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WO2016148905A1 - Flim permettant de différencier des ovocytes anciens et jeunes - Google Patents

Flim permettant de différencier des ovocytes anciens et jeunes Download PDF

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WO2016148905A1
WO2016148905A1 PCT/US2016/020245 US2016020245W WO2016148905A1 WO 2016148905 A1 WO2016148905 A1 WO 2016148905A1 US 2016020245 W US2016020245 W US 2016020245W WO 2016148905 A1 WO2016148905 A1 WO 2016148905A1
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flevi
nadh
embryo
fad
cells
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PCT/US2016/020245
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Timothy H. SANCHEZ
Manqi DENG
Daniel NEEDLEMAN
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President And Fellows Of Harvard College
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0609—Oocytes, oogonia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G01N2021/6413—Distinction short and delayed fluorescence or phosphorescence

Definitions

  • the invention relates to methods for assessing the metabolic condition of oocytes, and can be used, e.g., in assessment of oocytes in assisted reproductive technologies. Specifically, the invention relates to differentiating cells, such as oocytes that are more viable from the ones that are less viable, such as differentiating between older and younger oocytes. The methods can be also be used to select the most viable embryos for implantation or
  • ART Assisted Reproductive Technology
  • oocytes from aging women are often not optimal quality for IVF. Having a method to rank the oocytes based on their viability prior to IVF to allow IVF only on the most viable oocytes to be used in the procedure would be an important advance.
  • Minimally invasive, simple and reliable methods for assessing embryo or oocyte viability for assisted reproductive technologies would provide a significant advance for improving the safety of the mother and the fetus, and would also reduce the costs of the assisted reproductive technologies by reducing the number of times one has to try
  • the quality of an oocyte and/or embryo can refer to the health, metabolic activity, suitability for successful embryo (and/or fetal) development, and/or likelihood of successful embryo (and/or fetal) development.
  • the described methods are minimally invasive and allow ranking or scoring of the oocytes or embryos based on their metabolic activity, where one or more of the most metabolically active oocytes or embryos can be used in methods of in vitro fertilization.
  • an oocyte or embryo can "rate” or “score" the oocytes/embryos that are subjected to the analysis and allow selection of the oocyte or oocytes or embryo or embryos that have the best chance of viability in IVF.
  • the oocytes or embryos with the most optimal metabolic score can then be selected for in vitro fertilization or implantation, and/or the
  • oocytes/embryos with lowest metabolic activity can be discarded from in vitro fertilization or implantation.
  • oocyte quality depends on oocyte metabolic state. Oocyte metabolic state can be rapidly, non-invasively, and quantitatively measured by Fluorescence Lifetime Imaging Microscopy (FLEVI) of FAD or NADH. Two parameters (alpha and beta) were extracted from FLEVI measurements of NADH (or FAD) in oocytes.
  • FLEVI Fluorescence Lifetime Imaging Microscopy
  • alpha and beta were extracted from FLEVI measurements of NADH (or FAD) in oocytes.
  • mouse oocytes but similar calculations are expected to work for human oocytes, as the mammalian cells, such as mouse and human cells are relatively similar, particularly relating to their metabolic state, at this stage. Each point corresponds to data from a single oocyte.
  • Perturbing oocytes by specific metabolic inhibitors black crosses
  • nonspecific damage causes both parameters to increase.
  • Unperturbed oocytes (circles) exhibit a range of values of alpha and beta. These parameters are indicative of oocyte quality.
  • autofluorescence lifetime including fraction bound, short and long lifetimes, and average brightness to a reference distribution which provides a convenient way of selecting viable and non-viable oocytes/embryos.
  • the FLEVI process can also be used in previously cryopreserved oocytes and embryos to select the most viable ones from those.
  • the lifetime distribution of NADU/FAD can be approximated as a sum of two exponentials.
  • the parameter a is the ratio of the amplitude of the two exponentials
  • the parameter, ⁇ is the lifetime of the longer exponential.
  • Increase in both alpha and beta of NADH was found to be indicative of damage in the embryo/oocyte.
  • cells with increased alpha and beta values compared to a reference value are discarded from ART methods as they would be considered damaged and alpha and beta less than a reference value would be selected for ART methods as their metabolic activity is indicative of healthy activity.
  • the FLEVI measurements according to the methods of the invention can be performed with extremely low levels of illumination, which is far less than is currently used in in vitro fertilization clinics to determine the morphology of oocytes and embryos. Therefore, the FLEVI measurements performed according to the methods of the present invention will not perturb oocytes and embryos and are thus as non-invasive as possible.
  • a method for assessing the quality of an oocyte or an embryo comprising (a) exposing an embryo or a test cell or a plurality of test cells selected from an oocyte or an oocyte-associated cumulus cell or a cell from an embryo to a fluorescence lifetime imaging microscope (FLEVI) to acquire a fluorescence lifetime histogram of auto-fluorescence of endogenous NADH and FAD for the embryo or the test cell; (b) averaging the fluorescence lifetime histogram of NADH auto-fluorescence and FAD auto-fluorescence over the entire embryo, test cell or test cells, or the cytoplasm of the test cell or cells, or mitochondria of the test cell or cells to assay measurements for fraction bound, short and long lifetimes, and average brightness for both FAD and NADH (c) combining the measurements for fraction bound, short and long lifetimes, and average brightness for both FAD and NADH to obtain a metabolic score; (d) comparing the metabolic score between the plurality of test cells selected from an oocyte or an o
  • the cells can be fresh or previously cryopreserved.
  • One or more best scoring cells or embryos i.e., the cells with most optimal metabolic state can be selected for further processes, such as IVF, implantation or cryopreservation.
  • the method comprises a step of establishing the statistical significance by fitting lifetime histograms to a sum of two exponentials and the parameters of the measured cells are deemed to fall within or not fall within the range of parameters found in the healthy cells.
