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

摘要 最新的数字是，大约有150万个辅助生殖技术（ART）周期被执行。 每年全球约有35万婴儿出生。IVF过程中的关键步骤之一 这个过程是选择高质量的胚胎进行子宫移植。这一选择目前主要基于 根据胚泡的形态学标准和物理特征。虽然这些标准 被证明可用于提高植入率，评估个体的生殖潜力， 胚胎是不够的。因此，试管婴儿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身妊娠的风险。 新生儿残疾。随着全世界辅助生殖周期的数量不断增加， 我们迫切需要预测胚胎存活能力。制定更高质量和更客观的 评估胚胎质量和生存能力的方法更安全，更快，可以提供重要的 通过使单胎胚胎移植而不是植入多个胚胎， 以增加成功怀孕的可能性。鉴于形态学评估的局限性， 已经探索了几种用于评估胚胎存活力的技术。其中包括 测量胚胎培养基中的代谢物沿着基因组和蛋白质组分析， 胚胎本身光谱方法也已被用于测量化合物的量。 在着床前发育过程中产生的代谢物。然而，这些方法是时间- 消耗并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 应用相量FLIM（荧光寿命成像显微镜）方法，这是一个“非- 能够测量活体内内源性自发荧光代谢物的“侵入性”活体成像方法 进行体外培养的胚胎。该方法捕获代谢能量来源的信息 被植入前胚胎用作胚胎质量和存活力的读数。

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.