Assessment of the phasor Fluorescence Lifetime Imaging Microscopy (FLIM) Approach in an animal model

摘要 最新的数字是，进行了大约150万个辅助生殖技术(ART)周期 全球每年约有350,000名婴儿出生。试管婴儿过程中的关键步骤之一 过程是挑选高质量的胚胎进行子宫移植。这一选择目前主要基于 关于囊胚的明确形态标准和物理特征。虽然这样的标准已经 证明在提高着床率，评估个体的生殖潜力方面是有用的 仅有胚胎是不够的。因此，试管受精中心经常同时移植多个胚胎 这可能导致多胎妊娠，从而增加早产和死亡或终生的风险。 新生儿的残疾。随着世界范围内辅助生殖周期的增加，改进 在我们预测胚胎存活率的能力方面是迫切需要的。发展更具定性和客观性 评估胚胎质量和存活能力的更安全和更快的手段可以提供重要的 实现单胎胚胎移植而不是多个胚胎植入的体外受精研究进展 以增加成功怀孕的可能性。鉴于形态评估的局限性， 已经探索了几种评估胚胎活力的技术。其中包括 胚胎培养上清液中代谢物的测定及其基因组和蛋白质组学分析 胚胎本身。光谱方法也被用来测量 在植入前发育过程中产生的代谢物。然而，这些方法是时间- 这很耗时，需要训练有素的人员来分析复杂的数据。在这里，我们描述了 相量膜(荧光寿命成像显微镜)方法的应用，这是一种“非 可测量活体内内源性自身荧光代谢物的侵入性“活体成像”方法 进行体外培养的胚胎。该方法获取有关新陈代谢能源的信息 被植入前的胚胎用作胚胎质量和活力的读数。

Abstract The latest figure is that around 1.5 million Assisted Reproductive Technology (ART) cycles are performed each year worldwide, with an estimated 350,000 babies born. One of the critical steps during the IVF process is the selection of high-quality embryos for uterine transfer. This selection is currently based largely on defined morphological criteria and physical characteristics of the blastocyst. While such criteria have proven to be useful in improving implantation rates, assessment of the reproductive potential of individual embryos is not sufficient. Therefore, IVF centers often perform simultaneous transfers of multiple embryos that can result in multiple pregnancies, thus increasing the risk of preterm delivery and the death or lifelong disability of neonates. As the number of assisted reproduction cycles worldwide is increasing, improvements in our ability to predict embryo viability is urgently needed. Development of more qualitative and objective means for assessing embryo quality and viability that are safer and faster could provide significant advances in IVF by enabling singleton embryo transfers rather than the implantation of multiple embryos in order to increase the likelihood of a successful pregnancy. Given the limitations of morphologic evaluation, several technologies have been explored for the assessment of embryo viability. These include the measurement of metabolites in embryonic culture media along with genomic and proteomic profiling of the embryos themselves. Spectroscopic approaches have also been utilized to measure the amount of metabolites that arise during pre-implantation development. However, these approaches are time- consuming and require highly-trained personnel to analyze the complex data. Here we describe the application of a phasor-FLIM (Fluorescence-Lifetime Imaging Microscopy) approach, which is a “non- invasive” live imaging approach capable of measuring endogenous autofluorescent metabolites within living embryos undergoing in vitro culturing. The approach captures information on the metabolic energy sources utilized by pre-implantation embryos as readout of embryo quality and viability.