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

摘要

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.