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• W2912986577 abstract "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. 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. Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertility-and-sterility/posts/42041-27401 Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertility-and-sterility/posts/42041-27401 Soon after the application of IVF into clinical practice (1Steptoe P.C. Edwards R.G. Birth after the reimplantation of a human embryo.Lancet. 1978; 2: 366Abstract PubMed Google Scholar), embryo quality was understood to be pivotal to treatment outcome (2Speirs A.L. Lopata A. Gronow M.J. Kellow G.N. Johnston W.I. Analysis of the benefits and risks of multiple embryo transfer.Fertil Steril. 1983; 39: 468-471Abstract Full Text PDF PubMed Scopus (104) Google Scholar). As such, it quickly became a central objective in assisted reproductive technologies to identify methods of accurately assessing embryo quality (3O'Neill C. Saunders D.M. Assessment of embryo quality.Lancet. 1984; 324: 1035Abstract Scopus (39) Google Scholar). An association between embryo morphology and cleavage rate and IVF outcome has been observed (4Edwards R.G. Fishel S.B. Cohen J. Fehilly C.B. Purdy J.M. Slater J.M. et al.Factors influencing the success of in vitro fertilization for alleviating human infertility.J In Vitr Fertil Embryo Transf. 1984; 1: 3-23Crossref PubMed Scopus (428) Google Scholar), and sophisticated morphological algorithms to identify embryos that are more likely to implant have been developed (5Veeck L.L. An atlas of human gametes and conceptuses: an illustrated reference for assisted reproduction technology. Parthenon Publishing, New York1999Google Scholar, 6Gerris J. De Neubourg D. Mangelschots K. Van Royen E. Van De Meerssche M. Valkenburg M. Prevention of twin pregnancy after in-vitro fertilization or intracytoplasmic sperm injection based on strict embryo criteria: a prospective randomized clinical trial.Hum Reprod. 1999; 14: 2581-2587Crossref PubMed Scopus (409) Google Scholar, 7Van Royen E. Mangelschots K. De Neubourg D. Valkenburg M. Van De Meerssche M.V. Ryckaert G. et al.Characterization of a top quality embryo, a step towards single-embryo transfer.Hum Reprod. 1999; 14: 2345-2349Crossref PubMed Scopus (437) Google Scholar, 8Gardner D. Schoolcraft W. In vitro culture of human blastocyst.in: Towards reproductive certainty: infertility and genetics beyond. Parthenon Press, Carnforth1999: 378-388Google Scholar). However, the diagnostic accuracy of these approaches remained limited. More recently, time-lapse imaging has attempted to capture dynamic information regarding cleavage rate and morphology, in the hopes that this would be a strong predictor of viability; however, clinical trials have not demonstrated strong improvements in success rates (9Gardner D.K. Sakkas D. Human gametes and preimplantation embryos. Springer New York, New York2013Google Scholar, 10Armstrong S. Vail A. Mastenbroek S. Jordan V. Farquhar C. Time-lapse in the IVF-lab: how should we assess potential benefit?.Hum Reprod. 2015; 30: 3-8Crossref PubMed Scopus (53) Google Scholar, 11Armstrong S. Arroll N. Cree L.M. Jordan V. Farquhar C. Time-lapse systems for embryo incubation and assessment in assisted reproduction.Cochrane Database Syst Rev. 2015; 2: CD011320Google Scholar). In the absence of reliable assessment methods, clinicians resort to transferring multiple embryos to achieve higher success rates. However, as the risks associated with multiple pregnancies become increasingly more evident (12Adashi E.Y. Barri P.N. Berkowitz R. Braude P. Bryan E. Carr J. et al.Infertility therapy-associated multiple pregnancies (births): an ongoing epidemic.Reprod Biomed Online. 2003; 7: 515-542Abstract Full Text PDF PubMed Scopus (134) Google Scholar), practitioners have begun to prioritize achieving a healthy singleton birth at term (9Gardner D.K. Sakkas D. Human gametes and preimplantation embryos. Springer New York, New York2013Google Scholar, 13Gardner D.K. Wale P.L. Analysis of metabolism to select viable human embryos for transfer.Fertil Steril. 