WO2017058900A1 - Bioassay for mitochondrial resistance to cellular stress - Google Patents

Abstract

A method for selecting for cells that are resistant to stress by culturing in vitro a plurality of cells, exposing the cells to a stressor, and measuring the amount of stressor required to inhibit mitochondrial membrane potential of cells can also be used for selecting an oocyte that is competent for in vitro fertilization. Likewise, the method can be used for selecting an embryo isolated in vitro that is competent for subsequent implantation, and for optimizing the likelihood of a live birth after implantation of a human egg fertilized in vitro. Also described is a method of optimizing anti-oxidant treatment for a patient, such as a patient suffering from cancer or diabetes, or from a disorder associated with apoptosis, cellular stress, and/or oxidation.

Description

BIOASSAY FOR MITOCHONDRIAL RESISTANCE TO CELLULAR STRESS [0001] This application claims benefit of United States provisional patent application number 62/233,870, filed September 28, 2015, the entire contents of which are incorporated by reference into this application. TECHNICAL FIELD OF THE INVENTION [0002] The present invention relates to methods and reagents for use in assays for

mitochondrial resistance to cellular stress, and application of such assays to select stress- resistant cells, such as oocytes and embryos for use in in vitro fertilization and implantation, and to select optimal treatment agents. The invention additionally relates to compositions and reagent kits for performing these methods. BACKGROUND OF THE INVENTION [0003] In vitro fertilization (IVF) involves stimulating a woman's eggs to grow and retrieving them from the body with a needle under anesthesia. The eggs are then fertilized in the laboratory and grown in media specifically designed for appropriate embryo development. As the embryos develop, they are commonly cultured in different conditioned media. Eventually the highest quality embryo(s) are placed into the uterine cavity while any remaining high quality embryos may be frozen for future use. [0004] Embryos grown in this in vitro environment are often biopsied in order to remove one or two cells from the embryo. The genetic material inside these cells is tested for specific disorders, such as cystic fibrosis, or genetic competency by whole chromosome analysis. These methods of preimplantation genetic diagnosis (PGD) or screening (PGS) involve making an opening through the outer shell of the egg (zona pellucida) about two days before the biopsy is performed. This biopsy is an invasive and expensive process. Moreover, these methods do not detect all predictors of embryo competence, nor can they detect gamete competence prior to fertilization. [0005] There remains a need for improved methods to screen and diagnose gametes and embryos for competency that take into account non-genetic and non-chromosomal factors, and that minimize the risks and expense associated with embryo biopsy and IVF failure. SUMMARY OF THE INVENTION [0006] In one embodiment, the invention provides a method for selecting for cells that are resistant to stress. In one embodiment, the method comprises culturing in vitro a plurality of cells and exposing the cells to a stressor. The method further comprises measuring the amount of stressor required to inhibit mitochondrial membrane potential of cells by 50% relative to a reference level (IC₅₀). The cells exhibiting the greatest IC₅₀ after exposure to the stressor, and/or at or significantly higher than the reference level, are selected as resistant to stress. The invention additionally relates to compositions and reagent kits for performing these methods. [0007] In one embodiment, the invention provides a method for selecting an oocyte that is competent for in vitro fertilization. In one embodiment, the method comprises culturing in vitro a plurality of cumulus cells associated with an oocyte, wherein the cumulus cells have been isolated from the oocyte. The method further comprises exposing the cumulus cells to a stressor; and measuring the amount of stressor required to inhibit mitochondrial membrane potential of cumulus cells by 50% relative to a reference level (IC₅₀). The associated oocyte is selected as competent for in vitro fertilization when the IC₅₀ after exposure to the stressor is maintained at or significantly higher than the reference level. [0008] In one embodiment, the stressor is a mitochondrial membrane potential disruptor. One example of a mitochondrial membrane potential disruptor is carbonyl cyanide 3- chlorophenylhydrazone (CCCP). In a typical embodiment, the CCCP is used at a concentration of about 1 μM. As will be appreciated by those skilled in the art, the optimal range of CCCP concentration can be determined for each cell line. For example, Jurkat cells are more sensitive to the stressor, and thus the optimal concentration of CCCP is about 0.55 μM. Other

mitochondrial membrane potential stressors suitable for use with the methods described herein include 2,4-dinitrophenol (DNP) and carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP). For a given stressor and cell combination, one can determine the stressor

