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

项目总结 非整倍体筛查的植入前基因检测(PGT-A)是体外受精技术的一项重大进展，如 为移植前筛选非整倍体胚胎提供了一种方法。当前的PGT-A技术 使用下一代测序(NGS)，并可以确定存在于 滋养外胚层(TE)活检。NGS方法已经证实，40%-60%的体外受精来源的囊胚是 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(即马赛克 胚胎)。含有20%到40%非整倍体细胞的镶嵌胚泡现在被提供给一些 临床，如果没有整倍体胚泡的话。有些令人惊讶的是，相对较高的百分比(≈40% 有143个胚胎)的马赛克胚泡导致了活产。这些观察结果表明， 植入前胚胎选择性地删除内细胞团(ICM)的非整倍体细胞，从而导致 胎儿。用小鼠证明了ICM中非整倍体细胞的选择性耗尽 马赛克胚泡。这些研究表明，ICM中非整倍体细胞的数量，而不是在ICM中 在胚泡扩张过程中，TE降低。非整倍体细胞选择性形成的机制 从ICM中删除的情况未知。我们假设该机制涉及以下特征：1) TE和ICM中的非整倍体细胞都容易发生P53依赖性的细胞凋亡。 对于体细胞非整倍体细胞，TE内的细胞表达乳酸(MCT1)，而不表达ICM内的Na- 氢离子(NHE1)转运体。它们的表达使TE的整倍体和非整倍体细胞都能 在胚泡扩张过程中维持中性的细胞内pH，同时通过 糖酵解途径，3)糖酵解代谢增加胚胎液中乳酸和氢离子的含量 并引起ICM细胞内pH的轻微下降，4)细胞内pH的下降激活 先前存在的P53信号通路，导致ICM非整倍体细胞死亡。实验 在这项拨款提案中，将使用马赛克小鼠囊胚来检验我们的新假设，并将其组织成 三个具体目标。特异靶1将确定非整倍体细胞是否通过P53- 监测P53蛋白在囊胚扩张过程中的表达及其依赖机制 途径以及产生P53耗尽的非整倍体细胞。特定目标2将决定非整倍体是否 驻留在TE中的细胞由于其适应代谢变化的能力而对凋亡更具抵抗力 因为它们表达MCT1和NHE1。这些转运蛋白的作用将通过耗尽 MCT1和NHE1在非整倍体细胞中表达。具体目标3将决定是否补充培养 含有弱酸或葡萄糖和乳酸的培养基会降低细胞内的pH，并选择性地诱导 ICM中非整倍体细胞的凋亡。将通过以下方式监测这些治疗效果 TE和ICM中非整倍体细胞的数量及其细胞内pH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.