  • the parameters to be compared between the measurements and the healthy cells are alpha, defined as the ratio of the amplitude of the two exponentials, and beta, defined as the lifetime of the longer exponential.
  • the maximum value of alpha from the healthy cells is a specific value within the range 1.0 - 4.0 and the corresponding maximum value of beta of healthy cells is in the range 2000 ps - 3000 ps.
  • the FLEVI is performed using a wavelength of about 740 nm in two-photon fluorescence excitation and using an emission bandpass filtered centered around about 460 nm.
  • the FLEVI is performed using a wavelength of about 340 nm in one-photon fluorescence excitation and using an emission bandpass filtered centered around about 460 nm.
  • the FLEVI is performed in the frequency domain instead of the time domain.
  • the FLEVI is performed using a wavelength of about 900 nm in two-photon fluorescence excitation and using an emission bandpass filtered centered around about 550 nm.
  • the FLEVI is performed using a wavelength of about 450 nm in one-photon fluorescence excitation and using an emission bandpass filtered centered around about 550 nm.
  • the methods are performed in vitro. The noninvasive nature of the methods allow them to be performed without harming the embryos or oocytes.
  • Figure 1 shows data from FLEVI imaging of NADH in mouse oocytes. Non-linear microscopy was used to assess the quality of live oocytes and embryos. FLEVI curves from individual oocytes were fit to the sum of two exponentials. The parameter a, is the ratio of the amplitude of the two exponentials, the parameter, ⁇ , is the lifetime of the longer exponential in picoseconds. Each point corresponds to data from a single oocyte.
  • Unperturbed oocytes exhibit a range of values of a and ⁇ . Perturbing oocytes by specific metabolic inhibitors (circles) or non-specific damage (triangles) causes both parameters to increase.
  • Figure 2 shows fraction of FAD and NADH molecules bound to enzymes in young and old mouse oocytes.
  • Figure 3 shows long lifetime values for NADH and FAD (measured in nanoseconds) in young and old mouse oocytes.
  • Figure 4 shows fraction of FAD and NADH molecules bound to enzymes in young and old mouse oocytes.
  • Figure 5 shows an example of a "metabolic score" measurement. As can be seen, young eggs can be easily distinguished from the old eggs.
  • Figure 6 depicts a graph of two-photon measurements.
  • Figure 7 depicts a graph of one-photo measurements.
  • the metabolic state of an oocyte or an embryo can be directly assessed using FLEVI imaging of the autofluorescence of the cellular metabolites inside the oocyte or the embryo or using one or more cumulus cells that surround the oocyte or a cell biopsied from the embryo.
  • Oocytes and embryos with acceptable or best available metabolic status can then be selected for in vitro fertilization or implantation and oocytes and embryos having abnormal metabolic status can be discarded from further assisted reproductive methods.
  • the methods provide a way to analyze embryos/oocytes with a process that is much less invasive than any method we are aware of.
  • the embryos/oocytes can be fresh or previously cryopreserved. With the scoring method presented herein, one can also select the most viable embryos/oocytes for cryopreservation.
  • the methods for assessing embryo quality are useful for predicting which embryos have the greatest potential for implantation in order to: (i) increase pregnancy rates with assisted reproductive technologies; (ii) decrease multiple pregnancy rates by justifying transfer of the single "best" embryo; and (iii) appropriately select suitable embryos for cryopreservation and (iv) increase the efficiency of offspring from transgenic intervention.
  • the methods for assessing embryo quality are also useful for assessing the impact or effect of current invasive procedures on the oocyte and embryo, including: (i) intracytoplasmic sperm injection; and (ii) blastomere biopsy for pre-implantation cytogenetic diagnosis.
  • FLEVI FLEVI to detection of metabolic state of embryos and/or oocytes by direct analysis of the embryos or oocytes, or by analysis of cells from embryos or analysis of cumulus or granulosa cells surrounding the oocytes.
  • the currently described method measures FLIM parameters that include fraction bound, short and long lifetimes, and average brightness for both FAD and NADH; and combines these parameters into a comprehensive "metabolic score.”
  • FLEVI Fluorescence Lifetime Imaging Microscopy
  • the cells one can use at least three parameters, namely, short lifetime, long lifetime, and fraction bound to enzymes, that are extracted from FLEVI measurements of NADH and FAD in oocytes, embryos or oocyte-associated cumulus cells.
  • Binding to enzymes causes the lifetime of the fluorophore to shift significantly, explaining the strong correlation in all three parameters (see, e.g., Figures 2-4).
  • the metabolic score can be applied to compare between oocytes and/or embryos to select the oocytes/embryos for further use, either for IVF or implantation or cryopreservation, that show the best metabolic score indicating they have the best chance of survival.
  • oocyte refers to a female germ cell.
  • the oocyte analyzed according to the methods of the invention is obtained prior to fertilization, and the analysis is performed in vitro.
  • Pre-implantation embryo or "embryo” is used herein to refer typically to an in vitro fertilized oocyte with two pronuclei (up to and including a blastocyst) but which has not implanted in the lining of the female reproductive tract.
  • the pre-implantation embryo contains between about 2 and about 8 cells (i.e., the embryo is assessed between about 18 and about 70 hours post-fertilization), although these ranges may vary among species.
  • the quality assessment for a human or a mouse embryo is performed on an embryo containing between 2 and 8 cells.
  • Cell from an embryo refers to a single cell biopsied from the embryo.
  • Embryo biopsy is a procedure that involves removing one or more cells from the embryo before it is transferred to the mother's uterus. It is typically performed for testing the embryo for specific genetic disorders. The methods of the present invention can be performed to the cells prior to performing a genetic analysis.
  • cumulus oophorus cells cumulus granulosa cells, cumulus oophorous granulosa cells, granulosa- cumulus cells are used to make a distinction between these cells and the other functionally different subpopulation of granulosa cells at the wall of the Graafian follicle.