concentration required to inhibit mitochondrial membrane potential of the cells by 50% relative to a reference level (IC₅₀), facilitating assays suitable for detecting increases and/or decreases relative to the reference level. [0009] In one embodiment, the culture medium comprises a cationic carbocyanine dye that accumulates in mitochondria, such as JC-1, and mitochondrial membrane potential is indicated by change in fluorescence emitted by JC-1. JC-1 is a dye that accumulates in mitochondria. The dye exists as a monomer at low concentrations and yields green fluorescence, similar to fluorescein. At higher concentrations, the dye forms J-aggregates that exhibit a broad excitation spectrum and an emission maximum at ~590 nm. These characteristics make JC-1 a sensitive marker for mitochondrial membrane potential. Another dye with similar characteristics is JC-9 (ThermoFisher Scientific). [0010] In some embodiments, the reference level is obtained from the cumulus cells prior to the exposure to the stressor. In some embodiments, the method further comprises transferring mitochondria harvested from autogeneic stem cells into the oocyte. Typically, the autogeneic stem cells are selected on the basis of mitochondrial membrane potential resistance to a stressor. For example, the autogeneic stem cells can be selected by culturing a plurality of autogeneic stem cells in vitro; exposing the stem cells to a stressor; measuring the amount of stressor required to inhibit mitochondrial membrane potential of the stem cells by 50% relative to a reference level (IC₅₀); and selecting stem cells exhibiting the highest IC₅₀ after exposure to the stressor. [0011] The invention further provides a method of selecting an embryo isolated in vitro that is competent for subsequent implantation. In one embodiment, the method comprises culturing in vitro a sample of cells obtained by biopsy from the embryo; exposing the sample of cells to a stressor; and measuring mitochondrial membrane potential of the sample cells relative to a reference level; and selecting the embryo as competent for implantation when mitochondrial membrane potential of the sample cells is maintained at or significantly greater than the reference level upon exposure to the stressor. In one embodiment, a plurality of embryos is assayed for mitochondrial membrane potential resistance to exposure to the stressor, and one or more embryos whose sampled cells exhibit the greatest mitochondrial membrane potential upon exposure to the stressor is selected for implantation. The invention additionally provides a method for optimizing the likelihood of a live birth after implantation of a human egg fertilized in vitro. The method comprises performing the assay described above on one or more embryos developed after in vitro fertilization and implanting an embryo selected on the basis of mitochondrial membrane potential upon exposure to the stressor. [0012] In one embodiment, the invention provides a method for determining the effects of candidate (known and/or novel) antioxidant agents in improving mitochondrial membrane potential in patients with different type of cancers (such as, for example, leukemia) or viral infection (such as, for example, HIV infection). The method comprises exposing Jurkat cells to a mitochondrial membrane potential disruptor in the presence or absence of a candidate antioxidant agent, and then measuring mitochondrial membrane potential. Increased mitochondrial membrane potential after exposure to the disruptor in the presence of the agent relative to other agents or the absence of the candidate agent is indicative of protection from disruption of the mitochondrial membrane potential. Desirable antioxidant agents can be selected from those candidate agents able to protect mitochondrial membrane potential from disruption. [0013] Also provided is a method of optimizing anti-oxidant treatment for a patient. In one embodiment, the method comprises culturing in vitro a biopsy sample of cells obtained from the patient; exposing the sample of cells to a stressor in the presence of one or more candidate treatment agents; measuring the mitochondrial membrane potential of the sample cells in the presence of stressor relative to a reference level; and selecting an optimized anti-oxidant treatment agent for the patient when mitochondrial membrane potential of the sample cells is maintained at or significantly greater than the reference level upon exposure to the stressor and/or at a greater level than other candidate treatment agents. In one embodiment, the patient suffers from cancer, viral infection, or diabetes, or from a disorder associated with apoptosis, cellular stress, and/or oxidation. [0014] For any of the methods described herein, the measuring can be performed using an automated cell counting system, such as a Cellometer® (Nexcelom). DESCRIPTION OF THE DRAWINGS [0015] Fig.1. Schematic representation of the different components of mitochondria, including the cristae, the inner membrane, outer membrane, and matrix. [0016] Fig.2. Schematic illustration of electron transport chain and ATP production by mitochondria. [0017] Fig.3. Polarization of mitochondria membrane determined by change in the