2013; 99: 1062-1072Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), which can be best accomplished by single ET (14Practice Committees of SART and ASRMElective single-embryo transfer.Fertil Steril. 2012; 97: 835-842Google Scholar). A single ET strategy further increases the importance of precise preimplantation embryo assessment and selection tools. Embryo quality is determined by several factors. Chromosome copy number in embryonic cells is extremely important, as aneuploidy is highly associated with embryo failure (15Fragouli E. Alfarawati S. Spath K. Jaroudi S. Sarasa J. Enciso M. et al.The origin and impact of embryonic aneuploidy.Hum Genet. 2013; 132: 1001-1013Crossref PubMed Scopus (201) Google Scholar, 16Hassold T. Abruzzo M. Adkins K. Griffin D. Merrill M. Millie E. et al.Human aneuploidy: incidence, origin, and etiology.Environ Mol Mutagen. 1996; 175: 167-175Google Scholar, 17Sugiura-Ogasawara M. Ozaki Y. Katano K. Suzumori N. Kitaori T. Mizutani E. Abnormal embryonic karyotype is the most frequent cause of recurrent miscarriage.Hum Reprod. 2012; 27: 2297-2303Crossref PubMed Scopus (124) Google Scholar). Preimplantation genetic testing for aneuploidy has demonstrated some improvement in IVF success (18Scott R.T. Upham K.M. Forman E.J. Hong K.H. Scott K.L. Taylor D. et al.Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial.Fertil Steril. 2013; 100: 697-703Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, 19Forman E.J. Hong K.H. Ferry K.M. Tao X. Taylor D. Levy B. et al.In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial.Fertil Steril. 2013; 100: 100-107.e1Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 20Munne S. Kaplan B. Frattarelli J.L. Gysler M. Child T.J. Nakhuda G. et al.Global multicenter randomized controlled trial comparing single embryo transfer with embryo selected by preimplantation genetic screening using next-generation sequencing versus morphologic assessment.Fertil Steril. 2017; 108: e19Google Scholar). However, concerns exist regarding the consistency of these diagnostic methods (21Harper J. Jackson E. Sermon K. Aitken R.J. Harbottle S. Mocanu E. et al.Adjuncts in the IVF laboratory: where is the evidence for “add-on” interventions?.Hum Reprod. 2017; 32: 485-491Crossref PubMed Scopus (95) Google Scholar) and around the impact of mosaicism on diagnostic accuracy (22Capalbo A. Ubaldi F.M. Rienzi L. Scott R. Treff N. Detecting mosaicism in trophectodermbiopsies: current challenges and future possibilities.Hum Reprod. 2017; 32: 492-498PubMed Google Scholar, 23Vega M. Jindal S. Mosaicism: throwing the baby out with the bath water?.J Assist Reprod Genet. 2017; 34: 11-13Google Scholar). In addition, preimplantation genetic testing for aneuploidy does not provide information about metabolic or other nongenetic viability parameters. The mitochondrial DNA (mtDNA) copy number (24Fragouli E. Spath K. Alfarawati S. Kaper F. Craig A. Michel C.E. et al.Altered levels of mitochondrial DNA are associated with female age, aneuploidy, and provide an independent measure of embryonic implantation potential.Obstet Gynecol Surv. 2016; 71: 28-29Google Scholar) has been assessed as a proxy for the state of mitochondria, but results have not consistently shown a strong signal for predicting viability (25Victor A.R. Brake A.J. Tyndall J.C. Griffin D.K. Zouves C.G. Barnes F.L. et al.Accurate quantitation of mitochondrial DNA reveals uniform levels in human blastocysts irrespective of ploidy, age, or implantation potential.Fertil Steril. 2017; 107: 34-42.e3Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Today, at least 35%–40% of euploid embryos still fail to implant (18Scott R.T. Upham K.M. Forman E.J. Hong K.H. Scott K.L. Taylor D. et al.Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial.Fertil Steril. 2013; 100: 697-703Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar). Therefore, the need for additional approaches that may help improve implantation rates remains. As there are a number of potential risks associated with invasive methods, the demand for noninvasive assessment strategies is especially high (26Sanchez T. Seidler E.A. Gardner D.K. Needleman D. Sakkas D. Will noninvasive methods surpass invasive for assessing gametes and embryos?.Fertil Steril. 