  • Cumulus cells provide key products for the acquisition of developmental competence and differ from granulosa cells in their hormonal responses and growth factors they produce. The absence of cumulus cells or insufficient numbers of cumulus cells impairs embryo production. Denuded oocytes in culture cannot undergo normal fertilization with standard insemination. Cumulus cells are required for the successful maturation of oocytes.
  • the method uses one or more cumulus cells to select an oocyte for in vitro fertilization or for excluding an oocyte from in vitro fertilization.
  • the cumulus cells are obtained from around an oocyte prior to insemination or ICSI, and the analysis is performed in vitro.
  • the cells are typically placed in a well suitable for imaging and comprising cell culture medium at 37°C.
  • the "reference value" as referred to herein, is typically assessed using normal healthy cells of comparable origin, such as normal healthy oocytes, normal healthy embryos, cumulus cells around a normal healthy oocyte or cells from a normal healthy embryo.
  • the reference values are typically a range from averaged experiments, and are typically pre-determined although assays including a healthy reference cell of similar origin are also provided.
  • the methods of the invention use a device that is typically a self-contained, microscope setting, such as a box, such as a table-top microscope box that is typically used at in vitro fertilization clinics to select oocytes for fertilization and embryos for transfer.
  • a device that is typically a self-contained, microscope setting, such as a box, such as a table-top microscope box that is typically used at in vitro fertilization clinics to select oocytes for fertilization and embryos for transfer.
  • the methods are based on use of fluorescence lifetime imaging microscopy (FLEVI) of metabolic state of the oocytes or embryos.
  • the interior of the device comprises, or consists essentially of the microscope and all the peripherals, as well as an environmental chamber enclosing the microscope stage.
  • a small slot in the microscope exterior allows custom, multi-well plates containing granulosa cells, oocytes or embryos or cells biopsied from an embryo to be inserted onto the microscope stage.
  • a screen monitor such as a touch-screen monitor, for example located on the device's exterior, contains controls and displays acquired data.
  • the device can be used on oocytes and embryos as well as cumulus cells and cells isolated from an embryo that can be acquired by any procedure and placed in any media.
  • the methods are easily compatible with the current practices in in vitro fertilization clinics and other settings where assessment of embryos or oocytes is performed.
  • Acetyl CoA is completely oxidized in a cyclic series of oxidative reactions alternately referred to as the tricarboxylic acid (TCA) cycle, the Krebs cycle or the citric acid cycle.
  • TCA tricarboxylic acid
  • certain of the TCA cycle enzymes are also found in the cytosol, where the enzymes function in other metabolic pathways, all of the TCA cycle enzymes are located in the mitochondria.
  • the oxidation of acetyl CoA in one complete TCA cycle results in the production of two C0 2 molecules, one high energy phosphate bond (such as that present in GTP) and four reducing equivalents, i.e., three NADH and one FADH 2 from three NAD+ and one FAD, respectively.
  • the oocyte/embryo contains a steady-state concentration of NADH that is present in the cell(s) as a result of metabolic reactions taking place in the mitochondria (e.g., tricarboxylic acid cycle and electron transport) and in the cytosol (e.g., glycolysis).
  • mitochondria e.g., tricarboxylic acid cycle and electron transport
  • cytosol e.g., glycolysis
  • the method of the present invention allows direct analysis of NADH and/or FAD inside an
  • Fluorescence-lifetime imaging microscopy is an imaging technique for producing an image based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample.
  • FLEVI can be used as an imaging technique in confocal microscopy, two-photon excitation microscopy, and multiphoton tomography.
  • a fluorophore which is excited by a photon will drop to the ground state with a certain probability based on the decay rates through a number of different (radiative and/or nonradiative) decay pathways. To observe fluorescence, one of these pathways must be by spontaneous emission of a photon. This can be utilized for making non-intensity based measurements in chemical sensing.
  • Fluorescence lifetimes can be determined in the time domain by using a pulsed source.
  • Time-correlated single-photon counting is usually employed. More specifically, TCSPC records times at which individual photons are detected by something like a photo-multiplier tube (PMT) or an avalanche photo diode (APD) after a single pulse. The recordings are repeated for additional pulses, and after enough recorded events one is able to build a histogram of the number of events across all of these recorded time points. This histogram can then be fit to a function that contains parameters of interest, and thus the parameters can be accordingly be extracted. 16-64 multichannel PMT systems have been commercially available, whereas the recently demonstrated CMOS single-photon avalanche diode (SPAD)-TCSPC FLEVI systems can offer additional low-cost options.
  • PMT photo-multiplier tube
  • APD avalanche photo diode
  • Pulse excitation is still used in the gating method. Before the pulse reaches the sample, some of the light is reflected by a dichroic mirror and gets detected by a photodiode that activates a delay generator controlling a gated optical intensifier (GO I) that sits in front of your CCD detector.
  • the GOI only allows for detection for the fraction of time when it is open after the delay.
  • an adjustable delay generator one is able to collect fluorescence emission after multiple delay times encompassing the time range of the fluorescence decay of the sample.
  • fluorescence lifetimes can be determined in the frequency domain by a phase-modulated method.
  • the intensity of a continuous wave source is modulated at high frequency, by an acousto-optic modulator for example, which will modulate the fluorescence. Since the excited state has a lifetime, the fluorescence will be delayed with respect to the excitation signal, and the lifetime can be determined from the phase shift. Also, y- components to the excitation and fluorescence sine waves will be modulated, and lifetime can be determined from the modulation ratio of these y-components. Hence, 2 values for the lifetime can be determined from the phase-modulation method. Consequently, if the lifetimes that are extracted from the y-component and the phase do not match, it means that you have more than one lifetime species in your sample.
  • FLEVI has primarily been used in biology as a method to detect photosensitizers in cells and tumors as well as FRET in instances where ratiometric imaging is difficult.