fluorescence dye JC-1 from green (527 nm) to red (590 nm). [0018] Fig.4. Schematic representation of in vitro bioassay of mitochondria membrane potential resistance to the cellular stressor CCCP in cumulus cells. [0019] Fig.5. Mitochondria membrane potential in Jurkat cells detected by fluorescence microscope. Cells were preincubated with CCCP at different concentrations (0-1.5 mM) and then treated with JC-1. Fluorescence was detected in green and red channel. [0020] Fig.6. Determination of mitochondria membrane potential resistance of Jurkat cells by flow cytometry and Cellometer. Cells were preincubated with CCCP at different concentrations (0-2.0 mM) and then treated with JC-1. (A) Flow cytometry vs Cellometer, (B) IC₅₀ to CCCP determined by flow cytometry and (C) IC₅₀ to CCCP determined by Cellometer. [0021] Fig.7. Mitochondria membrane potential in cumulus cells detected by fluorescence microscopy. Cells were preincubated with CCCP at different concentrations (0-1.5 mM) and then treated with JC-1. Fluorescence was detected in green and red channel. [0022] Fig.8. Determination of mitochondria membrane potential resistance of cumulus cells by Cellometer. Cells were preincubated with CCCP at different concentrations (0-1.5 mM) and then treated with JC-1. Fluorescence was detected in green and red channel. [0023] Fig.9. Mitochondria membrane potential in cumulus cells vs total oocytes retrieved. (A) Basal levels of mitochondria membrane potential (without CCCP) and (B) Levels of

mitochondria membrane potential resistance to 1.0 mM CCCP. [0024] Fig.10. Mitochondria membrane potential in cumulus cells vs mature oocytes (M2). (A) Basal levels of mitochondria membrane potential (without CCCP) and (B) Levels of

mitochondria membrane potential resistance to 1.0 mM CCCP. [0025] Fig.11. Mitochondria membrane potential in cumulus cells vs 2PN Oocytes. (A) Basal levels of mitochondria membrane potential (without CCCP) and (B) Levels of mitochondria membrane potential resistance to 1.0 mM CCCP. [0026] Fig.12. Mitochondria membrane potential in cumulus cells vs total number 6-8 cell day 3 embryos. (A) Basal levels of mitochondria membrane potential (without CCCP) and (B) Levels of mitochondria membrane potential resistance to 1.0 mM CCCP. [0027] Fig.13. Mitochondria membrane potential in cumulus cells vs total number good/fair day 5 blastocysts. (A) Basal levels of mitochondria membrane potential (without CCCP) and (B) Levels of mitochondria membrane potential resistance to 1.0 mM CCCP. [0028] Fig.14. Mitochondria membrane potential resistance of cumulus cells obtained from pool of and individual cumulus cell-oocyte complexes by Cellometer. Cells were preincubated with CCCP at 1.0 mM and then treated with JC-1. Fluorescence was detected in green and red channel. (A) Pool, (B) individual complex. [0029] Fig.15. Mitochondria membrane potential resistance of cumulus cells obtained from pool of and individual cumulus cell-oocyte complexes by Cellometer. Cells were preincubated with CCCP at 1.0 mM and then treated with JC-1. Fluorescence was detected in green and red channel. (A) Pool, (B) individual complex.

DETAILED DESCRIPTION OF THE INVENTION [0030] The present invention is based on the discovery of a method for detecting oocyte competence, or mitochondrial resistance to stress, in a cell, including in a gamete, such as an oocyte or embryo in vitro. This method can be used to improve outcomes for in vitro fertilization. Currently, PGS (preimplantation genetic screening) is a method where an embryo undergoes biopsy and is assessed for genetic normalcy prior to replacement into the uterine cavity. This is an expensive method and does not guarantee a live birth even in circumstances where an embryo deemed genetically normal is transferred into the uterine cavity. [0031] The minimally invasive approach of the invention confers a greater advantage over traditional approaches. The invention obviates the risks to the embryo that can arise from conventional embryo biopsy, and the anguish of selecting oocytes for in vitro fertilization. The methods of the invention provide an inexpensive, reliable, and non-invasive way to evaluate an embryo prior to placement into the uterine cavity. In addition, oocytes retrieved from a patient that show insufficient mitochondria resistance to stress can be rendered competent by transferring mitochondrial from autogeneic stem cells. [0032] The invention can also be used to detect mitochondrial resistance to stress in other cells, such as unhealthy cells. For example, cells biopsied from a patient suffering from cancer, diabetes or other disease can be pre-tested in vitro to determine first the degree of