2017; 108: 730-737Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Embryo metabolic integrity is central to viability, and methods exist for assessing metabolism noninvasively. Attempts were made to measure glucose and pyruvate uptake by analyzing spent embryo media with microfluorometry (27Leese H.J. Hooper M.A.K. Edwards R.G. Ashwood-Smith M.J. Uptake of pyruvate by early human embryos determined by a non-invasive technique.Hum Reprod. 1986; 1: 181-182PubMed Google Scholar). Gardner and Leese found that viable embryos had a significantly higher rate of glucose consumption than nonviable ones (28Gardner D.K. Leese H.J. Assessment of embryo viability prior to transfer by the noninvasive measurement of glucose uptake.J Exp Zool. 1987; 242: 103-105Crossref PubMed Scopus (170) Google Scholar), again highlighting the importance of metabolism. Additionally, spent embryo culture media amino acid concentration has been associated with IVF outcome (29Brison D.R. Houghton F.D. Falconer D. Roberts S.A. Hawkhead J. Humpherson P.G. et al.Identification of viable embryos in IVF by non-invasive measurement of amino acid turnover.Hum Reprod. 2004; 19: 2319-2324Crossref PubMed Scopus (315) Google Scholar). However, efforts to translate this into a clinical tool failed due to technical complexities and the need for highly specialized equipment (13Gardner D.K. Wale P.L. Analysis of metabolism to select viable human embryos for transfer.Fertil Steril. 2013; 99: 1062-1072Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Similarly, metabolomic assessments performing spectroscopic analysis on spent media have been attempted with some initial success in proof-of-concept studies (30Seli E. Sakkas D. Scott R. Kwok S.C. Rosendahl S.M. Burns D.H. Noninvasive metabolomic profiling of embryo culture media using Raman and near-infrared spectroscopy correlates with reproductive potential of embryos in women undergoing in vitro fertilization.Fertil Steril. 2007; 88: 1350-1357Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 31Scott R. Seli E. Miller K. Sakkas D. Scott K. Burns D.H. Noninvasive metabolomic profiling of human embryo culture media using Raman spectroscopy predicts embryonic reproductive potential: a prospective blinded pilot study.Fertil Steril. 2008; 90: 77-83Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar); however, subsequent randomized controlled trials failed to show a benefit (32Hardarson T. Ahlstrm A. Rogberg L. Botros L. Hillensj T. Westlander G. et al.Non-invasive metabolomic profiling of day 2 and 5 embryo culture medium: a prospective randomized trial.Hum Reprod. 2012; 27: 89-96Crossref PubMed Scopus (121) Google Scholar, 33Vergouw C.G. Kieslinger D.C. Kostelijk E.H. Botros L.L. Schats R. Hompes P.G. et al.Day 3 embryo selection by metabolomic profiling of culture medium with near-infrared spectroscopy as an adjunct to morphology: a randomized controlled trial.Hum Reprod. 2012; 27: 2304-2311Crossref PubMed Scopus (87) Google Scholar). Metabolic imaging via fluorescence lifetime imaging microscopy (FLIM) is a new, noninvasive approach to measuring the biochemical status of embryos. It is a fluorescence technique (34Lakowicz J.R. Principles of fluorescence spectroscopy.3d ed. Springer, 2006Crossref Scopus (17811) Google Scholar) focusing on NADH and FAD. Because these molecules are naturally fluorescent and integral to cellular respiration (35Ghukasyan V.V. Heikal A.A. Natural biomarkers for cellular metabolism: biology, techniques, and applications. CRC Press, 2014Crossref Scopus (39) Google Scholar), they provide a means of directly probing cellular mitochondrial metabolic status. This technique has been previously validated for distinguishing metabolic states in other biological systems, such as cancer cells (36Yu Q. Heikal A.A. Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level.J Photochem Photobiol B. 2009; 95: 46-57Crossref PubMed Scopus (224) Google Scholar), cell lines (37Niesner R. Peker B. Schlüsche P. Gericke K.-H. Noniterative biexponential fluorescence lifetime imaging in the investigation of cellular metabolism by means of NAD(P)H autofluorescence.Chemphyschem. 2004; 5: 1141-1149Crossref PubMed Scopus (102) Google Scholar), animal tissues (38Vishwasrao H.D. Heikal A. Kasischke K. Webb W.W. Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy.J Biol Chem. 2005; 280: 25119-25126Crossref PubMed Scopus (261) Google Scholar), and during germ cell differentiation in Caenorhabditis elegans and stem cells differentiation (39Stringari C. Cinquin A. Cinquin O. Digman M.A. Donovan P.J. Gratton E. Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue.Proc Natl Acad Sci U S A. 2011; 108: 13582-13587Crossref PubMed Scopus (307) Google Scholar). Preliminary animal studies on oocytes and embryos indicate sensitivity to metabolic differences that are relevant in fertility. Recently, Sanchez et al. (40Sanchez T. Wang T. Pedro M.V. Zhang M. Esencan E. Sakkas D. et al.Metabolic imaging with the use of fluorescence lifetime imaging microscopy (FLIM) accurately detects mitochondrial dysfunction in mouse oocytes.Fertil Steril. 2018; 110: 1387-1397Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) showed that FLIM measurements were able to sensitively distinguish between metabolic states that are known to be different: [1] old versus young mice (40Sanchez T. Wang T. Pedro M.V. Zhang M. Esencan E. Sakkas D. et al.Metabolic imaging with the use of fluorescence lifetime imaging microscopy (FLIM) accurately detects mitochondrial dysfunction in mouse oocytes.Fertil Steril. 2018; 110: 1387-1397Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and [2] oocytes from wildtype and knockout mice for the gene, Clpp, a mutation affecting metabolic function and fertility (41Gispert S. Parganlija D. Klinkenberg M. Dröse S. Wittig I. Mittelbronn M. et al.Loss of mitochondrial peptidase clpp leads to infertility, hearing loss plus growth retardation via accumulation of CLPX, mtDNA and inflammatory factors.Hum Mol Genet. 2013; 22: 4871-4887Crossref PubMed Scopus (125) Google Scholar, 42Wang T. Babayev E. Jiang Z. Li G. Zhang M. Esencan E. et al.Mitochondrial unfolded protein response gene Clpp is required to maintain ovarian follicular reserve during aging, for oocyte competence, and development of pre-implantation embryos.Aging Cell. 2018; 17: 1-13Crossref Scopus (50) Google Scholar). Glycolysis (Fig. 1) is the metabolic pathway that converts glucose (a 6-carbon molecule [6C]) into two molecules of pyruvate (3-carbon molecule; [3C]). Glycolysis takes place in the cytoplasm and does not require oxygen. The free energy released in this process is used to gain two net molecules of ATP and to convert two molecules of NAD+ to NADH. Pyruvate molecules produced during glycolysis can be transported into the mitochondrial matrix and oxidized into acetyl CoA (AcCoA), leading to the formation of NADH (one for each pyruvate molecule converted to AcCoA) and facilitating the start of the Krebs cycle for additional energy production. For each acetyl group that enters the Krebs cycle, three additional molecules of NADH, one FADH2, and one GTP are produced. The NADH and FADH2 molecules can then be used to create additional ATP through oxidative phosphorylation (OXPHOS) (43Akram M. Citric acid cycle and role of its intermediates in metabolism.Cell Biochem Biophys. 