  • the technique was developed in the late 1980s and early 1990s (Bugiel et al. 1989. Konig 1989) before being more widely applied in the late 1990s (Oida T, Sako Y, Kusumi A (March 1993). "Fluorescence lifetime imaging microscopy (flimscopy). Methodology development and application to studies of endosome fusion in single cells". Biophys. J. 64 (3): 676-85). In cell culture, it has been used to study EGF receptor signaling (W outers FS, Bastiaens PI (October 1999).
  • FLFM imaging using pulsed illumination has been used to study Ras (Harvey CD, Yasuda R, Zhong H, Svoboda K (July 2008). "The spread of ras activity triggered by activation of a single dendritic spine”. Science 321 (5885): 136-40), CaMKII, Rac, and Ran (The design of Forester (fluorescence) resonance energy transfer (FRET)-based molecular sensors for Ran GTPase, in press P. Kalab, J. Soderholm, Methods (2010) family proteins). FLFM has also been used in clinical multiphoton tomography to detect intradermal cancer cells as well as pharmaceutical and cosmetical compounds.
  • the cells in the present method can be analyzed in any suitable cell culture medium used for embryo/oocyte/cumulus cell/cell from embryo.
  • the present methods avoid subjecting the cells to any extraordinary medium changes.
  • there is no need to change the metabolic state of the cell like e.g., in the '081 patent, no additional time is needed for the analysis, making the analysis fast and convenient.
  • the measurement consists of acquiring a single FLEVI image per cell, which can be obtained rapidly and non-invasively or minimally invasively. Typically, it takes about 1-5 minutes, sometimes 30 seconds to 2 minutes to load a sample containing the cells to be studied and only seconds to acquire the data after which the oocyte/embryo that has been analyzed can either be selected for further fertilization or implantation or discarded as not optimally fit for these procedures.
  • NADH nicotinamide adenine dinucleotide
  • FAD dinucleotide
  • the acquired data are typically subsequently averaged over the entire cell, because subcellular information is unnecessary, producing one FLEVI curve per cell.
  • mouse embryos and oocytes While we have used mouse embryos and oocytes in our examples provided herein, the assay will not need to be altered when analyzing human cells.
  • the metabolic state of human and mouse embryos and oocytes are comparable during these stages of development and thus the results obtained with mouse oocytes/embryos can be directly applied to human cells as well.
  • the method of the invention comprises exposing a test cell to a
  • the test cell can be an oocyte or an oocyte-associated cumulus cell or an embryo or a cell from the embryo.
  • test cell Before the exposure, the test cell can be in or be placed in any normal cell culture medium used in maintaining the embryos/oocytes at the clinic. Culture media for embryo development should meet the metabolic needs of pre-implantation embryos by addressing amino acid and energy requirements based on the specific developmental stage of the embryo.
  • Culture media like Earle, Ham's F10, Tyrode's T6 and Whitten's WM1 were based on different salts and were constructed to support the development of somatic cells and cell lines in culture. These culture media, known as physiological salt solutions were used by Robert Edwards for his first successful In Vitro Fertilization (IVF). These media were formulated for use with or without serum supplementation, depending on the cell type being cultured.
  • the Ham's Nutrient Mixtures were originally developed to support growth of several clones of Chinese hamster ovary (CHO) cells, as well as clones of HeLa and mouse L-cells.
  • HTF Human Tubal Fluid
  • Gl supports the in-vitro development of the fertilized oocyte, the zygote, to the 8-cell stage, and G2 from 8-cells to blastocyst.
  • the typical composition of the embryo culture medium includes: culture media containing a phosphate buffer or Hepes organic buffer are used for procedures that involve handling of gametes outside of the incubator, flushing of follicles and micromanipulation.
  • the pH and osmolality for most culture media utilize a bicarbonate/C0 2 buffer system to keep pH in the range of 7.2-7.4.
  • the osmolanty of the culture medium should be in the range of 275-290 mosmol/kg. Similar conditions should optimally be maintained while imaging the cells according to the methods of the invention.
  • the human oocyte is temperature-sensitive and a humidified incubator with a temperature setting of 37.0-37.5°C should be used for oocyte fertilization and embryo culture. Similar temperature should optimally be maintained while imaging the cells according to the methods of the invention.
  • Embryos should be cultured under paraffin oil, which prevents evaporation of the medium preserving a constant osmolarity.
  • the oil also minimizes fluctuations of pH and temperature when embryos are taken out of the incubator for microscopic assessment.
  • Paraffin oil can be toxic to gametes and embryos; therefore, batches of oil must be screened and tested on mouse embryos before use in culture of human embryos. The oil does not need to be removed to perform the FLEVI analysis of the invention.
  • the medium is also composed of 99% water. Purity of the water is important, and is typically achieved by ultrafiltration.
  • Albumin or synthetic serum are typically added in concentrations of 5 to 20% (w/v or v/v, respectively).
  • the commercial media typically includes synthetic serum in which the composition is well known.
  • Commercial IVF media typically comprises, for example, one or more of the following components: synthetic serum, recombinant albumin, salt solution in MTF, NaCl, KC1, KH 2 P0 4 , CaCl 2 2H 2 0, MgS0 4 7H20, NaHC0 3 , and carbohydrates.
  • Carbohydrates are present in the female reproductive tract. Their concentrations vary throughout the length of the oviduct and in the uterus, and are also dependent on the time of the cycle.
  • Amino acids supplement of the culture medium with amino acids is necessary for embryo development.
  • Media that support the development of zygotes up to 8-cells are often further supplemented with non-essential amino acids.
  • Media that support the development of 8-cell embryos up to the blastocyst stage are typically supplemented with essential amino acids: Cystine, histadine, isolucine, leucine, lysine, methionine, valine, argentine, glutamine, phenylalanine, therionine, tryptophane.
  • Aminoglycoside Gram-negative bacteria disturbs protein synthesis.
  • the anti -bacterial effect of penicillin is attributed to its ability to inhibit the synthesis of peptidoglycan, unique glycoproteins of the bacterial cell wall.