mitochondrial potential and its resistance to a stressor, and second to identify an anti-oxidant agent or cocktail of anti-oxidants that could revert the mitochondria membrane potential into a healthy state to overcome disease and/or symptoms using the assays described herein. These methods can lead to selection of the optimal treatment anti-oxidant-agent(s) for an individual patient (personalized medicine), as well as to methods of identifying new therapeutic agents. [0033] Mitochondria potential in living cells is currently determined by confocal microscopy and flow cytometry (FACS) by quantifying the ratio of red/green fluorescence of the dye JC-1. When the ratio of red/green fluorescence is higher, the state of the mitochondria is healthier. Both techniques are extremely laborious and expensive. Calibration of confocal microscopy and FACS machine and preparation of cells are time consuming. More important, a minimum number of cells (10,000-100,000 cells) are necessary to obtain reliable data of mitochondria potential. Because mitochondria potential in cells has to be measured immediately after their treatment with the fluorescence dye, it is not possible to fix cells and measure mitochondria potential later on. Therefore, equipment to measure mitochondria potential in real time needs to be accessible in the same lab used for the processing of the samples. [0034] In the mitochondrial potential resistance bioassay described herein, mitochondria potential is determined by a simple, image-based instrument optimized for the analysis of fluorescent cell-based assays, such as a Cellometer® Vision CBA Image Cytometry system. [0035] One advantage of the invention is the measurement of mitochondria potential resistance to a mitochondrial potential disrupter (e.g., CCCP) instead of basal mitochondria potential in untreated samples. By determining the value of mitochondria potential in the presence of disruptor at a concentration that disrupts mitochondria potential to 50% of the value of the untreated cells (IC₅₀), the sensitivity and specificity of the bioassay is significantly increased. Definitions [0036] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified. [0037] As used herein,“reference level”^means any reference material known to be

representative of normal or unaltered cells after exposure to a stressor that disrupts