2014; 68: 475-478Crossref PubMed Scopus (322) Google Scholar). The process of OXPHOS is mediated by the electron transport chain (ETC) located in the inner mitochondrial membrane and involves five protein complexes (Fig. 1). NADH and FADH2, are oxidized by complex I (NADH-coenzyme Q oxidoreductase) and complex II (succinate-Q oxidoreductase) of the ETC, respectively. The added electrons at complexes I and II are then relayed along the ETC and help generate a proton gradient between the mitochondrial intermembranous space (higher) and the mitochondrial matrix (lower). Finally, the movement of protons through the ATP synthase (complex V), along the proton gradient (from the mitochondrial intermembranous space to the mitochondrial matrix), results in the generation of ATP. Overall, the ETC oxidizes NADH to NAD+, and FADH2 to FAD, generating three and two ATPs per molecule, respectively (44Alberts B. Johnson A. Lewis J. Morgan D. Raff M. Roberts K. et al.Molecular biology of the cell.6th ed. Garland Science, New York2014Crossref Google Scholar). While glycolysis generates only a net total of two ATP molecules per glucose molecule, the Krebs cycle and ETC result in the synthesis of an additional 36 ATPs for each glucose metabolized (Fig. 1). NAD+ and FAD play a vital role in energy metabolism in eukaryotic cells by accepting hydride equivalents to form reduced NADH and FADH2. These furnish reducing equivalents to the mitochondrial ETC to fuel OXPHOS. NADH is a product of both the glycolysis (in the cytoplasm) and the Krebs cycle (in the mitochondrial matrix), while FADH2 is only produced in the Krebs cycle (Fig. 1). Since the mitochondrial membrane is not permeable to NAD+ (45Barile M. Passarella S. Danese G. Quagliariello E. Rat liver mitochondria can synthesize nicotinamide adenine dinucleotide from nicotinamide mononucleotide and ATP via a putative matrix nicotinamide mononucleotide adenylyltransferase.Biochem Mol Biol Int. 1996; 38: 297-306PubMed Google Scholar), the reduced form of NADH generated in the cytoplasm can be transported into the mitochondrial matrix via either the malate-aspartate shuttle or the glycerol-3-phosphate shuttle of the inner mitochondrial membrane (46Pittelli M. Formentini L. Faraco G. Lapucci A. Rapizzi E. Cialdai F. et al.Inhibition of nicotinamide phosphoribosyltransferase: cellular bioenergetics reveals a mitochondrial insensitive NAD pool.J Biol Chem. 2010; 285: 34106-34114Crossref PubMed Scopus (146) Google Scholar). Importantly, even in the presence of an excess of glucose, inadequate NAD+ could block glycolysis and NADH production, leading to cell death (47Ying W. Alano C.C. Garnier P. Swanson R.A. NAD+ as a metabolic link between DNA damage and cell death.J Neurosci Res. 2005; 79: 216-223Crossref PubMed Scopus (153) Google Scholar, 48Alano C.C. Garnier P. Ying W. Higashi Y. Kauppinen T.M. Swanson R.A. NAD+ depletion is necessary and sufficient for poly(ADP-ribose) polymerase-1-mediated neuronal death.J Neurosci. 2010; 30: 2967-2978Crossref PubMed Scopus (342) Google Scholar). When insufficient oxygen is available to support OXPHOS, pyruvate generated from glycolysis can be converted into lactate by lactate dehydrogenase through a process called lactate fermentation (Fig. 1). Fermentation allows the recycling of NADH back into NAD+ so that glycolysis can continue. This process does not require oxygen and occurs in muscle when the need for energy surpasses what OXPHOS can produce. NADH and FAD are fluorescent molecules, which means that shining light on them of one wavelength can cause them to transition to an excited state and emit light of another wavelength as they relax back to their ground state (34Lakowicz J.R. Principles of fluorescence spectroscopy.3d ed. Springer, 2006Crossref Scopus (17811) Google Scholar). Fluorescence microscopy takes advantage of this property to specifically visualize fluorescent molecules by selectively controlling the wavelength of the exciting light and using optical filters to only view light emitted by the molecule of interest (Fig. 2A). NADH and FAD have absorption and emission spectra that are highly distinct from each other (Fig. 2B), and from other cellular components (49Zipfel W.R. Williams R.M. Christie R. Nikitin A.Y. Hyman B.T. Webb W.W. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation.Proc Natl Acad Sci U S A. 2003; 100: 7075-7080Crossref PubMed Scopus (1531) Google Scholar), making it possible to study the behavior of these two molecules in vivo. Since the pioneering work of Chance and collaborators nearly 60 years ago (50Chance B. Schoener B. Oshino R. Oxidation-reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals.J Biol Chem. 1979; 254: 4764-4771Abstract Full Text PDF PubMed Google Scholar), fluorescence microscopy of NADH and FAD has been widely used to characterize the metabolic state of mitochondria, cells, and tissues (38Vishwasrao H.D. Heikal A. Kasischke K. Webb W.W. Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy.J Biol Chem. 2005; 280: 25119-25126Crossref PubMed Scopus (261) Google Scholar, 51Heikal A. Intracellular coenzymes as natural biomarkers for metabolic activities and mitochondrial anomalies.Biomark Med. 2010; 4: 241-263Crossref PubMed Scopus (341) Google Scholar, 52Walsh A.J. Cook R.S. Manning H.C. Hicks D.J. Lafontant A. Arteaga C.L. et al.Optical metabolic imaging identifies glycolytic levels, subtypes, and early-treatment response in breast cancer.Cancer Res. 2013; 73: 6164-6174Crossref PubMed Scopus (213) Google Scholar, 53Quinn K.P. Sridharan G.V. Hayden R.S. Kaplan D.L. Lee K. Georgakoudi I. Quantitative metabolic imaging using endogenous fluorescence to detect stem cell differentiation.Sci Rep. 2013; 3: 3432Crossref PubMed Scopus (176) Google Scholar). Fluorescence microscopy of NADH and FAD provides morphological information, including allowing the visualization of mitochondria, in which both molecules are highly enriched. The measured fluorescence intensity also reflects the activity of the pathways that these molecules are engaged in because the brightness of the fluorescence signal from these molecules is proportional to their concentration. While such intensity measurements are highly informative, they suffer from two major limitations: [1] the concentration of NADH and FAD reflects the relative balance of biochemical pathways, so very different physiological states can give rise to similar measured values; [2] the observed intensity depends on the details of the experimental setup in ways that are difficult to calibrate, making quantitative measurements highly challenging. Additional metabolic information can be extracted by using FLIM to measure the distribution of times NADH and FAD spend in their excited states (54Becker W. Fluorescence lifetime imaging—techniques and applications.J Microsc. 2012; 247: 119-136Crossref PubMed Scopus (572) Google Scholar, 55Heikal A.A. A multiparametric imaging of cellular coenzymes for monitoring metabolic and mitochondrial activities.in: Geddes C. Reviews in fluorescence 2010. Springer, New York2012Google Scholar), which strongly depends on the microenvironment of the fluorophores: most importantly, engagement with enzymes leads to a drastic shift in the time NADH and FAD spend in their excited state (35Ghukasyan V.V. Heikal A.A. Natural biomarkers for cellular metabolism: biology, techniques, and applications. CRC Press, 2014Crossref Scopus (39) Google Scholar, 56Blinova K. Levine R.L. Boja E.S. Griffiths G.L. Shi Z.D. Ruddy B. et al.Mitochondrial NADH fluorescence is enhanced by complex I binding.Biochemistry. 2008; 47: 9636-9645Crossref PubMed Scopus (81) Google Scholar). Thus, FLIM enables measurements reflecting the concentration of NADH and FAD (from intensity) and the extent to which those molecules are engaged with enzymes (from the time they spend in the excited state). There are a variety of different methods for performing FLIM measurements (54Becker W. Fluorescence lifetime imaging—techniques and applications.J Microsc. 2012; 247: 119-136Crossref PubMed Scopus (572) Google Scholar). Of these, time-correlated single photon counting (TCSPC) has a number of advantages in terms of photon economy, signal-to-noise, and error analysis, making it well suited for robust, quantitative measurements (57Becker W. The bh TCSPC handbook.7th ed. Becker & Hickl Gmbh, Berlin, Germany2017Google Scholar). TCSPC-FLIM uses a laser that generates a high frequency of very short pulses for excitation (Fig. 2C). The power of the laser is kept low enough such that only about one in 100 laser pulses results in the fluorescence molecule producing a photon that can be detected. A sensitive detector enables these individual photons to be counted, and, for each photon, fast electronics allow the precise