School of BioSciences, University of Melbourne, Parkville, Victoria, Australia.
Department of Anatomy & Physiology, University of Melbourne, Parkville, Victoria, Australia.
Hum Reprod. 2022 Aug 25;37(9):1994-2011. doi: 10.1093/humrep/deac153.
What is the effect of the ketone β-hydroxybutyrate (βOHB) on preimplantation mouse embryo development, metabolism, epigenetics and post-transfer viability?
In vitro βOHB 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.
A maternal KD in humans elevates gamete and offspring βOHB 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 βOHB 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 βOHB at KD-relevant concentrations, and the developmental competence, physiology, epigenetic state and post-transfer viability of in vitro cultured βOHB-exposed embryos was assessed.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Mouse embryos were cultured in vitro with or without βOHB 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 βOHB exposure on embryo development (blastocyst formation rate, morphokinetics and blastocyst total, inner cell mass and trophectoderm (TE) cell number), physiology (redox state, βOHB metabolism, glycolytic metabolism), epigenetic state (histone 3 lysine 27 β-hydroxybutyrylation, H3K27bhb) and post-transfer viability (implantation rate, fetal and placental development) was assessed.
All βOHB concentrations tested slowed embryo development (P < 0.05), and βOHB at KD-relevant serum levels (2 mM) delayed morphokinetic development, beginning at syngamy (P < 0.05). Compared with unexposed controls, βOHB 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 βOHB by embryos, mediated via monocarboxylate transporters, was detected throughout preimplantation development. Supraphysiological (20 mM; P < 0.001), but not physiological (0.25-4 mM) βOHB elevated H3K27bhb levels. Preimplantation βOHB exposure at serum KD levels (2 mM) reduced post-transfer viability. Implantation and fetal development rates of βOHB-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 βOHB was detected, whereby female fetuses from βOHB-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 βOHB exposure; the impact of in vivo exposure was not assessed. The concentrations of βOHB utilized were modelled on blood/serum levels as the true oviduct and uterine concentrations are currently unknown.
These findings indicate that the development, physiology and viability of mouse embryos is detrimentally impacted by preimplantation exposure to βOHB within a physiological range. Maternal diets which increase βOHB 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 βOHB 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.
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β-羟丁酸（βOHB）对植入前小鼠胚胎发育、代谢、表观遗传学和移植后活力的影响是什么？
在体外，以与生酮饮食（KD）相关的血清浓度暴露于βOHB 会显著损害植入前小鼠胚胎的发育，以性别特异性的方式诱导异常的糖酵解代谢，并降低移植后的胎儿活力。
人类的母体 KD 在受孕和妊娠期间会提高配子和后代的 βOHB 暴露水平，而在啮齿动物中，这与妊娠时间延长以及后代器官发生、产后生长和行为改变有关，表明存在发育编程效应。在体外，βOHB 在高于生理浓度（8-80mM）下的暴露会干扰植入前小鼠胚胎的发育。
研究设计、规模、持续时间：利用小鼠胚胎发育和活力模型进行了这项基于实验室的研究。胚胎培养介质中添加了与 KD 相关的浓度的 βOHB，评估了体外培养的βOHB 暴露胚胎的发育能力、生理机能、表观遗传状态和移植后活力。
参与者/材料、设置、方法：在 0.1mM（代表妊娠期间的血清水平）、0.25mM（标准饮食消耗）、2mM（KD 消耗）和 4mM（糖尿病酮症酸中毒）的条件下，将小鼠胚胎在含有或不含有βOHB 的培养基中进行体外培养。βOHB 暴露对胚胎发育（囊胚形成率、形态动力学和囊胚总细胞数、内细胞团和滋养外胚层（TE）细胞数）、生理机能（氧化还原状态、βOHB 代谢、糖酵解代谢）、表观遗传状态（组蛋白 3 赖氨酸 27 β-羟丁酸化，H3K27bhb）和移植后活力（着床率、胎儿和胎盘发育）的影响进行了评估。
所有测试的βOHB 浓度都减缓了胚胎发育（P<0.05），而 KD 相关血清水平（2mM）的βOHB 延迟了形态动力学发育，从合子形成（syngamy）开始（P<0.05）。与未暴露的对照组相比，βOHB 暴露降低了囊胚总细胞数和 TE 细胞数（≥0.25mM；P<0.05），降低了囊胚葡萄糖消耗（2mM；P<0.01），增加了乳酸产生（0.25mM；P<0.05）和糖酵解通量（0.25 和 2mM；P<0.01）。胚胎通过单羧酸转运体消耗βOHB，在整个植入前发育过程中都能检测到。高水平的（20mM；P<0.001）但不是生理水平的（0.25-4mM）βOHB 会升高 H3K27bhb 水平。植入前的βOHB 暴露在 KD 水平（2mM）时降低了移植后的活力。βOHB 处理胚胎的植入和胎儿发育率比对照组低 50%（P<0.05），结果胎儿的头臀长（crown-rump length）更短（P<0.01），胎盘直径（placental diameter）更小（P<0.05）。βOHB 还表现出强烈的性别特异性效应，与雌性对照胎儿相比，βOHB 处理的胚胎中的雌性胎儿体重更轻（P<0.05），头臀长更短（P<0.05），并且耳发育速度加快（P<0.08）。
局限性、谨慎的原因：本研究仅在利用体外βOHB 暴露的小鼠模型中评估了胚胎发育、生理机能和活力；未评估体内暴露的影响。βOHB 的浓度是根据血液/血清水平建模的，因为目前还不知道真正的输卵管和子宫内的浓度。
这些发现表明，βOHB 在生理范围内的植入前暴露会损害小鼠胚胎的发育、生理机能和活力。增加βOHB 水平的母体饮食，如 KD，可能会影响植入前胚胎的发育，并因此损害随后的活力和长期健康。因此，我们的初步观察结果需要在更大的人群中进行后续研究。此外，还需要分析不同营养状态下人类和啮齿动物输卵管和子宫内液体中的 βOHB 浓度。
研究资金/利益冲突：这项工作得到了墨尔本大学和 Norma Hilda Schuster（née Swift）奖学金的资助。作者没有利益冲突。
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