mitochondria potential to 50% of the value of the cells (IC₅₀). In some embodiments, the reference level is obtained from the same cells assayed prior to exposure to a stressor. In other embodiments, a standard reference level has been developed from assaying a representative number of similar cells. [0038] As used herein,“at or significantly greater”^in the context of mitochondrial membrane potential resistance being at or significantly greater than the reference level upon exposure to the stressor”^means within about 10% of the reference level. It can also mean a statistically significant difference. [0039] If the reference level shows a 50% reduction of mitochondrial membrane potential values upon exposure to a stressor (IC₅₀), then a group of cells that show a higher IC₅₀ in comparison with the reference level would be considered to show greater mitochondrial membrane potential resistance to stress. For example, if the amount of stressor required to produce the same response relative to the reference level is 20% higher than the IC₅₀ determined in the reference level, those cells have a 20% higher resistance to the stressor. Conversely, a group of cells that show a lower value in comparison with the reference level would be considered to have lower mitochondrial membrane potential resistance (the amount of stressor to produce the same response than of the reference level is lower than the IC₅₀ determined as the reference level. If the IC₅₀ is 20% lower, those cells that have a 20% lower resistance to the stressor than the reference level. [0040] As used herein,“a” or“an” means at least one, unless clearly indicated otherwise. Methods of the Invention [0041] Described herein is a very sensitive in vitro mitochondrial bioassay that can be used to quantify the degree of mitochondrial resistance to a mitochondrial potential disrupter. This assay was first successfully developed in an immortalized line of human T lymphocytes, Jurkat cells, widely used for mitochondria membrane potential determinations. Jurkat cells have been used to study acute T cell leukemia, T cell signaling, and the expression of various chemokine receptors susceptible to viral entry, particularly HIV. [0042] The invention provides methods for selecting for cells that are resistant to stress. In one embodiment, the method comprises culturing in vitro a plurality of cells and exposing the cells to a stressor. The method further comprises measuring the amount of stressor required to inhibit mitochondrial membrane potential of cells by 50% relative to a reference level (IC₅₀). The cells exhibiting the greatest IC₅₀ after exposure to the stressor, and/or at or significantly higher than the reference level, are selected as resistant to stress. The assay can be used to isolate cells exhibiting the greatest resistance to stressor, and/or to select cells whose resistance to stressor is above a pre-determined threshold. [0043] In one embodiment, the culture medium comprises a cationic carbocyanine dye that accumulates in mitochondria, such as JC-1, and mitochondrial membrane potential is indicated by change in fluorescence emitted by JC-1. JC-1 is a dye that accumulates in mitochondria. The dye exists as a monomer at low concentrations and yields green fluorescence, similar to fluorescein. At higher concentrations, the dye forms J-aggregates that exhibit a broad excitation spectrum and an emission maximum at ~590 nm. These characteristics make JC-1 a sensitive marker for mitochondrial membrane potential. Another dye with similar characteristics is JC-9 (ThermoFisher Scientific). [0044] In some embodiments, the reference level is obtained from the cumulus cells prior to the exposure to the stressor. In some embodiments, the method further comprises transferring mitochondria harvested from autogeneic stem cells into the oocyte. Typically, the autogeneic stem cells are selected on the basis of mitochondrial membrane potential resistance to a stressor. For example, the autogeneic stem cells can be selected by culturing a plurality of autogeneic stem cells in vitro; exposing the stem cells to a stressor; measuring the amount of stressor required to inhibit mitochondrial membrane potential of the stem cells by 50% relative to a reference level (IC₅₀); and selecting stem cells exhibiting the highest IC₅₀ after exposure to the stressor. [0045] For any of the methods described herein, the measuring can be performed using an automated cell counting system, such as a Cellometer® (Nexcelom). [0046] The following summarizes an exemplary embodiment of the invention. After processing of the sample with JC-1, 20μl of sample is pipetted into the Cellometer counting chamber. The chamber is inserted into the Vision CBA instrument. The Mitochondrial Membrane Potential (JC- 1) Assay is selected from the drop-down menu. The specific Sample ID is entered, and“count” is selected. In less than 2 minutes, the Vision CBA takes multiple cell images and analyzes the images based on pre-set parameters for the Mitochondrial Membrane Potential Assay selected. When cell imaging and counting is complete, the initial results table displays total cells counted, concentration, and mean diameter. The bright field image can be viewed to verify cell morphology and identify cell clumps. The JC-1 dye accumulates in the mitochondria of healthy cells as aggregates which are fluorescent red in color. Upon onset of cell death, the

