Noninvasive metabolic profiling of cumulus cells, oocytes, and embryos via fluorescence lifetime imaging microscopy: a mini-review.

A major challenge in ART is to select high-quality oocytes and embryos. The metabolism of oocytes and embryos has long been linked to their viability, suggesting the potential utility of metabolic measurements to aid in selection. Here, we review recent work on noninvasive metabolic imaging of cumulus cells, oocytes, and embryos. We focus our discussion on fluorescence lifetime imaging microscopy (FLIM) of the autofluorescent coenzymes NAD(P)H and flavine adenine dinucleotide (FAD+), which play central roles in many metabolic pathways. FLIM measurements provide quantitative information on NAD(P)H and FAD+ concentrations and engagement with enzymes, leading to a robust means of characterizing the metabolic state of cells. We argue that FLIM is a promising approach to aid in oocyte and embryo selection.

Metabolic state of human blastocysts measured by fluorescence lifetime imaging microscopy.

STUDY QUESTION: Can non-invasive metabolic imaging via fluorescence lifetime imaging microscopy (FLIM) detect variations in metabolic profiles between discarded human blastocysts? SUMMARY ANSWER: FLIM revealed extensive variations in the metabolic state of discarded human blastocysts associated with blastocyst development over 36 h, the day after fertilization and blastocyst developmental stage, as well as metabolic heterogeneity within individual blastocysts. WHAT IS KNOWN ALREADY: Mammalian embryos undergo large changes in metabolism over the course of preimplantation development. Embryo metabolism has long been linked to embryo viability, suggesting its potential utility in ART to aid in selecting high quality embryos. However, the metabolism of human embryos remains poorly characterized due to a lack of non-invasive methods to measure their metabolic state. STUDY DESIGN, SIZE, DURATION: We conducted a prospective observational study. We used 215 morphologically normal human embryos from 137 patients that were discarded and donated for research under an approved institutional review board protocol. These embryos were imaged using metabolic imaging via FLIM to measure the autofluorescence of two central coenzymes, nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD+), which are essential for cellular respiration and glycolysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Here, we used non-invasive FLIM to measure the metabolic state of human blastocysts. We first studied spatial patterns in the metabolic state within human blastocysts and the association of the metabolic state of the whole blastocysts with stage of expansion, day of development since fertilization and morphology. We explored the sensitivity of this technique in detecting metabolic variations between blastocysts from the same patient and between patients. Next, we explored whether FLIM can quantitatively measure metabolic changes through human blastocyst expansion and hatching via time-lapse imaging. For all test conditions, the level of significance was set at P < 0.05 after correction for multiple comparisons using Benjamini-Hochberg's false discovery rate. MAIN RESULTS AND THE ROLE OF CHANCE: We found that FLIM is sensitive enough to detect significant metabolic differences between blastocysts. We found that metabolic variations between blastocyst are partially explained by both the time since fertilization and their developmental expansion stage (P < 0.05), but not their morphological grade. Substantial metabolic variations between blastocysts from the same patients remain, even after controlling for these factors. We also observe significant metabolic heterogeneity within individual blastocysts, including between the inner cell mass and the trophectoderm, and between the portions of hatching blastocysts within and without the zona pellucida (P < 0.05). And finally, we observed that the metabolic state of human blastocysts continuously varies over time. LIMITATIONS, REASONS FOR CAUTION: Although we observed significant variations in metabolic parameters, our data are taken from human blastocysts that were discarded and donated for research and we do not know their clinical outcome. Moreover, the embryos used in this study are a mixture of aneuploid, euploid and embryos of unknown ploidy. WIDER IMPLICATIONS OF THE FINDINGS: This work reveals novel aspects of the metabolism of human blastocysts and suggests that FLIM is a promising approach to assess embryo viability through non-invasive, quantitative measurements of their metabolism. These results further demonstrate that FLIM can provide biologically relevant information that may be valuable for the assessment of embryo quality. STUDY FUNDING/COMPETING INTEREST(S): Supported by the Blavatnik Biomedical Accelerator Grant at Harvard University. Becker and Hickl GmbH and Boston Electronics sponsored research with the loaning of equipment for FLIM. D.J.N. is an inventor on patent US20170039415A1. TRIAL REGISTRATION NUMBER: N/A.

Metabolic imaging via fluorescence lifetime imaging microscopy for egg and embryo assessment.

Current strategies for embryo assessment in the assisted reproductive technology laboratories rely primarily on morphologic parameters that have limited accuracy for determining embryo viability. Even with the addition of invasive diagnostic interventions such as preimplantation genetic testing for aneuploidy alone or in combination with mitochondrial DNA copy number assessment, at least one third of embryos fail to implant. Therefore, at a time when the clinical benefits of single ET are widely accepted, improving viability assessment of embryos is ever more important. Building on the previous work demonstrating the importance of metabolic state in oocytes and embryos, metabolic imaging via fluorescence lifetime imaging microscopy offers new and potentially useful diagnostic method by detecting natural fluorescence of FAD and NADH, the two electron transporters that play a central role in oxidative phosphorylation. Recent studies demonstrate that fluorescence lifetime imaging microscopy can detect oocyte and embryo metabolic function and dysfunction in a multitude of experimental models and provide encouraging evidence for use in scientific investigation and possibly for clinical application.

Metabolic imaging with the use of fluorescence lifetime imaging microscopy (FLIM) accurately detects mitochondrial dysfunction in mouse oocytes.

OBJECTIVE: To determine whether metabolic imaging with the use of fluorescence lifetime imaging microscopy (FLIM) identifies metabolic differences between normal oocytes and those with metabolic dysfunction. DESIGN: Experimental study. SETTING: Academic research laboratories. PATIENT(S): None. INTERVENTION(S): Oocytes from mice with global knockout of Clpp (caseinolytic peptidase P; n = 52) were compared with wild-type (WT) oocytes (n = 55) as a model of severe oocyte dysfunction. Oocytes from old mice (1 year old; n = 29) were compared with oocytes from young mice (12 weeks old; n = 35) as a model of mild oocyte dysfunction. MAIN OUTCOME MEASURE(S): FLIM was used to measure the naturally occurring nicotinamide adenine dinucleotide dehydrogenase (NADH) and flavin adenine dinucleotide (FAD) autofluorescence in individual oocytes. Eight metabolic parameters were obtained from each measurement (4 per fluorophore): short (τ1) and long (τ2) fluorescence lifetime, fluorescence intensity (I), and fraction of the molecule engaged with enzyme (F). Reactive oxygen species (ROS) levels and blastocyst development rates were measured to assess illumination safety. RESULT(S): In Clpp-knockout oocytes compared with WT, FAD τ1 and τ2 were longer and I was higher, NADH τ2 was longer, and F was lower. In old oocytes compared with young ones, FAD τ1 was longer and I was lower, NADH τ1 and τ2 were shorter, and I and F were lower. FLIM did not affect ROS levels or blastocyst development rates. CONCLUSION(S): FLIM parameters exhibit strong differentiation between Clpp-knockout versus WT, and old versus young oocytes. FLIM could potentially be used as a noninvasive tool to assess mitochondrial function in oocytes.

Determining physical principles of subcellular organization.

Recent advances have transformed our understanding of cell biology, but we are still unable to predict the behaviors of these systems. One difficulty is that we lack an understanding of the physical principles of subcellular organization. Combining quantitative experiments with new theoretical insights may allow such principles to be developed.