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.

OBJECTIVE: To determine whether metabolic imaging using 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. MAIN INTERVENTIONS: Oocytes from mice with global knockout of Clpp (caseinolytic peptidase P; n=52) were compared to wild type (WT) oocytes (n=55) as a model of severe oocyte dysfunction. Oocytes from old mice (1-year old; n=29) were compared to oocytes from young mice (12-week-old; n=35) as a model of mild oocyte dysfunction. MAIN OUTCOME MEASURE(S): FLIM was used to measure the naturally occurring NADH and 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). ROS levels and blastocyst development rates were measured to assess illumination safety. RESULTS: In Clpp-knockout oocytes compared to WT, FAD τ1 and τ2 were longer (p<0.0001) and I was higher (p<0.01), NADH τ2 was longer (p<0.0001), and F was lower (p<0.0001). In older oocytes compared to young ones, FAD τ1 was longer (p<0.001) and I was lower (p<0.01), while NADH τ1and τ2were shorter (p<0.0001 for both), I and F were lower (p<0.0001 and p<0.05, respectively). FLIM did not affect ROS levels or blastocyst development rates. CONCLUSIONS: FLIM parameters exhibit strong differentiation between Clpp-knockout vs WT, and old vs young oocytes. FLIM could potentially be used as a non-invasive tool to assess mitochondrial function in oocytes.

STUDY QUESTION Can non-invasive imaging with fluorescence lifetime imaging microscopy (FLIM) detect metabolic differences in euploid versus aneuploid human blastocysts? SUMMARY ANSWER FLIM has identified significant metabolic differences between euploid and aneuploid blastocysts. WHAT IS KNOWN ALREADY Prior studies have demonstrated that FLIM can detect metabolic differences in mouse oocytes and embryos and in discarded human blastocysts. STUDY DESIGN, SIZE, DURATION This was a prospective observational study from August 2019 to February 2020. Embryo metabolic state was assessed using FLIM to measure the autofluorescence metabolic factors nicotinamide adenine dinucleotide dehydrogenase together with nicotinamide adenine phosphate dinucleotide dehydrogenase (NAD(P)H) and flavin adenine dinucleotide (FAD). Eight metabolic FLIM parameters were obtained from each blastocyst (four for NAD(P)H and four for FAD): short (T1) and long (T2) fluorescence lifetime, fluorescence intensity (I) and fraction of the molecules engaged with enzymes (F). The redox ratio (NAD(P)H-I)/(FAD-I) was also calculated for each image. PARTICIPANTS/MATERIALS, SETTING, METHODS This study was performed at a single academically affiliated centre where there were 156 discarded frozen blastocysts (n = 17 euploids; 139 aneuploids) included. Ploidy status was determined by pre-implantation genetic testing for aneuploidy (PGT-A). Discarded human blastocysts were compared using single FLIM parameters. Additionally, inner cell mass (ICM) and trophectoderm (TE) were also evaluated. Multilevel models were used for analysis. A post-hoc correction used Benjamini–Hochberg’s false discovery rate, at a q-value of 0.05. MAIN RESULTS AND THE ROLE OF CHANCE Comparing euploid (n = 17) versus aneuploid (n = 139) embryos, a significant difference was seen in NAD(P)H-F (P &lt; 0.04), FAD-I (P &lt; 0.04) and redox ratio (P &lt; 0.05). Euploid ICM (n = 15) versus aneuploid ICM (n = 119) also demonstrated significantly different signatures in NAD(P)H-F (P &lt; 0.009), FAD-I (P &lt; 0.03) and redox ratio (P &lt; 0.03). Similarly, euploid TE (n = 15) versus aneuploid TE (n = 119) had significant differences in NAD(P)H-F (P &lt; 0.0001) and FAD-I (P &lt; 0.04). LIMITATIONS, REASONS FOR CAUTION This study utilized discarded human blastocysts, and these embryos may differ metabolically from non-discarded human embryos. The blastocysts analysed were vitrified after PGT-A biopsy and it is unclear how the vitrification process may affect the metabolic profile of blastocysts. Our study was also limited by the small number of rare donated euploid embryos available for analysis. Euploid embryos are very rarely discarded due to their value to patients trying to conceive, which limits their use for research purposes. However, we controlled for the imbalance with the bootstrap resampling analysis. WIDER IMPLICATIONS OF THE FINDINGS These findings provide preliminary evidence that FLIM may be a useful non-invasive clinical tool to assist in identifying the ploidy status of embryos. STUDY FUNDING/COMPETING INTEREST(S) The study was 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. There are no other conflicts of interest to declare. TRIAL REGISTRATION NUMBER N/A