mitochondrial membrane potential is compromised and the JC-1 dye remains in the cytoplasm in a monomeric form that fluoresces green. [0047] The export function is used to generate results in FCS Express 4 Software (DeNovo® Software) using optimized Vision CBA data layouts. Mitochondrial membrane potential is displayed as a colorized scatter plot showing percent compromised and percent healthy cells based on red and green fluorescence. The gating can be manually optimized for cell size and to eliminate the interfering fluorescence of debris and red blood cells. Associated data tables update automatically to reflect revised percentages. Users can choose to print a second report containing both bright field and fluorescent images. Data tables and cell images can be easily exported for use in presentations and publications. After removing the disposable Cellometer cell-counting chamber, the Vision CBA is ready to analyze the next sample. No washing or instrument set-up is required. Processing of the samples takes less than 30 minutes and the quantification process can be done in less than 2 minutes. Furthermore, the mitochondrial potential resistance bioassay is quite sensitive and only requires a minimum of 100 cells for quantification. Methods For Detecting Mitochondrial Resistance to Stress and Cellular Competence [0048] In one embodiment, the stressor is a mitochondrial membrane potential disruptor. One example of a mitochondrial membrane potential disruptor is carbonyl cyanide 3- chlorophenylhydrazone (CCCP). In a typical embodiment, the CCCP is used at a concentration of about 1 μM. As will be appreciated by those skilled in the art, the optimal range of CCCP or other stressor concentration can be determined for each cell line. For example, Jurkat cells are more sensitive to the stressor, and thus the optimal concentration of CCCP is about 0.55 μM. Other examples of mitochondrial membrane potential stressors suitable for use with the methods described herein include 2,4-dinitrophenol (DNP) and carbonyl cyanide p- triflouromethoxyphenyl-hydrazone (FCCP). For a given stressor and cell combination, one can determine the stressor concentration required to inhibit mitochondrial membrane potential of the cells by 50% relative to a reference level (IC₅₀), facilitating assays suitable for detecting increases and/or decreases relative to the reference level. [0049] Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), is a chemical inhibitor of oxidative phosphorylation. CCCP is a nitrile, hydrazone and protonophore that causes the gradual destruction of living cells and death of an organism by affecting the protein synthesis reactions in mitochondria. CCCP causes an uncoupling of the proton gradient that is established during the normal activity of electron carriers in the electron transport chain. The chemical acts essentially as an ionophore and reduces the ability of ATP synthase to function optimally. CCCP is widely used as positive control of most of the assays for mitochondria potential assays. Thus, preincubation of cells in CCCP at 1.0 μM concentration for 5 minutes at 37^ is sufficient to severely damage the mitochondria membrane leaving to low values of mitochondria membrane potential. [0050] In one embodiment, this in vitro bioassay consists of preincubating living cells (e.g. Jurkat cells) for 5 minutes in DMEM culture medium / 10 % FCS / 5 % CO2 at 37°C in the presence of different concentrations of CCCP (0.1-2 μM) and another 15 minutes with 2.0 μM JC-1 under the same culture conditions. After washing cells using the same culture medium indicated above, mitochondria membrane potential indicated by a fluorescence wavelength shift from green (530 nm) to red (590 nm) was measured using a Cellometer® automated cell counting system. The degree of mitochondria membrane potential resistance to CCCP was expressed as IC₅₀ (concentration of CCCP that inhibits 50% of the basal mitochondrial membrane potential). The greater the CCCP IC₅₀ value, the greater the mitochondrial membrane potential resistance to CCCP. Methods For Improving Outcome of In vitro Fertilization [0051] Mitochondrial resistance as a predictor of oocyte developmental competence can be used to guide clinical strategies by providing a means of identifying optimal embryo quality during IVF. Determining mitochondrial resistance to stress of a single oocyte complex and tracing embryogenesis of the same oocyte with time lapse photography via embryoscopy (Kovacs 2014) will allow for the optimal embryo to be chosen for implantation. [0052] Other methods that can be used to complement the screening of IVF embryos are increasingly used during fertility treatments to ensure that the embryos transferred have the correct number of chromosomes. However, even when a chromosomally normal embryo is transferred, about one-third fail to produce a pregnancy. One approach is based on the quantification of mitochondrial DNA found in the outermost layer of cells in a five-day old embryo. However, this method is risky because it requires puncturing of the embryo.