arrival time of the photon to be determined (Fig. 2D). The arrival times are combined to form a histogram, which represents how long the fluorophores remain in the excited state (Fig. 2E). Simple fluorophores, such as fluorescein, exhibit an exponential distribution of times in the excited state. In contrast, the histogram of times in the excited state for NADH and FAD are double exponentials, one corresponding to the population of molecules engaged with enzymes and the other corresponding to the population of molecules not engaged with enzymes. By fitting the histogram of photon arrival times to a double exponential, it is possible to measure the fraction of NADH and FAD molecules engaged with enzymes (Fig. 2E). The value of the characteristic lifetime associated with these two states depends on the detailed local environment of NADH and FAD, and FLIM provides information on that as well. FLIM is a microscopy-based technique, producing histograms of times in the excited state for each pixel in an image. Thus, FLIM of NADH and FAD can provide metabolic information with subcellular resolution, limited only by the signal-to-noise of the measurement. In addition, FLIM measurements are relatively robust and are not prone to the experimental artifacts that plague intensity measurements. Since the metabolism of embryos and oocytes is central to their viability, and since FLIM provides a means of noninvasively and quantitatively measuring metabolism, FLIM is a promising technique for assessing oocyte and embryo viability. We have recently carried out a number of studies on mouse oocytes and embryos to evaluate the safety and potential utility of FLIM within this context (40Sanchez T. Wang T. Pedro M.V. Zhang M. Esencan E. Sakkas D. et al.Metabolic imaging with the use of fluorescence lifetime imaging microscopy (FLIM) accurately detects mitochondrial dysfunction in mouse oocytes.Fertil Steril. 2018; 110: 1387-1397Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). FLIM of NADH and FAD of embryos and oocytes allows their structure to be visualized (Fig. 3A). As NADH is highly concentrated in the mitochondria (58Stein L.R. Imai S.I. The dynamic regulation of NAD metabolism in mitochondria.Trends Endocrinol Metab. 2012; 23: 420-428Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar), and FAD is almost entirely localized within the mitochondria (59Dumollard R. Marangos P. Fitzharris G. Swann K. Duchen M. Carroll J. Sperm-triggered [Ca2+] oscillations and Ca2+ homeostasis in the mouse egg have an absolute requirement for mitochondrial ATP production.Development. 2004; 131: 3057-3067Crossref PubMed Scopus (188) Google Scholar), both intensity images reflect the distribution of mitochondria. Since aberrations in mitochondrial localization have been associated with mitochondrial dysfunction (60Nagai S. Mabuchi T. Hirata S. Shoda T. Kasai T. Yokota S. et al.Correlation of abnormal mitochondrial distribution in mouse oocytes with reduced developmental competence.Tohoku J Exp Med. 2006; 210: 137-144Crossref PubMed Scopus (104) Google Scholar), these images alone may be useful for screening metabolically challenged oocytes and embryos. High-resolution images from FLIM of NADH and FAD can be automatically segmented using image processing (61Gonzalez R.C. Digital image processing.4th ed. Pearson, 2018Google Scholar) and feature recognition algorithms (62Breiman L. Random forests.Mach Learn. 2001; 45: 1-32Google Scholar), allowing mitochondrial and cytoplasmic regions to be separately integrated (Fig. 3B). Combining photon arrival times from all pixels in each region into a single histogram leads to high signal-to-noise measurements, which can be fit by a double exponential model (Fig. 3C). Each of these fits provides four metabolic parameters: fraction engaged (F), short lifetime (τ1), long lifetime (τ2), and average intensity (I). With four parameters each from mitochondrial NADH, cytosolic NADH, and mitochondrial FAD (there is no appreciable cytosolic FAD), a single metabolic acquisition yields up to 12 parameters for measuring embryo or