体外受精技术（IVF）子代的代谢性疾病风险的机制研究及防治策略

In vitro fertilisation (IVF) has revolutionised human reproduction and enabled millions of previously infertile couples to have children. However, clinical data collected in recent years indicates that children conceived by IVF are at increased risk of developing type 2 diabetes and cardiovascular disease. These findings have been mirrored in IVF mouse models. In 2014, we reported for the first time that young IVF adults (<26 years) display peripheral insulin resistance, which is considered a hallmark of these metabolic diseases. We observed similar results in genetically identical, young adult C57Bl6/J mice that were generated utilising state of the art embryo culture. Our pilot data indicates there is a reduction in mitochondrial content in the blastocysts as a result of IVF. This proposal will now build on these preliminary findings to assess the mitochondrial metabolism in the oocytes, 2-cell embryos and blastocysts, metabolically active tissues of IVF offspring produced by different treatments, to examine the phenotypes of glucose metabolism and the levels of transcription, DNA methylation and protein expression of mitochondrial related genes in IVF offspring. Therefore, this proposal will determine if a delay in mitochondrial development in IVF mouse blastocysts is translated into metabolically active tissues of IVF offspring (muscle, liver and adipose tissue) and results in the insulin resistant phenotype; whether the metabolic phenotype observed in IVF is mainly related to the process of embryo culture; whether the impaired mitochondrial development and metabolic phenotype of IVF can be rescued by reducing the doses of ovarian stimulation including natural IVF or minimal stimulation, shortening the culture period such as 2-cell embryo transfer, or adding an antioxidant melatonin to mouse embryo culture. Findings from this body of work will drive pre-clinical studies to mitigate and/or eliminate the metabolic health risks of IVF for future generations.

体外受精技术（IVF）使数百万不孕症夫妇成功生育后代，但近年来的研究表明IVF子代的代谢性疾病如2型糖尿病的风险可能增加。本项目旨在之前的研究基础上在C57BL/J小鼠模型中检测不同治疗方式对卵细胞、2细胞胚胎和囊胚的线粒体代谢及胚胎发育的影响，分析子代的糖代谢和不同发育阶段代谢活跃组织的线粒体代谢、相关基因的转录、甲基化及表达分析，并了解在培养液中添加抗氧化剂褪黑素对胚胎发育及子代糖代谢表型的影响。本项目将明确IVF所致的胚胎线粒体发育异常是否延续至子代的代谢活跃组织或器官，并导致胰岛素抵抗；是否主要由体外培养所致；是否可以通过减少超排卵药物的剂量如自然周期IVF或微刺激IVF、缩短体外培养的时间如2细胞胚胎移植或者在培养液中添加抗氧化剂褪黑素等方式改善胚胎线粒体发育并消除IVF子代的胰岛素抵抗的风险。本研究将为人类临床前期试验提供理论依据，以实现在IVF子代中消除代谢性疾病风险的目标。

IVF（in vitro fertilization）是治疗不孕症常用的方法，至今约有800万婴儿通过IVF助孕后出生。但是大量研究发现IVF后代低出生体重，并且成年后肥胖和罹患代谢性疾病（例如2型糖尿病和心血管疾病）的风险增加。体外受精过程产生的大量氧自由基和代谢产物将对卵母细胞产生不良影响，干扰配子受精和胚胎发育，这可能是IVF胚胎发育迟缓甚至阻滞和后代不良代谢表型的重要原因。有研究通过向培养基中添加抗氧化剂来提高胚胎的质量。在人类前瞻性随机研究中，褪黑素可以改善卵的质量，增加成熟卵的数量，并可以通过口服的方式提高优质胚胎的比率。在小鼠实验中，在培养基中补充褪黑素可以上调线粒体DNA合成的基因转录，并增加线粒体DNA的拷贝数和生物合成。在本项目研究中，我们构建了小鼠模型，用于移植的囊胚是通过自然交配（对照组）或在有或没有褪黑素（10-6M）的IVF（分别为mIVF和IVF组）临床级受精和培养基中产生的，以分析褪黑素对IVF后代发育和代谢的影响。通过研究，我们发现IVF胚胎Fbxl7表达下调和发育迟缓，其在子代肝脏表达降低是导致IVF子代糖代谢紊乱的重要原因，而褪黑素可以通过上调肝脏Fbxl7的表达进而显著改善子代的表型。作为泛素化蛋白家族的成员，Fbx17通过PI3K-AKT和MAPK途径影响葡萄糖和脂质代谢。通过进一步的机制研究，我们还发现Fbxl7在IVF小鼠肝脏中受转录因子Foxa2调节。Foxa2也是肝特异性基因（如白蛋白和转甲状腺素蛋白）的转录激活因子，并在代谢调节和胰腺及肝脏分化中发挥重要作用。此外，我们还证明Fblx7通过靶向其下游基因Rhoa，在糖脂代谢中发挥调控作用，Rhoa的作用是在信号转导级联反应中充当分子开关，受到上游信号的刺激之后激活或关闭相应的信号通路，参与各种营养物质的代谢和分解。.总之，我们确定IVF导致的小鼠子代的肝脏组织Fbxl7表达异常与子代糖代谢异常的发生有关，并证实通过培养液中添加抗氧化剂褪黑素上调Fbxl7可以部分逆转子代的表型，这将推动临床前期实验以减轻IVF子代的远期糖尿病风险。