Furthermore, quantification of mitochondrial DNA by PCR or DNA sequencing is time- consuming. [0053] The invention thus provides a method for optimizing the likelihood of a live birth after implantation of human egg fertilized in vitro. In one embodiment, the invention provides a method for selecting an oocyte that is competent for in vitro fertilization. In one embodiment, the method comprises culturing in vitro a plurality of cumulus cells associated with an oocyte, wherein the cumulus cells have been isolated from the oocyte. The method further comprises exposing the cumulus cells to a stressor; and measuring the amount of stressor required to inhibit mitochondrial membrane potential of cumulus cells by 50% relative to a reference level (IC₅₀). The associated oocyte is selected as competent for in vitro fertilization when the IC₅₀ after exposure to the stressor is maintained at or significantly higher than the reference level. [0054] The invention further provides a method of selecting an embryo isolated in vitro that is competent for subsequent implantation. In one embodiment, the method comprises culturing in vitro a sample of cells obtained by biopsy from the embryo; exposing the sample of cells to a stressor; and measuring mitochondrial membrane potential of the sample cells relative to a reference level; and selecting the embryo as competent for implantation when mitochondrial membrane potential of the sample cells is maintained at or significantly greater than the reference level upon exposure to the stressor. The method optionally comprises subsequently placing the selected embryo into the uterine cavity of a patient. [0055] In one embodiment, a plurality of embryos is assayed for mitochondrial membrane potential resistance to exposure to the stressor, and one or more embryos whose sampled cells exhibit the greatest mitochondrial membrane potential upon exposure to the stressor is selected for implantation. The invention additionally provides a method for optimizing the likelihood of a live birth after implantation of a human egg fertilized in vitro. The method comprises performing the assay described above on one or more embryos developed after in vitro fertilization and implanting an embryo selected on the basis of mitochondrial membrane potential upon exposure to the stressor. [0056] The invention provides an in vitro mitochondrial membrane potential resistance assay for use in cumulus cells from IVF patients subjected to ovarian stimulation. The data presented in the Examples below demonstrate a strong correlation between the resistance to oxidative stress in the cumulus cell mitochondria with total/mature oocytes as well as day 1 and 3 and oocyte developmental competence in vivo (Fig.4). Mitochondrial resistance as a predictor of oocyte developmental competence can be used to guide clinical strategies to improve women’s reproductive health by providing means of (1) identifying optimal embryo quality during IVF; (2) diagnosing mitochondrial-related oxidative stress from metabolic diseases and/or maternal aging; and (3) treating such oxidative stress with antioxidants that restore the balance between cellular energy utilization and human oocyte development. [0057] The advantages of the invention are many. The rising age of women choosing to start a family leads to poorer oocyte health. Health care providers will be able to determine the health of the cumulus-oocyte complex based on its mitochondrial resistance. Since the cumulus cells are already stripped from the oocyte in a normal IVF procedure, performing the bioassay on the cumulus cells to determine their mitochondrial resistance will not pose any health risks to the women. This is in contrast to methods such as time-lapse photos of the embryo or extraction of embryonic fluid, which may pose health risks to the developing embryo. This will improve the cost and efficiency of the IVF procedure, as health care providers may select only the healthiest embryo for implantation in the womb that is most likely to develop successfully based on the mitochondrial health of the cumulus oocyte-complex from which the embryo originates. This eliminates the need for placing more than one embryo into the womb. This will also avoid the risk of multiple pregnancies that can arise when more than one embryo is placed into the womb. Carrying more than one baby at a time increases the risk of premature birth and low birth weight. The improved cost and efficiency of IVF will make the procedure more readily available to a wider range of women who aim to be pregnant, and have not been able to achieve success through other methods. Also, the potential to treat oxidative stress with antioxidants can provide new medicines to boost oocyte health, and thus fertility. Science will benefit from a better understanding of how resistance to oxidative stress relates to oocyte health and development and could further develop in-vivo treatments through antioxidants. Bioassay for mitochondrial potential resistance to stress in the treatment of mitochondria related disorders [0058] Damage and subsequent dysfunction in mitochondria is an important factor in a range of human diseases due to their influence in cell metabolism. Mitochondrial disorders often present themselves as neurological disorders, including autism. They can also manifest as myopathy, diabetes, multiple endocrinopathy, and a variety of other systemic disorders. Diseases caused by mutation in the mtDNA include Kearns-Sayre syndrome, MELAS syndrome and Leber's hereditary optic neuropathy. In the vast majority of cases, these diseases are transmitted by a female to her children, as the zygote derives its mitochondria and hence its mtDNA from the ovum. Diseases such as Kearns-Sayre syndrome, Pearson's syndrome, and progressive external ophthalmoplegia are thought to be due to large-scale mtDNA rearrangements, whereas other diseases such as MELAS syndrome, Leber's hereditary optic neuropathy, myoclonic epilepsy with ragged red fibers (MERRF), and others are due to point mutations in mtDNA. [0059] In other diseases, defects in nuclear genes lead to dysfunction of mitochondrial proteins. This is the case in Friedreich's ataxia, hereditary spastic paraplegia, and Wilson's disease. These diseases are inherited in a dominance relationship, as applies to most other genetic diseases. A variety of disorders can be caused by nuclear mutations of oxidative

phosphorylation enzymes, such as coenzyme Q10 deficiency and Barth syndrome.

