A maternal ketogenic diet alters oviduct fluid nutrients and embryo histone acetylation in mice.

In brief: A ketogenic diet (KD) elevates blood ß-hydroxybutyrate to concentrations that are known to perturb the development, metabolism, histone acetylation and viability of preimplantation mouse embryos in culture. This study shows that a maternal KD changes available nutrient levels in the oviduct, leading to altered embryo development and epigenetic state in vivo. Abstract: A ketogenic diet elevates blood ß-hydroxybutyrate to concentrations that perturb the development, metabolism, histone acetylation (H3K27ac) and viability of preimplantation mouse embryos in vitro. However, whether a ketogenic diet alters ß-hydroxybutyrate concentrations within female reproductive fluid is unknown. This study aimed to quantify glucose and ß-hydroxybutyrate within mouse blood and oviduct fluid following standard diet and ketogenic diet consumption and to assess whether a maternal periconceptional ketogenic diet impacts in vivo embryo development and blastocyst H3K27ac. Female C57BL/6 × CBA mice were fed a standard or ketogenic diet (n = 24 each) for 24-27 days. Glucose and ß-hydroxybutyrate were quantified in blood via an electronic monitoring system and in oviduct fluid via ultramicrofluorescence. The developmental grade of flushed blastocysts was recorded, and blastocyst cell number and H3K27ac were assessed via immunofluorescence. A maternal ketogenic diet elevated ß-hydroxybutyrate in day 24 blood (P < 0.001) and oviduct fluid (P < 0.05) compared with a standard diet, whereas glucose was unchanged. A periconceptional ketogenic diet did not impact blastocyst cell number; however, it significantly delayed blastocyst development (P < 0.05) and reduced trophectoderm-specific H3K27ac (P < 0.05) compared with standard diet-derived embryos. Maternal ketogenic diet consumption is, therefore, associated with reproductive tract nutrient changes and altered embryonic development and epigenetics in vivo. Future studies to assess whether periconceptional/gestational ketogenic diet consumption impacts human preimplantation, fetal, and long-term offspring development and health are warranted.

Preimplantation embryo exposure to ketone bodies exerts sex-specific effects on mouse fetal and placental transcriptomes.

RESEARCH QUESTION: Does in vitro exposure of preimplantation mouse embryos to the ketone bodies ß-hydroxybutyrate (ßOHB) and acetoacetate (AcAc) impact post-transfer fetal and placental gene expression? DESIGN: Blastocysts cultured in vitro with or without 2 mmol/l ßOHB alone ('ßOHB') or combined with 0.8 mmol/l AcAc ('Keto') underwent embryo transfer. Transcriptional profiles of sexed placenta, liver and brain at gestational day 14.5 were examined via RNA sequencing and DAVID functional analysis. RESULTS: A sexually dimorphic response to in vitro ketone exposure was observed. Both ßOHB and Keto exposure down-regulated genes related to oxidative phosphorylation specifically in female liver. ßOHB down-regulated female placental steroid biosynthetic processes, while Keto treatment up-regulated genes relevant to blood vessel formation and cell migration in male placenta. Brain transcriptomes were minimally affected. X-linked genes and chromatin modifiers were identified as differentially expressed in both liver and placenta, alluding to a sex-specific regulatory mechanism. CONCLUSIONS: Transient preimplantation ketone exposure perturbs sex-specific fetal liver and placental gene expression, demonstrating a developmental programming effect that warrants future investigation of the postnatal metabolic health of male and female offspring.

Acetoacetate and ß-hydroxybutyrate reduce mouse embryo viability via differential metabolic and epigenetic mechanisms.

RESEARCH QUESTION: Does the ketone acetoacetate (AcAc) alone, or combined with ß-hydroxybutyrate (ßOHB), impact mouse embryo development, metabolism, histone acetylation and viability? DESIGN: Pronucleate mouse oocytes were cultured in vitro in G1/G2 media supplemented with ketones (AcAc or AcAc + ßOHB) at concentrations representing those in maternal serum during pregnancy (0.04 mmol/l AcAc, 0.1 mmol/l ßOHB), standard diet consumption (0.1 mmol/l AcAc, 0.25 mmol/l ßOHB), ketogenic diet consumption (0.8 mmol/l AcAc, 2 mmol/l ßOHB) and diabetic ketoacidosis (2 mmol/l AcAc, 4 mmol/l ßOHB). Day 5 blastocysts were assessed for cell allocation, glucose metabolism and histone acetylation. Day 4 blastocysts exposed to 0.8 mmol/l AcAc + 2 mmol/l ßOHB were transferred to standard-fed recipient females, and E14.5 fetal and placental development assessed. RESULTS: Exposure to 2 mmol/l AcAc or 0.8 mmol/l AcAc + 2 mmol/l ßOHB did not impair blastocyst development, but significantly increased glucose consumption (P = 0.001 each), lowered glycolytic flux (P = 0.01, P < 0.001) and elevated trophectoderm (TE) histone 3 lysine 27 acetylation (H3K27ac; P < 0.001 each) compared with unexposed controls. Preimplantation AcAc + ßOHB exposure reduced post-implantation fetal development by 25% (P = 0.037), and delayed female-specific fetal limb development (P = 0.019) and estimated fetal age (P = 0.019) compared with controls. CONCLUSION: Preimplantation exposure to ketones affects underlying metabolism and histone acetylation in blastocysts that are associated with persistent, female-specific perturbations in fetal development. A periconceptional diet that elevates ketone concentrations may impair human embryonic viability.

