Metabolic changes in the trophectoderm induce the selective elimination of aneuploid cells by apoptosis

项目摘要 用于非整倍体筛查的植入前基因检测（PGT-A）是IVF技术的一项重大进步， 本发明提供了一种在移植前针对非整倍体胚胎进行选择的方法。目前的PGT-A技术 使用下一代测序（NGS），可以确定存在于细胞中的非整倍体细胞的百分比。 滋养外胚层（TE）活检。NGS方法已经证实，40-60%的IVF衍生囊胚是 非整倍体，大多数非整倍体囊胚由整倍体和非整倍体细胞组成（即嵌合体 胚胎）。具有20%至40%非整倍体细胞的镶嵌胚泡现在被提供用于在一些实施例中的移植。 诊所，如果整倍体囊胚不可用。令人惊讶的是，相对较高的百分比（约40%） 143个胚胎）的镶嵌囊胚导致活产。这些观察表明， 植入前胚胎选择性地删除内细胞团（ICM）的非整倍体细胞， 胎儿使用小鼠实验证明了ICM中非整倍体细胞的选择性清除 镶嵌囊胚。这些研究表明，非整倍体细胞的数量在ICM中，而不是在 TE在囊胚扩张期间减少。非整倍体细胞被选择性地 从ICM中删除的信息尚不清楚。我们假设这种机制涉及以下特征：1） TE和ICM中的非整倍体细胞都倾向于经历p53依赖性凋亡， 对于体细胞非整倍体细胞，2）TE内的细胞而不是ICM表达乳酸盐（MCT 1）和钠- 氢离子（NHE 1）转运蛋白。它们的表达使TE的整倍体和非整倍体细胞都能够 在胚泡扩张期间维持中性细胞内pH，同时通过 糖酵解途径，3）糖酵解代谢增加囊胚腔液的乳酸和氢离子含量 并导致ICM细胞的细胞内pH轻微降低，以及4）细胞内pH的降低激活 预先存在的p53信号通路，其导致ICM的非整倍体细胞的死亡。实验 在这项拨款申请中，我们将使用嵌合小鼠囊胚来测试我们的新假设，并组织成 三个具体目标。特异性目的1将确定非整倍体细胞是否通过p53- 通过监测p53蛋白组分的表达， 途径以及产生p53耗尽的非整倍体细胞。具体目标2将确定是否非整倍体 TE中的细胞由于其适应代谢变化的能力而对凋亡具有更强的抵抗力 因为它们表达MCT 1和NHE 1。这些转运蛋白的作用将通过消耗 非整倍体细胞中的MCT 1和NHE 1。具体目标3将决定是否补充培养物 含有弱酸或葡萄糖和乳酸盐的培养基将降低细胞内pH，并选择性地诱导细胞内pH升高。 ICM的非整倍体细胞凋亡。这些治疗的有效性将通过确定 TE和ICM中非整倍体细胞数量及其细胞内pH值的变化。

PROJECT SUMMARY Preimplantation genetic testing for aneuploidy screening (PGT-A) is a major advancement in IVF technology as it provides a method for selecting against aneuploid embryos prior to transfer. The current PGT-A technology uses next generation sequencing (NGS) and can determine the percentage of aneuploid cells present in a trophectoderm (TE) biopsy. The NGS approach has confirmed that 40-60% of IVF derived blastocysts are aneuploid with most aneuploid blastocysts being composed of euploid and aneuploid cells (i.e. mosaic embryos). Mosaic blastocysts with between 20 and 40% aneuploid cells are now offered for transfer in some clinics, if euploid blastocysts are not available. Somewhat surprisingly, a relatively high percentage (≈ 40% of 143 embryos) of mosaic blastocysts have resulted in live births. These observations suggest that the preimplantation embryo selectively deletes aneuploid cells of the inner cell mass (ICM), which gives rise to the fetus. That the selective depletion of aneuploid cells from the ICM occurs was demonstrated using mouse mosaic blastocysts. These studies demonstrated that the number of aneuploid cells in the ICM but not in the TE decreases during blastocyst expansion. The mechanism by which the aneuploid cells are selectively deleted from the ICM is not known. We hypothesize that this mechanism involves the following features: 1) aneuploid cells in both the TE and ICM are predisposed to undergo p53-dependent apoptosis, as is the case for somatic aneuploid cells, 2) cells within the TE but not the ICM express lactate (MCT1) and sodium- hydrogen ion (NHE1) transporters. Their expression enable both the euploid and aneuploid cells of the TE to maintain a neutral intracellular pH during blastocyst expansion while metabolizing glucose through the glycolytic pathway, 3) glycolytic metabolism increases lactate and hydrogen ion content of the blastocoel fluid and causes a slight decrease in intracellular pH of ICM cells and 4) the decrease in intracellular pH activates the preexisting p53 signaling pathway, which leads to a demise of the aneuploid cells of the ICM. Experiments in this grant proposal will use mosaic mouse blastocysts to test our novel hypothesis and are organized into three specific aims. Specific aim 1 will determine whether aneuploid cells undergo apoptosis through a p53- dependent mechanism during blastocyst expansion by monitoring the expression of the components of the p53 pathway as well as generating p53 depleted aneuploid cells. Specific aim 2 will determine whether aneuploid cells that reside in TE are more resistant to apoptosis due to their ability to adapt to metabolic changes because they express MCT1 and NHE1. The role for these transporters will be demonstrated by depleting MCT1 and NHE1 in the aneuploid cells. Specific aim 3 will determine whether supplementing the culture media with weak acids or glucose and lactate will decrease the intracellular pH and selectively induce apoptosis of aneuploid cells of the ICM. The effectiveness of these treatments will be monitor by determining changes in the number of aneuploid cells in the TE and ICM and their intracellular pH.