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

滋养外胚层的代谢变化诱导细胞凋亡选择性消除非整倍体细胞

基本信息

批准号：
9924594
负责人：
JOHN J PELUSO
金额：
$ 8.2万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-05-06 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9924594
关键词：
Abnormal Cell Acids Affect Age Age-Years Aneuploid Cells Aneuploidy Antimitotic Agents Apoptosis Apoptotic Applications Grants Biological Models Biopsy Birth Cells Child Chromosome abnormality Clinic Couples Culture Media Data Development Dyes Embryo Fertilization in Vitro Fetus Genes Glucose Human In Situ Hybridization In Vitro Infertility Inner Cell Mass Lactose Liquid substance Live Birth Measures Mediating Metabolic Metabolism Methods Modeling Modification Monitor Mosaicism Mus NHE1 Oocytes Oxazolidinediones Pathway interactions Phosphotransferases Preimplantation Diagnosis Proteins Protons Resistance Role Signal Transduction Small Interfering RNA Sodium Stains TP53 gene Technology Testing Treatment Effectiveness United States Woman blastocyst child bearing differential expression embryo cell experimental study immunocytochemistry insight mouse model next generation sequencing novel organic acid p53 Signaling Pathway preimplantation preservation screening single-cell RNA sequencing statistics success transcriptome

项目摘要

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.

项目概要用于非整倍体筛查的植入前基因检测 (PGT-A) 是 IVF 技术的重大进步，因为它提供了一种在移植前选择非整倍体胚胎的方法当前的 PGT-A 技术。使用下一代测序 (NGS)，可以确定细胞中存在的非整倍体细胞的百分比 NGS 方法已证实 40-60% 的 IVF 衍生囊胚是滋养外胚层 (TE) 活检。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）具有 20% 至 40% 非整倍体细胞的嵌合囊胚现在可用于移植。令人惊讶的是，如果没有整倍体囊胚，比例相对较高（约 40%）。 143 个胚胎）的嵌合囊胚已导致活产。植入前胚胎选择性地删除内细胞团（ICM）的非整倍体细胞，从而产生使用小鼠证明了 ICM 中非整倍体细胞的选择性消耗。这些研究表明，ICM 中的非整倍体细胞数量较多，但在 ICM 中则不然。囊胚扩张期间 TE 降低非整倍体细胞选择性的机制。从ICM中删除我们不得而知，这个机制涉及以下功能：1） TE 和 ICM 中的非整倍体细胞都容易发生 p53 依赖性细胞凋亡，情况就是如此对于体细胞非整倍体细胞，2) TE 内但 ICM 内不表达乳酸 (MCT1) 和钠的细胞它们的表达使 TE 的整倍体和非整倍体细胞能够在囊胚扩张过程中维持中性细胞内pH，同时通过糖酵解途径，3) 糖酵解代谢增加囊胚腔液中的乳酸和氢离子含量并导致 ICM 细胞的细胞内 pH 值略有下降，4) 细胞内 pH 值的下降激活先前存在的 p53 信号通路，导致 ICM 实验的非整倍体细胞死亡。在这项拨款提案中，将使用镶嵌小鼠囊胚来测试我们的新假设，并将其组织成三个具体目标。具体目标 1 将确定非整倍体细胞是否通过 p53- 发生凋亡。通过监测 p53 成分的表达来实现囊胚扩张过程中的依赖机制途径以及生成 p53 耗尽的非整倍体细胞。具体目标 2 将确定是否为非整倍体。 TE 中的细胞由于能够适应代谢变化而更能抵抗细胞凋亡因为它们表达 MCT1 和 NHE1，这些转运蛋白的作用将通过消耗来证明。非整倍体细胞中的MCT1和NHE1将决定是否补充培养物。弱酸或葡萄糖和乳酸介质会降低细胞内pH值并选择性诱导 ICM的非整倍体细胞的凋亡将通过确定来监测这些治疗的有效性。 TE 和 ICM 中非整倍体细胞数量及其细胞内 pH 值的变化。