  • Streptomycin and gentamycin belong to the aminoglycoside group of antibiotics which exert their antibacterial effect by inhibiting bacterial protein synthesis.
  • the use of genthamicine is still controversial and it is not being used by every laboratory.
  • the culture medium also comprises EDTA which is used as a chelator in medium that supports the embryo from the zygote stage to 8-cells and prevents abnormal glycolysis.
  • the methods of the invention are not dependent on the type of the medium.
  • the cells should remain in the medium and conditions they are cultured to avoid additional stress to them during the FLEVI analysis.
  • the method further comprises averaging the fluorescence lifetime histogram of NADH auto-fluorescence or FAD auto-fluorescence or both over the entire test cell, or the cytoplasm of the test cell, or mitochondria of the test cell.
  • the method also comprises comparing the averaged fluorescence lifetime histogram from the test cell to an averaged fluorescence lifetime histogram reference value to determine if the measured averaged fluorescence lifetime histogram from the test cell differs statistically from that of the reference value.
  • the comparing is typically made using a non-human machine typically using a computer executable software which includes a comparison between a reference value and the value from each individual cell FLEVI analyses.
  • the FLEVI curves of NADH crom cells exhibit a double exponential decay with a long lifetime (about 2.5 nanoseconds (ns)) corresponding to protein bound NADH and a short lifetime (about 0.4 ns) corresponding to free NADH.
  • the FLEVI curve is a double exponential with the relative fraction of the long and short lifetimes reflecting the relative fraction of protein bound and free NADH. This provides a direct readout of the metabolic state of the cell (Lacowciz et al., 1992).
  • the long lifetime might vary from 1-3 (nanoseconds) ns and the short life time might vary from 0.2 - 0.7 ns.
  • NADH absorption and fluorescence spectra of NADH (the reduced form) have been well characterized at different levels of organization, i.e., in solution, mitochondria and cell suspensions, tissue slices, and organs in vitro and in vivo.
  • NADH has an optical absorption band at about 300 to 380 nm and a fluorescence emission band at 420 to 480 nm.
  • the spectra are considered the same, although there are small differences in the shape and maxima of the spectra for different environments and measurement conditions.
  • the intensity of the fluorescence band is proportional to the concentration of mitochondrial NADH (the reduced form), particularly when measured in vivo from a tissue (see, e.g., review by Avraham Mayevsky and Gennady G. Rogatsky, Am J Physiol Cell Physiol February 2007 vol. 292 no. 2 C615-C640).
  • the direct autofluorescence of NADH can be analyzed with FLEVI using a wavelength of about 740 nm in two-photon fluorescence excitation and using an emission bandpass filter centered around about 460 nm.
  • the direct autofluorescence of NADH can be analyzed with FLEVI using a wavelength of from about 720 nm to about 760 nm in two-photon fluorescence excitation and using an emission bandpass filter centered around from about 400 nm to about 485 nm.
  • the direct autofluorescence of NADH can be analyzed with FLEVI using a wavelength of from about 720 nm to about 760 nm in two-photon fluorescence excitation and using an emission bandpass filter of about 50 nm bandwidth centered around from about 400 nm to about 485 nm.
  • the direct autofluorescence of NADH can be analyzed with FLEVI using a wavelength of from about 720 nm to about 760 nm in two-photon fluorescence excitation and using an emission bandpass filter centered around about 460 nm. In some aspects of all the embodiments of the invention, the direct autofluorescence of NADH can be analyzed with FLEVI using a wavelength of from about 720 nm to about 760 nm in two-photon fluorescence excitation and using an emission bandpass filter of about 50 nm bandwidth centered around about 460 nm.
  • the direct autofluorescence of NADH can be analyzed with FLEVI using a wavelength of about 740 nm in two-photon fluorescence excitation and using an emission bandpass filter centered around about 460 nm. In some aspects of all the embodiments of the invention, the direct autofluorescence of NADH can be analyzed with FLEVI using a wavelength of about 740 nm in two-photon fluorescence excitation and using an emission bandpass filter of about 50 nm bandwidth centered around about 460 nm.
  • the direct autofluorescence of NADH can be analyzed with FLEVI using a wavelength of about 750 nm in two-photon fluorescence excitation and using an emission bandpass filter centered around about 460 nm. In some aspects of all the embodiments of the invention, the direct autofluorescence of NADH can be analyzed with FLEVI using a wavelength of about 750 nm in two-photon fluorescence excitation and using an emission bandpass filter of about 50 nm bandwidth centered around about 460 nm.
  • the direct autofluorescence of NADH can be analyzed with FLEVI using a wavelength of from 720 nm to 760 nm in two-photon fluorescence excitation and using an emission bandpass filter centered around from 400 nm to 485 nm.
  • the direct autofluorescence of NADH can be analyzed with FLEVI using a wavelength of from 720 nm to 760 nm in two-photon fluorescence excitation and using an emission bandpass filter of about 50 nm bandwidth centered around from 400 nm to 485 nm.
  • the direct autofluorescence of NADH can be analyzed with FLEVI using a wavelength of from 720 nm to 760 nm in two- photon fluorescence excitation and using an emission bandpass filter centered around 460 nm. In some aspects of all the embodiments of the invention, the direct autofluorescence of NADH can be analyzed with FLEVI using a wavelength of from 720 nm to 760 nm in two- photon fluorescence excitation and using an emission bandpass filter of 50 nm bandwidth centered around 460 nm.
  • the direct autofluorescence of NADH can be analyzed with FLEVI using a wavelength of 740 nm in two-photon fluorescence excitation and using an emission bandpass filter centered around 460 nm. In some aspects of all the embodiments of the invention, the direct autofluorescence of NADH can be analyzed with FLEVI using a wavelength of 740 nm in two-photon fluorescence excitation and using an emission bandpass filter of 50 nm bandwidth centered around 460 nm.
