim test. (D) Locomotor activity in the light and dark cycles. Box plots include all values with outliers, median, individual values, first and last quartiles, whiskers drawn within the 1.5 interquartile range value. Line graphs (D) shows mean±SEM. Statistics A-C: Anova p > 0.05.

Fig 6. A partial gestational ketogenic diet had no lasting impact on offspring’s reproductive success.

(A) Latency to give birth (left) and number of weaned pups (right) by diet. (B) Offspring sex ratio by diet. A: T-test, p < 0.05; B: χ² test, p > 0.05. Box plots include all values with outliers, median, individual values, first and last quartiles, whiskers drawn within the 1.5 interquartile range value.

Fig 7. A partial gestational KD impacted male lifespan and mass during late-life.

(A) Body mass dynamics across time by diet: Repeated ANOVA, p < 0.05. (B) Survival rate of the offspring by sex and diet: Log-rank test: χ² = 7.47, p = 0.006.