Fluorescence lifetime imaging microscopy (FLIM) detects differences in metabolic signatures between euploid and aneuploid human blastocysts.

Can non-invasive imaging with fluorescence lifetime imaging microscopy (FLIM) detect metabolic differences in euploid versus aneuploid human blastocysts? FLIM has identified significant metabolic differences between euploid and aneuploid blastocysts. Prior studies have demonstrated that FLIM can detect metabolic differences in mouse oocytes and embryos and in discarded human blastocysts. 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. 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. Comparing euploid (n = 17) versus aneuploid (n = 139) embryos, a significant difference was seen in NAD(P)H-F (P < 0.04), FAD-I (P < 0.04) and redox ratio (P < 0.05). Euploid ICM (n = 15) versus aneuploid ICM (n = 119) also demonstrated significantly different signatures in NAD(P)H-F (P < 0.009), FAD-I (P < 0.03) and redox ratio (P < 0.03). Similarly, euploid TE (n = 15) versus aneuploid TE (n = 119) had significant differences in NAD(P)H-F (P < 0.0001) and FAD-I (P < 0.04). 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. These findings provide preliminary evidence that FLIM may be a useful non-invasive clinical tool to assist in identifying the ploidy status of embryos. 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. N/A.

Shah JS ，Venturas M ，Sanchez TH ，Penzias AS ，Needleman DJ ，Sakkas D ... -  《-》

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

Can non-invasive metabolic imaging via fluorescence lifetime imaging microscopy (FLIM) detect variations in metabolic profiles between discarded human blastocysts? 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. 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. 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. 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. 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. 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. 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. 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. N/A.

Venturas M ，Shah JS ，Yang X ，Sanchez TH ，Conway W ，Sakkas D ，Needleman DJ ... -  《-》

Metabolic imaging of human embryos is predictive of ploidy status but is not associated with clinical pregnancy outcomes: a pilot trial.

Sakkas D ，Gulliford C ，Ardestani G ，Ocali O ，Martins M ，Talasila N ，Shah JS ，Penzias AS ，Seidler EA ，Sanchez T ... -  《-》

Non-invasive, label-free optical analysis to detect aneuploidy within the inner cell mass of the preimplantation embryo.

Can label-free, non-invasive optical imaging by hyperspectral autofluorescence microscopy discern between euploid and aneuploid cells within the inner cell mass (ICM) of the mouse preimplantation embryo? Hyperspectral autofluorescence microscopy enables discrimination between euploid and aneuploid ICM in mouse embryos. Euploid/aneuploid mosaicism affects up to 17.3% of human blastocyst embryos with trophectoderm biopsy or spent media currently utilized to diagnose aneuploidy and mosaicism in clinical in vitro fertilization. Based on their design, these approaches will fail to diagnose the presence or proportion of aneuploid cells within the foetal lineage ICM of some blastocyst embryos. The impact of aneuploidy on cellular autofluorescence and metabolism of primary human fibroblast cells and mouse embryos was assessed using a fluorescence microscope adapted for imaging with multiple spectral channels (hyperspectral imaging). Primary human fibroblast cells with known ploidy were subjected to hyperspectral imaging to record native cell fluorescence (4-6 independent replicates, euploid n = 467; aneuploid n = 969). For mouse embryos, blastomeres from the eight-cell stage (five independent replicates: control n = 39; reversine n = 44) and chimeric blastocysts (eight independent replicates: control n = 34; reversine n = 34; 1:1 (control:reversine) n = 30 and 1:3 (control:reversine) n = 37) were utilized for hyperspectral imaging. The ICM from control and reversine-treated embryos were mechanically dissected and their karyotype confirmed by whole genome sequencing (n = 13 euploid and n = 9 aneuploid). Two models were employed: (i) primary human fibroblasts with known karyotype and (ii) a mouse model of embryo aneuploidy where mouse embryos were treated with reversine, a reversible spindle assembly checkpoint inhibitor, during the four- to eight-cell division. Individual blastomeres were dissociated from control and reversine-treated eight-cell embryos and either imaged directly or used to generate chimeric blastocysts with differing ratios of control:reversine-treated cells. Individual blastomeres and embryos were interrogated by hyperspectral imaging. Changes in cellular metabolism were determined by quantification of metabolic co-factors (inferred from their autofluorescence signature): NAD(P)H and flavins with the subsequent calculation of the optical redox ratio (ORR: flavins/[NAD(P)H + flavins]). Autofluorescence signals obtained from hyperspectral imaging were examined mathematically to extract features from each cell/blastomere/ICM. This was used to discriminate between different cell populations. An increase in the relative abundance of NAD(P)H and decrease in flavins led to a significant reduction in the ORR for aneuploid cells in primary human fibroblasts and reversine-treated mouse blastomeres (P < 0.05). Mathematical analysis of endogenous cell autofluorescence achieved separation between (i) euploid and aneuploid primary human fibroblast cells, (ii) control and reversine-treated mouse blastomeres cells, (iii) control and reversine-treated chimeric blastocysts, (iv) 1:1 and 1:3 chimeric blastocysts and (v) confirmed euploid and aneuploid ICM from mouse blastocysts. The accuracy of these separations was supported by receiver operating characteristic curves with areas under the curve of 0.97, 0.99, 0.87, 0.88 and 0.93, respectively. We believe that the role of chance is low as mathematical features separated euploid from aneuploid in both human fibroblasts and ICM of mouse blastocysts. N/A. Although we were able to discriminate between euploid and aneuploid ICM in mouse blastocysts, confirmation of this approach in human embryos is required. While we show this approach is safe in mouse, further validation is required in large animal species prior to implementation in a clinical setting. We have developed an original, accurate and non-invasive optical approach to assess aneuploidy within the ICM of mouse embryos in the absence of fluorescent tags. Hyperspectral autofluorescence imaging was able to discriminate between euploid and aneuploid human fibroblast and mouse blastocysts (ICM). This approach may potentially lead to a new diagnostic for embryo analysis. K.R.D. is supported by a Mid-Career Fellowship from the Hospital Research Foundation (C-MCF-58-2019). This study was funded by the Australian Research Council Centre of Excellence for Nanoscale Biophotonics (CE140100003) and the National Health and Medical Research Council (APP2003786). The authors declare that there is no conflict of interest.

Tan TCY ，Mahbub SB ，Campbell JM ，Habibalahi A ，Campugan CA ，Rose RD ，Chow DJX ，Mustafa S ，Goldys EM ，Dunning KR ... -  《-》

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

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? 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. 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. 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. 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. 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). 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. 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. 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.

Sanchez T ，Venturas M ，Aghvami SA ，Yang X ，Fraden S ，Sakkas D ，Needleman DJ ... -  《-》