  • the direct autofluorescence of NADH can be analyzed with FLEVI using a wavelength of 750 nm in two-photon fluorescence excitation and using an emission bandpass filter centered around 460 nm. In some aspects of all the embodiments of the invention, the direct autofluorescence of NADH can be analyzed with FLEVI using a wavelength of 750 nm in two-photon fluorescence excitation and using an emission bandpass filter of 50 nm bandwidth centered around 460 nm.
  • the direct autofluorescence of NADH can also be analyzed with FLEVI using a wavelength of from about 300 nm to about 380 nm in one-photon fluorescence excitation and using an emission bandpass filtered centered around about 550 nm.
  • the direct autofluorescence of NADH can also be analyzed with FLEVI using a wavelength of from about 300 nm to about 380 nm in one-photon fluorescence excitation and using an emission bandpass filter of about 80 nm bandwidth centered around about 550 nm.
  • the direct autofluorescence of NADH can also be analyzed with FLEVI using a wavelength of about 340 nm in one-photon fluorescence excitation and using an emission bandpass filter centered around about 460 nm. In some aspects of all the embodiments of the invention, the direct autofluorescence of NADH can also be analyzed with FLEVI using a wavelength of about 340 nm in one-photon fluorescence excitation and using an emission bandpass filter of about 80 nm bandwidth centered around about 460 nm.
  • the direct autofluorescence of NADH can also be analyzed with FLEVI using a wavelength of from 300 nm to 380 nm in one-photon fluorescence excitation and using an emission bandpass filtered centered around 550 nm.
  • the direct autofluorescence of NADH can also be analyzed with FLIM using a wavelength of from 300 nm to 380 nm in one-photon fluorescence excitation and using an emission bandpass filter of 80 nm bandwidth centered around 550 nm.
  • the direct autofluorescence of NADH can also be analyzed with FLFM using a wavelength of 340 nm in one-photon fluorescence excitation and using an emission bandpass filter centered around 460 nm. In some aspects of all the embodiments of the invention, the direct autofluorescence of NADH can also be analyzed with FLFM using a wavelength of 340 nm in one-photon fluorescence excitation and using an emission bandpass filter of 80 nm bandwidth centered around 460 nm.
  • the direct autofluorescence of FAD can be analyzed with FLFM using a wavelength of from about 810 nm to about 950 nm in two-photon fluorescence excitation and using an emission bandpass filter centered around from about 550 nm to about 600 nm. In some aspects of all the embodiments of the invention, the direct autofluorescence of FAD can be analyzed with FLFM using a wavelength of from about 810 nm to about 950 nm in two-photon fluorescence excitation and using an emission bandpass filter of about 80 nm bandwidth centered around from about 550 nm to about 600 nm.
  • the direct autofluorescence of FAD can be analyzed with FLFM using a wavelength of from about 810 nm to about 950 nm in two-photon fluorescence excitation and using an emission bandpass filter centered around about 550 nm. In some aspects of all the embodiments of the invention, the direct autofluorescence of FAD can be analyzed with FLFM using a wavelength of from about 810 nm to about 950 nm in two-photon fluorescence excitation and using an emission bandpass filter of about 80 nm bandwidth centered around about 550 nm.
  • the direct autofluorescence of FAD can be analyzed with FLFM using a wavelength of about 845 nm in two-photon fluorescence excitation and using an emission bandpass filter centered around about 550 nm. In some aspects of all the embodiments of the invention, the direct autofluorescence of FAD can be analyzed with FLFM using a wavelength of about 845 nm in two-photon fluorescence excitation and using an emission bandpass filter of about 80 nm bandwidth centered around about 550 nm.
  • the direct autofluorescence of FAD can be analyzed with FLFM using a wavelength of about 900 nm in two-photon fluorescence excitation and using an emission bandpass filter centered around about 550 nm. In some aspects of all the embodiments of the invention, the direct autofluorescence of FAD can be analyzed with FLEVI using a wavelength of about 900 nm in two-photon fluorescence excitation and using an emission bandpass filter of about 80 nm bandwidth centered around about 550 nm.
  • the direct autofluorescence of FAD can be analyzed with FLEVI using a wavelength of from 810 nm to 950 nm in two-photon fluorescence excitation and using an emission bandpass filter centered around from 550 nm to 600 nm.
  • the direct autofluorescence of FAD can be analyzed with FLEVI using a wavelength of from 810 nm to 950 nm in two-photon fluorescence excitation and using an emission bandpass filter of 80 nm bandwidth centered around from 550 nm to 600 nm.
  • the direct autofluorescence of FAD can be analyzed with FLEVI using a wavelength of from 810 nm to 950 nm in two-photon fluorescence excitation and using an emission bandpass filter centered around 550 nm. In some aspects of all the embodiments of the invention, the direct autofluorescence of FAD can be analyzed with FLEVI using a wavelength of from 810 nm to 950 nm in two-photon fluorescence excitation and using an emission bandpass filter of 80 nm bandwidth centered around 550 nm.
  • the direct autofluorescence of FAD can be analyzed with FLEVI using a wavelength of 845 nm in two-photon fluorescence excitation and using an emission bandpass filter centered around 550 nm.
  • the direct autofluorescence of FAD can be analyzed with FLEVI using a wavelength of 845 nm in two-photon fluorescence excitation and using an emission bandpass filter of 80 nm bandwidth centered around 550 nm.
  • the direct autofluorescence of FAD can be analyzed with FLEVI using a wavelength of 900 nm in two-photon fluorescence excitation and using an emission bandpass filter centered around 550 nm. In some aspects of all the embodiments of the invention, the direct autofluorescence of FAD can be analyzed with FLEVI using a wavelength of 900 nm in two-photon fluorescence excitation and using an emission bandpass filter of 80 nm bandwidth centered around 550 nm.
  • the direct autofluorescence of FAD can be analyzed with FLEVI using a wavelength of from about 400 nm to about 450 nm in one-photon fluorescence excitation and using an emission bandpass filter centered around about 550 nm.
