Metabolic imaging methods for assessment of oocytes and embryos

Abstract

The invention provides novel non-invasive in vitro to methods for assessing the metabolic condition of oocytes and/or embryos with fluorescence lifetime imaging microscope, that can be used, for example, in assessment of oocytes and embryos in assisted reproductive technologies.

1 . A method for assessing the quality of an oocyte or an embryo, the method comprising
 (a) exposing a test cell selected from the oocyte or an oocyte-associated cumulus cell or the embryo or a cell from the embryo to a fluorescence lifetime imaging microscope (FLIM) to acquire a fluorescence lifetime histogram of auto-fluorescence of endogenous NADH or FAD for the test cell;   (b) averaging the fluorescence lifetime histogram of NADH auto-fluorescence or FAD auto-fluorescence or both over the entire test cell, or the cytoplasm the test cell, or mitochondria of the test cell;   (c) 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; and   (d) if the averaged fluorescence lifetime histogram from the test cell does not differ statistically from the reference value then selecting the oocyte for in vitro fertilization or embryo for implantation and if the averaged fluorescence lifetime histogram from the test cell differs statistically from the reference value then excluding the oocyte from in vitro fertilization or embryo from implantation.   
     
     
         2 . The method of  claim 1  comprising the 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. 
     
     
         3 . The method of  claim 1 , wherein 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. 
     
     
         4 . The method of  claim 1 , wherein 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. 
     
     
         5 . The method of  claim 1 , wherein the FLIM 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. 
     
     
         6 . The method of  claim 1 , wherein the FLIM 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. 
     
     
         7 . The method of  claim 1 , wherein the FLIM is performed in the time domain. 
     
     
         8 . The method of  claim 1 , wherein the FLIM is performed in the frequency domain. 
     
     
         9 . The method of  claim 1 , wherein the FLIM 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. 
     
     
         10 . The method of  claim 1 , wherein the FLIM 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.