Combined noninvasive metabolic and spindle imaging as potential tools for embryo and oocyte assessment

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.

Keywords: embryo assessment; flavin adenine dinucleotide; fluorescence; metabolism; mitochondria; nicotinamide adenine dinucleotide; noninvasive; oocyte; spindle.

Figure 1

Morphological features imaged with fluorescence lifetime imaging microscopy and second harmonic generation imaging. (A) Left: a standard bright-field image of a mouse blastocyst shows gross morphological features. Middle: fluorescence lifetime imaging microscopy (FLIM) measurements of nicotinamide adenine dinucleotide (NADH) generate intensity images that visualize subcellular structures, including nuclei (white arrow, 25-μm scale bar). Right: 3D reconstruction from multiple focal planes (100-μm scale bar). (B) Left: Bright-field image of an oocyte. Second to left: a flavin adenine dinucleotide (FAD) FLIM image of the same oocyte. Second to right: a second harmonic generation (SHG) image of the same oocyte, which clearly shows the oocyte’s spindle. Right: Overlay image of SHG (magenta) and FAD (grey scale) (25-μm scale bar). (C) Time lapse FLIM (NADH) and spindle measurements capture key embryo dynamics, such as cell division (25-μm scale bar).

Figure 2

FLIM allows for mitochondrial imaging and separate segmentation of mitochondrial and cytoplasmic signals. (A) MitoTracker images (left) of a two-cell embryo shows co-localization with brighter regions of NADH (center) (25-μm scale bars). A machine-learning-based algorithm performed on autofluorescence images successfully segments mitochondria and cytoplasm (right). (B) MitoTracker and FAD autofluoresence images of a one-cell embryo also show co-localization. For FAD images, only mitochondrial regions were segmented. (C) For each region, all photon arrival times were binned into a separate histogram, which were normalized to produce a probability distribution, which represents the probability of being in an excited state after excitation. Fitting these curves to a two-exponential decay model produces a total of 12 parameters for characterizing the metabolic state of an embryo or oocyte (right).

Figure 3

Metabolic and SHG imaging of oocytes detects effects of metabolic perturbations. Oocytes were exposed to 10 mM oxamate and then to 1 μM rotenone. (A) Oxamate exposure (indicated with a dashed orange line) caused a visible drop in cytosolic NADH FLIM intensity, whereas rotenone exposure (purple line) caused an increase in mitochondrial NADH intensity. Bar is 30 μm. (B) For the same oocyte, oxamate slightly increased FAD intensity, while rotenone decreased it. Simultaneous SHG imaging, overlaid in magenta, revealed that spindles were not disrupted by oxamate, but were disintegrated by rotenone. (C) Percentage-change time courses for all eight metabolic parameters. Colored traces represent individual oocyte trajectories (n = 25), and average curves are shown in black with SE bars. Vertical dashed lines indicate oxamate (orange) and rotenone (purple) exposures. Red bars in the NADH intensity panel correspond to the time stamps of the images shown in (A) and (B). (D) FLIM parameters were compared before and after oxamate exposure, and the three FLIM parameters with the largest separation were plotted on 3D plots, yielding complete separation. This comparison was also performed for (E) oxamate versus oxamate + rotenone, and (F) no perturbation versus oxamate + rotenone.

Figure 4

High time-resolution measurements of metabolic state during mouse embryo development. (A) Embryos were imaged together in 9-microwell dishes and individually tracked and analyzed to generate time plots for all FLIM parameters. Bar is 30 μm. (B) Time plots for NADH and FAD intensities and fractions engaged for the individual embryo shown in (A). Distinct changes are evident in multiple parameters, especially around the onset of blastocyst formation (~44 h after the 1st division, which is represented here as t = 0 h). Corresponding time points for the four images are displayed with red dashes on the plots. (C) The observed trends were robust and reproducible among all healthy embryos. These plots display individual embryo trajectories as thin-colored lines (n = 39), which were synchronized by 1st division. Averaged metabolic curves from all 39 embryos are displayed as thick black lines with SE bars. (D) FLIM parameters were compared between the 1st division and morula stages, and the three FLIM parameters with the largest separation were plotted as 3D plots, yielding complete separation. This comparison was also performed for (E) compaction versus expanded blastocyst and (F) 1st division versus expanded blastocyst.

Figure 5

Live birth safety study. To evaluate the safety of FLIM illumination on embryos, we implanted illuminated and nonilluminated embryos into pseudo-pregnant mice, later measuring birth rates and pup weights as metrics for possible damage. Sample numbers indicate number of mice and number of pups, respectively. No significant differences were observed.