  • the direct autofluorescence of FAD can be analyzed with FLEVI using a wavelength of from about 400 nm to about 450 nm in one-photon fluorescence excitation and using an emission bandpass filter of about 80 nm bandwidth centered around about 550 nm.
  • the direct autofluorescence of FAD can be analyzed with FLEVI using a wavelength of about 450 nm in one-photon fluorescence excitation and using an emission bandpass filter of about 80 nm bandwidth centered around about 550 nm. In some aspects of all of the embodiments of the invention, the direct autofluorescence of FAD can be analyzed with FLEVI using a wavelength of about 450 nm in one-photon fluorescence excitation and using an emission bandpass filter centered around about 550 nm.
  • the direct autofluorescence of FAD can be analyzed with FLEVI using a wavelength of from 400 nm to 450 nm in one-photon fluorescence excitation and using an emission bandpass filter centered around 550 nm. In some aspects of all of the embodiments of the invention, the direct autofluorescence of FAD can be analyzed with FLEVI using a wavelength of from 400 nm to 450 nm in one-photon fluorescence excitation and using an emission bandpass filter of 80 nm bandwidth centered around 550 nm. In some aspects of all of the embodiments of the invention, the direct autofluorescence of FAD can be analyzed with FLEVI using a
  • the direct autofluorescence of FAD can be analyzed with FLEVI using a wavelength of 450 nm in one-photon fluorescence excitation and using an emission bandpass filter centered around 550 nm.
  • the embryo analyzed according to the methods of the invention is a pre- implantation embryo, and the analysis is performed in vitro. Similarly, a cell obtained from an embryo is obtained from a pre-implantation embryo and the cell biopsy is performed in vitro.
  • the method comprises a sequential analysis of FAD and NADH.
  • the analysis typically only takes a short time, such as 30 seconds to 5 minutes and can be multiplexed and automated.
  • a method for assessing the quality of an oocyte or an embryo comprising (a) exposing, in a medium, granulosa cell(s), oocyte, the embryo or cell(s) from the embryo to a fluorescence lifetime imaging microscopy (FLEVI) to acquire a fluorescence lifetime histogram of auto-fluorescence of NADH in the granulosa cells, oocyte, the embryo or cell(s) from the embryo; (b) averaging the fluorescence lifetime histogram of auto-fluorescence of NADH over the entire granulosa cell, oocyte, the embryo or cell(s) from the embryo; (c) fitting the averaged fluorescence lifetime histogram of auto- fluorescence of NADH to a sum of two exponentials; and (d) selecting for in vitro
  • FLEVI fluorescence lifetime imaging microscopy
  • the cell can be an embryo, oocyte, or a cell extracted from an embryo or a cumulus cell surrounding an oocyte.
  • the method further comprises a step of in vitro fertilizing the oocyte when one detects a cell, either oocyte or one or more cumulus cell from around the oocyte where the alpha is less than an alpha reference value and the beta is less than a beta reference value, or implanting the embryo when one detects a cell from the embryo or an embryo where the alpha is less than an alpha reference value and the beta is less than a beta reference value.
  • the alpha reference value is between 1 and 4, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, or 2-3, or 2-4, or 1- 2, or 1-3.
  • the alpha reference value is 1.7.
  • the beta reference value is 2000 ps - 3000 ps, or 2000-2500, or 2500-3000, or 2000-2250, or 2000-2750. In some aspects of all the embodiments the beta reference value is 2250 ps (picoseconds).
  • Figure 1 provides an example of suitable alpha and beta values for evaluation of oocytes.
  • a typical analysis of the metabolic health of an oocyte includes calculating comprehensive "metabolic scores" for oocytes or embryos.
  • the scores represent the metabolic health of the oocyte or embryo, and therefore the general viability.
  • One way to calculate the score comprises or consists of deriving from all the parameters obtained in one FLEVI measurement, namely, (1) fraction bound, (2) short and long lifetimes, and (3) average brightness for both FAD and NADH. Combination of the parameters may provide a more accurate assessment of the metabolic state of the target oocyte or embryo.
  • the metabolic state of oocytes and embryos in both humans and mice is practically identical. Therefore, the reference values obtained from the metabolic state FLEVI analysis in a mouse cumulus cell, oocyte and/or embryo, can be directly applied to human oocyte and embryo analysis. The reference values can also be obtained from human cumulus cell, oocyte and/or embryo.
  • the FLEVI of NADH is performed using a wavelength of about 740 nm in two-photon excitation and using an emission bandpass filter centered around about 460 nm.
  • This kind of time-lapse data would be richer, and thus can provide more information about the cells.
  • the described method is typically performed using a system that contains nearly all of the components of a traditional microscope.
  • the method can be combined with analysis of morphological time-lapse data as well.
  • the methods described herein can relate to obtaining, e.g., an average metabolic score for a subject and/or a sample obtained from the subject.
  • a plurality of test cells from a clinical sample can be assayed according the methods described herein such that an individual metabolic score is obtained for each of the test cells and the individual metabolic scores averaged to obtain an average metabolic score for the plurality of test cells.
  • the, e.g., standard deviation, of the average metabolic score can be obtained and/or reported.
  • Such average metabolic scores can permit a determination of whether the average egg obtained from a subject will be suitable for, e.g., IVF and thus whether the subject should continue with IVF or how aggressive an approach to IVF should be considered for the individual subject.
  • the average metabolic score can be compared to reference values, e.g., metabolic scores for subjects who have previously attempted IVF and a probability of future success thereby provided.
  • the FLEVI data is analyzed by fitting the lifetime histograms to a model. In some embodiments of any of the aspects, the FLEVI data is analyzed by phasor analysis.
  • the absence of a given treatment can include, for example, a decrease by at least about 10%>, at least about 20%, at least about 25%, at least about 30%), at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%), at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%), at least about 99% , or more.
  • “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level.
  • “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
  • the terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount.
  • the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%), or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%), or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100%) as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • a "increase” is a statistically significant increase in such level.
