Short-Term Exposure with Long-Term Effects: The Ketone Body, Beta-Hydroxybutyrate, Alters Mouse Embryo Developmental Programming and Reduces Viability

Abstract

Background: Diet-induced nutritional changes in the maternal reproductive tract stimulate embryonic adaptations to maintain growth and survival, directly impacting child and adult health via developmental programming. This is plausibly facilitated by an interrelationship between metabolism and epigenetic control of gene expression, termed ‘metaboloepigenetics’, resulting in heritable modifications that affect long-term offspring health and viability. The ketogenic diet (KD) is increasingly popular amongst sub-fertile women trying to conceive, and results in elevated maternal ketone levels, including beta-hydroxybutyrate (bOHB). The impact of high bOHB levels on embryonic development, physiology, and developmental programming remains unknown. Aim: To assess how bOHB affects preimplantation mouse embryo development, metaboloepigenetic regulation, and post-transfer viability. Method: Preimplantation mouse embryos were cultured in vitro with or without 2 mM bOHB, representing serum concentrations with KD consumption. Day 5 blastocyst cell number and lineage allocation was assessed via differential nuclear stain, metabolism of bOHB and carbohydrates was assessed by ultramicrofluorescence, and acetylation of histone 3 lysine 9 (H3K9ac) and lysine 27 (H3K27ac) were assessed by immunofluorescence. Day 4 blastocysts were transferred to pseudo-pregnant females for analysis of embryonic day 14.5 placental and fetal development. Results: A reduction in total and trophectoderm cell number (P<0.05) was observed, indicating reduced viability. Blastocysts were shown to consume bOHB in vitro, with uptake increasing with bOHB concentration. Further, bOHB increased glycolytic rate (P<0.01), while epigenetic analyses revealed H3K9ac and H3K27ac were unaffected by bOHB. Of significance, post-transfer implantation rates were reduced (P<0.05), concurrent with smaller placental diameter (P<0.01) and fetal crown-rump length (P<0.05), indicating developmental programming alterations. Conclusion: These data infer that bOHB-stimulated metabolic alterations are a contributing factor to ketone-induced developmental programming, which may incorporate epigenetic components other than H3K9ac or H3K27ac. Therefore, consumption of a KD during conception and pregnancy may have negative impacts on the long-term viability and health of offspring.