The transgenerational impact of high fat diet and diabetic preg-nancy on embryonic transcriptomics and mitochondrial health

Overnutrition increases comorbidities such as gestational diabetes during pregnancy that can have detrimental consequences for both parent and progeny. We previously reported that high fat (HF) diet and late-gestation diabetes (DM) incite mitochondrial dysfunction, oxidative stress, and cardiometabolic disease in first generation (F1) rat offspring, partially through epigenomic and transcriptomic programming. Oogonia, which become the second generation (F2), are also exposed which could incite generational risk. This study aimed to determine whether the F2 transcriptome already has genomic variation at the preimplantation embryo stage, and whether variations normalize, persist or compound in the third generation (F3). F0 female rats were fed a control or HF diet, then DM was induced in HF-fed dams on embryonic day (E) 14 exposing F1 offspring and F2 gametogonia to hyperlipidemia, hyperglycemia and fetal hyperinsulinemia during the last third of pregnancy. F1 pups were reared by healthy dams and bred to produce F2 embryos (F2e) and F2 pups. F2 offspring were bred to produce F3 embryos (F3e). Embryos were assessed by a novel grading method, live cell imaging, and single-cell RNA sequencing. Embryo grades were not different, but HF+DM F2e had more cells while F3e had fewer cells and overall fewer embryos. HF+DM F2e had similar mitochondria quantity but a down regulation of genes involved in lipid metabolism and more oxidative stress, consistent with mitochondrial dysfunction. They also had an upregulation of chromatin remodeling genes. The predicted developmental effect is accelerated embryo ageing and epigenetic drift. In contrast, HF+DM F3e had an adaptive stress response leading to increased mitochondria quantity and an up regulation of genes involved in mitochondrial respiration, metabolism, and genomic repair that led to a predicted developmental effect of delayed embryo maturation. Although pathways vary, both generations have metabolically linked differentially expressed genes that influence cell fate and developmental pathways. In conclusion, HF+DM pregnancy can program the early embryonic transcriptome for three generations, despite an intergenerational healthy diet. scRNAseq of F2 and F3 generation rat blastomeres from F0 controls or HF fed dams with late-gestation diabetes