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

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.

 
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Award ID(s):
2013874
NSF-PAR ID:
10405237
Author(s) / Creator(s):
; ; ;
Publisher / Repository:
Oxford University Press
Date Published:
Journal Name:
Human Reproduction
Volume:
38
Issue:
5
ISSN:
0268-1161
Page Range / eLocation ID:
p. 799-810
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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  1. Abstract STUDY QUESTION

    Is the combined use of fluorescence lifetime imaging microscopy (FLIM)-based metabolic imaging and second harmonic generation (SHG) spindle imaging a feasible and safe approach for noninvasive embryo assessment?

    SUMMARY ANSWER

    Metabolic imaging can sensitively detect meaningful metabolic changes in embryos, SHG produces high-quality images of spindles and the methods do not significantly impair embryo viability.

    WHAT IS KNOWN ALREADY

    Proper metabolism is essential for embryo viability. Metabolic imaging is a well-tested method for measuring metabolism of cells and tissues, but it is unclear if it is sensitive enough and safe enough for use in embryo assessment.

    STUDY DESIGN, SIZE, DURATION

    This study consisted of time-course experiments and control versus treatment experiments. We monitored the metabolism of 25 mouse oocytes with a noninvasive metabolic imaging system while exposing them to oxamate (cytoplasmic lactate dehydrogenase inhibitor) and rotenone (mitochondrial oxidative phosphorylation inhibitor) in series. Mouse embryos (n = 39) were measured every 2 h from the one-cell stage to blastocyst in order to characterize metabolic changes occurring during pre-implantation development. To assess the safety of FLIM illumination, n = 144 illuminated embryos were implanted into n = 12 mice, and n = 108 nonilluminated embryos were implanted into n = 9 mice.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    Experiments were performed in mouse embryos and oocytes. Samples were monitored with noninvasive, FLIM-based metabolic imaging of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) autofluorescence. Between NADH cytoplasm, NADH mitochondria and FAD mitochondria, a single metabolic measurement produces up to 12 quantitative parameters for characterizing the metabolic state of an embryo. For safety experiments, live birth rates and pup weights (mean ± SEM) were used as endpoints. For all test conditions, the level of significance was set at P < 0.05.

    MAIN RESULTS AND THE ROLE OF CHANCE

    Measured FLIM parameters were highly sensitive to metabolic changes due to both metabolic perturbations and embryo development. For oocytes, metabolic parameter values were compared before and after exposure to oxamate and rotenone. The metabolic measurements provided a basis for complete separation of the data sets. For embryos, metabolic parameter values were compared between the first division and morula stages, morula and blastocyst and first division and blastocyst. The metabolic measurements again completely separated the data sets. Exposure of embryos to excessive illumination dosages (24 measurements) had no significant effect on live birth rate (5.1 ± 0.94 pups/mouse for illuminated group; 5.7 ± 1.74 pups/mouse for control group) or pup weights (1.88 ± 0.10 g for illuminated group; 1.89 ± 0.11 g for control group).

    LIMITATIONS, REASONS FOR CAUTION

    The study was performed using a mouse model, so conclusions concerning sensitivity and safety may not generalize to human embryos. A limitation of the live birth data is also that although cages were routinely monitored, we could not preclude that some runt pups may have been eaten.

    WIDER IMPLICATIONS OF THE FINDINGS

    Promising proof-of-concept results demonstrate that FLIM with SHG provide detailed biological information that may be valuable for the assessment of embryo and oocyte quality. Live birth experiments support the method’s safety, arguing for further studies of the clinical utility of these techniques.

    STUDY FUNDING/COMPETING INTEREST(S)

    Supported by the Blavatnik Biomedical Accelerator Grant at Harvard University and by the Harvard Catalyst/The Harvard Clinical and Translational Science Center (National Institutes of Health Award UL1 TR001102), by NSF grants DMR-0820484 and PFI-TT-1827309 and by NIH grant R01HD092550-01. T.S. was supported by a National Science Foundation Postdoctoral Research Fellowship in Biology grant (1308878). S.F. and S.A. were supported by NSF MRSEC DMR-1420382. Becker and Hickl GmbH sponsored the research with the loaning of equipment for FLIM. T.S. and D.N. are cofounders and shareholders of LuminOva, Inc., and co-hold patents (US20150346100A1 and US20170039415A1) for metabolic imaging methods. D.S. is on the scientific advisory board for Cooper Surgical and has stock options with LuminOva, Inc.

     
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    Methods

    Here, we used autofluorescence imaging of endogenous metabolic co-factors via multiphoton microscopy and FLIM in conjunction with oxygen consumption rate and extracellular acidification rate through Seahorse extracellular flux assays to detect changes in cellular metabolism in quiescent and classically activated macrophages in response to cytokine stimulation.

    Results

    Based on our Seahorse XFP flux analysis, M0 and M1 macrophages exhibit comparable trends in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Autofluorescence imaging of M0 and M1 macrophages was not only able to show acute changes in the optical redox ratio from pre-differentiation (0 hours) to 72 hours post-cytokine differentiation (M0: 0.320 to 0.258 and M1: 0.316 to 0.386), mean NADH lifetime (M0: 1.272 ns to 1.379 ns and M1: 1.265 ns to 1.206 ns), and A1/A2 ratio (M0: 3.452 to ~ 4 and M1: 3.537 to 4.529) but could also detect heterogeneity within each macrophage population.

    Conclusions

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