ß-hydroxybutyrate reduces blastocyst viability via trophectoderm-mediated metabolic aberrations in mice.

STUDY QUESTION: What is the effect of the ketone ß-hydroxybutyrate (ßOHB) on preimplantation mouse embryo development, metabolism, epigenetics and post-transfer viability? SUMMARY ANSWER: In vitro ßOHB exposure at ketogenic diet (KD)-relevant serum concentrations significantly impaired preimplantation mouse embryo development, induced aberrant glycolytic metabolism and reduced post-transfer fetal viability in a sex-specific manner. WHAT IS KNOWN ALREADY: A maternal KD in humans elevates gamete and offspring ßOHB exposure during conception and gestation, and in rodents is associated with an increased time to pregnancy, and altered offspring organogenesis, post-natal growth and behaviour, suggesting a developmental programming effect. In vitro exposure to ßOHB at supraphysiological concentrations (8-80 mM) perturbs preimplantation mouse embryo development. STUDY DESIGN, SIZE, DURATION: A mouse model of embryo development and viability was utilized for this laboratory-based study. Embryo culture media were supplemented with ßOHB at KD-relevant concentrations, and the developmental competence, physiology, epigenetic state and post-transfer viability of in vitro cultured ßOHB-exposed embryos was assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mouse embryos were cultured in vitro with or without ßOHB at concentrations representing serum levels during pregnancy (0.1 mM), standard diet consumption (0.25 mM), KD consumption (2 mM) and diabetic ketoacidosis (4 mM). The impact of ßOHB exposure on embryo development (blastocyst formation rate, morphokinetics and blastocyst total, inner cell mass and trophectoderm (TE) cell number), physiology (redox state, ßOHB metabolism, glycolytic metabolism), epigenetic state (histone 3 lysine 27 ß-hydroxybutyrylation, H3K27bhb) and post-transfer viability (implantation rate, fetal and placental development) was assessed. MAIN RESULTS AND THE ROLE OF CHANCE: All ßOHB concentrations tested slowed embryo development (P < 0.05), and ßOHB at KD-relevant serum levels (2 mM) delayed morphokinetic development, beginning at syngamy (P < 0.05). Compared with unexposed controls, ßOHB exposure reduced blastocyst total and TE cell number (≥0.25 mM; P < 0.05), reduced blastocyst glucose consumption (2 mM; P < 0.01) and increased lactate production (0.25 mM; P < 0.05) and glycolytic flux (0.25 and 2 mM; P < 0.01). Consumption of ßOHB by embryos, mediated via monocarboxylate transporters, was detected throughout preimplantation development. Supraphysiological (20 mM; P < 0.001), but not physiological (0.25-4 mM) ßOHB elevated H3K27bhb levels. Preimplantation ßOHB exposure at serum KD levels (2 mM) reduced post-transfer viability. Implantation and fetal development rates of ßOHB-treated embryos were 50% lower than controls (P < 0.05), and resultant fetuses had a shorter crown-rump length (P < 0.01) and placental diameter (P < 0.05). A strong sex-specific effect of ßOHB was detected, whereby female fetuses from ßOHB-treated embryos weighed less (P < 0.05), had a shorter crown-rump length (P < 0.05), and tended to have accelerated ear development (P < 0.08) compared with female control fetuses. LIMITATIONS, REASONS FOR CAUTION: This study only assessed embryo development, physiology and viability in a mouse model utilizing in vitro ßOHB exposure; the impact of in vivo exposure was not assessed. The concentrations of ßOHB utilized were modelled on blood/serum levels as the true oviduct and uterine concentrations are currently unknown. WIDER IMPLICATIONS OF THE FINDINGS: These findings indicate that the development, physiology and viability of mouse embryos is detrimentally impacted by preimplantation exposure to ßOHB within a physiological range. Maternal diets which increase ßOHB levels, such as a KD, may affect preimplantation embryo development and may therefore impair subsequent viability and long-term health. Consequently, our initial observations warrant follow-up studies in larger human populations. Furthermore, analysis of ßOHB concentrations within human and rodent oviduct and uterine fluid under different nutritional states is also required. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the University of Melbourne and the Norma Hilda Schuster (nee Swift) Scholarship. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.