  • a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus.
  • Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • the terms, "individual,” “patient” and “subject” are used interchangeably herein.
  • the subject is a mammal.
  • the mammal can be a human, non- human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of infertility.
  • a subject can be male or female.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. fertility related conditions) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition.
  • a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition.
  • a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.
  • a "subject in need" of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
  • the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder.
  • the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is,
  • treatment includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable.
  • treatment also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
  • test cell denotes a cell taken or isolated from a biological organism, e.g., a oocyte from a subject.
  • the test sample can be freshly collected or a previously collected sample.
  • the methods, assays, and systems described herein can further comprise a step of obtaining a test cell from a subject.
  • the subject can be a human subject.
  • statically significant or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • the term "consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.
  • a method for assessing the quality of an oocyte or an embryo comprising
  • a method for assessing the quality of an oocyte or an embryo comprising (a) exposing an embryo or a test cell or a plurality of test cells selected from an oocyte or an oocyte-associated cumulus cell or a cell from an embryo to a fluorescence lifetime imaging microscope (FLFM) to acquire a fluorescence lifetime histogram of auto- fluorescence of endogenous NADH and FAD for the embryo or the test cell; (b) averaging the fluorescence lifetime histogram of NADH auto-fluorescence and FAD auto-fluorescence over the entire embryo, test cell or test cells, or the cytoplasm of the test cell or cells, or mitochondria of the test cell or cells to assay measurements for fraction bound, short and long lifetimes, and average brightness for both FAD and NADH
  • FLFM fluorescence lifetime imaging microscope
  • Oocyte metabolic state can be rapidly, non-invasively, and quantitatively measured by Fluorescence Lifetime Imaging Microscopy (FLFM) of NADH and FAD.
  • FLFM Fluorescence Lifetime Imaging Microscopy
  • Three parameters are extracted from FLFM measurements of NADH and FAD in oocytes. Binding to enzymes causes the lifetime of the fluorophore to shift significantly, explaining the strong correlation in all three parameters.
  • each point corresponds to data from a single oocyte. 20 oocytes originating from old mice and 12 oocytes originating from young mice were tested. Gray dots represent oocytes originating from old mice and red dots represent oocytes originating from young mice.
  • the preliminary data was acquired on a FLFM system.
  • the microscope consists of a ti-sapphire femtosecond laser (Spectra-Physics), an inverted microscope base (Nikon), a scan head (Becker & Hickl), a hybrid PMT detector (Hamamatsu), and electronics for time correlated single photon counting (Becker & Hickl). This microscope was assembled to acquire the preliminary data.
  • Oocytes were placed in a medium on the microscope stage and imaged. A single image of each oocyte was analyzed by averaging the FLFM data over the entire oocyte. The acquired fluorescence lifetime histogram from NADH, averaged over the entire oocyte, was fit to a sum of two exponentials.
  • the parameter alpha is the ratio of the amplitude of the two exponentials
  • the parameter, beta is the lifetime of the longer exponential (in
  • oocyte quality depends on oocyte metabolic state. Oocyte metabolic state can be rapidly, non-invasively, and quantitatively measured by Fluorescence Lifetime Imaging Microscopy (FLFM) of FAD or NADH. Two parameters (alpha and beta) are extracted from FLFM measurements of NADH (or FAD) in oocytes.
  • FLFM Fluorescence Lifetime Imaging Microscopy
  • alpha and beta are extracted from FLFM measurements of NADH (or FAD) in oocytes.
  • Perturbing oocytes by specific metabolic inhibitors black crosses
  • non-specific damage causes both parameters to increase.
  • Unperturbed oocytes (circles) exhibit a range of values of alpha and beta. These parameters are indicative of oocyte quality.
  • chromosome screening is highly predictive of the reproductive potential of human embryos: a prospective, blinded, non-selection study. Fertil Steril 2012; 97:870-5.
  • chromosome screening is highly predictive of the reproductive potential of human embryos: a prospective, blinded, non-selection study. Fertil Steril 2012; 97:870-5. [00173] Vergouw CG, Kieslinger DC, Kostelijk EH, Botros LL, Schats R, Hompes
  • Figure 6 depicts a graph of absorption(2 photon)-emission spectra graph for
  • NADH and FAD Lines indicate the laser illumination wavelengths for NADH (orange) and FAD (blue).
  • the emission filters which isolate the fluorescence are shown on the left.
  • ranges suitable for use in the methods described herein can include:
  • NADH excitation - 720-760nm. Above 760 nm, NADH excitation decreases

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Abstract

L'invention concerne de nouveaux procédés in vitro non invasifs pour évaluer l'état métabolique d'ovocytes et/ou d'embryons avec un microscope à imagerie de fluorescence résolue en temps, qui peuvent être employés, par exemple, dans l'évaluation d'ovocytes et d'embryons dans des technologies de procréation médicalement assistée. Le procédé permet de faire la différence entre des ovocytes plus anciens et des ovocytes plus jeunes.
PCT/US2016/020245 2015-03-19 2016-03-01 Flim permettant de différencier des ovocytes anciens et jeunes WO2016148905A1 (fr)

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CN110969616A (zh) * 2019-12-13 2020-04-07 北京推想科技有限公司 评价卵母细胞质量的方法及装置
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CN110114653A (zh) * 2016-10-10 2019-08-09 瑞克斯旺种苗集团公司 用于拾取和收集植物物质的方法和系统
CN110969616A (zh) * 2019-12-13 2020-04-07 北京推想科技有限公司 评价卵母细胞质量的方法及装置
CN110969616B (zh) * 2019-12-13 2020-10-27 北京推想科技有限公司 评价卵母细胞质量的方法及装置
WO2022212950A1 (fr) * 2021-04-02 2022-10-06 Optiva Fertility, Inc. Procédés d'intelligence artificielle pour prédire la viabilité d'embryons sur la base de procédés de microscopie

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