oocyte metabolic state (Fig. 3D shows eight metabolic parameters extracted from the NADH measurement). These parameters are highly sensitive to differences and deficiencies in the metabolic state of oocytes and embryos. In a recent proof-of-concept study, mouse oocytes with significant metabolic dysfunction due to a mutation in a mitochondrial stress response gene exhibited highly significantly different FLIM parameter values compared with wild-type (normal) oocytes (40Sanchez T. Wang T. Pedro M.V. Zhang M. Esencan E. Sakkas D. et al.Metabolic imaging with the use of fluorescence lifetime imaging microscopy (FLIM) accurately detects mitochondrial dysfunction in mouse oocytes.Fertil Steril. 2018; 110: 1387-1397Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Within the same experimental system, mtDNA copy number was only marginally different between the groups. FLIM was also used to compare oocytes from old (1-year-old) versus young mice as a model for mild metabolic dysfunction and showed highly significant differences; mtDNA copy number was not significantly different between the groups (40Sanchez T. Wang T. Pedro M.V. Zhang M. Esencan E. Sakkas D. et al.Metabolic imaging with the use of fluorescence lifetime imaging microscopy (FLIM) accurately detects mitochondrial dysfunction in mouse oocytes.Fertil Steril. 2018; 110: 1387-1397Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Furthermore, FLIM metabolic parameters change over the course of preimplantation embryo development, as the embryo's metabolism reconfigures. Parameters also undergo a large shift in response to mitochondria poisons and changing culture media and oxygen tension (unpublished). Taken together, these results show that FLIM of NADH and FAD can detect biologically relevant differences in the metabolism of oocytes and embryos. Metabolic imaging with FLIM serves as a powerful research tool for elucidating fundamental aspects of embryo and oocyte metabolism. Studies aimed at determining how oocytes and preimplantation embryos respond to environmental cues such as changes in nutrient and gas content in the culture environment would largely benefit from this sensitive assay. We can also be cautiously optimistic for a potential application in clinical IVF, despite the failure of previous attempts at exploiting metabolic and metabolomics parameters as an embryo viability test. One advantage of FLIM is that the metabolic assessment is done directly in the cell, without being affected by the dilution and variation associated with spent culture media analyses. Nevertheless, clinical application will require a number of challenging steps, including development of sophisticated algorithms for viability prediction, nonselection studies to determine the diagnostic accuracy of the technique, and randomized clinical trials to demonstrate benefit. Metabolism is a key determinant of cell survival, and metabolic parameters could be exploited to improve our understanding of oocyte and embryo viability. Within this context, metabolic imaging via FLIM offers new and potentially useful diagnostic potential by detecting natural fluorescence of FAD and NADH, the two electron transporters that play a central role in OXPHOS. FLIM has been used for metabolic imaging of a variety of systems and most recently has been shown to effectively assess oocyte metabolic state in mouse models of severe and mild metabolic dysfunction. It is likely that FLIM technology will be very useful for experimental studies aimed at improving our understanding of oocyte and embryo metabolism. In addition, FLIM could potentially be implemented as a noninvasive embryo viability test in assisted reproduction, pending appropriate studies." @default.
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• W2912986577 title "Metabolic imaging via fluorescence lifetime imaging microscopy for egg and embryo assessment" @default.
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• W2912986577 doi "https://doi.org/10.1016/j.fertnstert.2018.12.014" @default.
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