Environmental influences may interact with hereditary predispositions and cause mitochondrial disease. For example, there may be a link between pesticide exposure and the later onset of Parkinson's disease. Other pathologies with etiology involving mitochondrial dysfunction include schizophrenia, bipolar disorder, dementia, Alzheimer's disease, Parkinson's disease, epilepsy, stroke, cardiovascular disease, retinitis pigmentosa, and diabetes mellitus. [0060] Mitochondria-mediated oxidative stress plays a role in cardiomyopathy in Type 2 diabetics. Increased fatty acid delivery to the heart increases fatty acid uptake by

cardiomyocytes, resulting in increased fatty acid oxidation in these cells. This process increases the reducing equivalents available to the electron transport chain of the mitochondria, ultimately increasing reactive oxygen species (ROS) production. ROS increases uncoupling proteins (UCPs) and potentiate proton leakage through the adenine nucleotide translocator (ANT), the combination of which uncouples the mitochondria. Uncoupling then increases oxygen consumption by the mitochondria, compounding the increase in fatty acid oxidation. This creates a vicious cycle of uncoupling; furthermore, even though oxygen consumption increases, ATP synthesis does not increase proportionally because the mitochondria are uncoupled. Less ATP availability ultimately results in an energy deficit presenting as reduced cardiac efficiency and contractile dysfunction. To compound the problem, impaired sarcoplasmic reticulum calcium release and reduced mitochondrial reuptake limits peak cytosolic levels of the important signaling ion during muscle contraction. The decreased intra-mitochondrial calcium

concentration increases dehydrogenase activation and ATP synthesis. So, in addition to lower ATP synthesis due to fatty acid oxidation, ATP synthesis is impaired by poor calcium signaling, causing cardiac problems for diabetics. [0061] The use of the in vitro mitochondrial potential resistant bioassay to explore novel antioxidants compounds that could reverse mitochondria dysfunction could further develop in- vivo treatments for such disorders. Methods of screening for new anti-aging reagents [0062] The free radical theory of aging hypothesizes that oxidative stress through oxygen- derived free radicals are responsible for the age-related damage at the cellular and tissue levels. For example, oxidative stress plays a central role in initiating and driving events that cause skin aging at the cellular level. Oxidative stress breaks down protein (collagen), alters cellular renewal cycles, damages DNA, and promotes the release of pro-inflammatory mediators which trigger the generation of inflammatory skin diseases. One of the most important factors in accelerated skin aging is solar ultraviolet radiation. Epidemiological and clinical studies have identified excessive sun exposure as a primary causal factor in various skin diseases including, premature aging, inflammatory conditions, melanoma and non-melanoma skin cancers. A series of deleterious biochemical reactions occur within the skin when it is exposed to excess UV radiation; this process is referred to as photoaging. [0063] Chronic sun exposure damages the dermal connective tissue and alters normal skin metabolism. In addition to depressing immunity, and stimulating oxidative stress and

inflammation, UV radiation increases the production of matrix metalloproteinases (MMPS), enzymes that degrade collagen. The destruction of collagen is a major contributor to the loss of skin suppleness and structure that occurs with advancing age. Antioxidants applied locally into the damage tissue are widely used as antiaging reagents. Thus, the in vitro mitochondrial potential resistance bioassay could be utilized in human skin cells (patient’s own cells) to discover new antioxidants alone or in combination as anti-aging reagents. Such compounds could be used by injection in facial and other tissues for tissue repair due to oxidative stress, or incorporated into cosmetic creams, vitamin supplements and food. Methods of screening for new anti-cancer reagents [0064] Conventional chemotherapy using cytotoxic agents tends to damage both tumor and normal cells and cause significant toxic side effects, which compromise the therapeutic outcomes. The emergence of targeted therapy which is based on the concept of specifically targeting critical molecules unique to cancer cells may provide promising means of selectively killing malignant cells. However, cancer may be caused by multiple genetic alterations and environmental factors. As such, there are many potential targets in cancer cells but no single critical target can be readily identified in most cancer types, perhaps with an exception of Bcr- Abl in CML. This situation makes the development of targeted therapy a challenging task. [0065] Mitochondria structural and functional alterations associated with malignant

transformation seem to be a common phenomenon observed in many types of cancers. Utilizing these differences to preferentially target mitochondria of cancer cells may be a logical strategy to achieve therapeutic selectivity. Several classes of small molecules that directly target mitochondria at specific sites or indirectly impact the metabolic alterations in cancer cells with dysfunctional mitochondria seem to exhibit promising anticancer activity. Since many of these small molecules have mainly been tested in cancer cell lines and in animal tumor models, clinical trials are needed to further test their potential for use in clinical treatment of cancer patients. [0066] Many compounds that target mitochondria or impact the metabolic alterations have been associated with mitochondrial dysfunction in cancer cells. The modes of action of these compounds and their clinical trial status are also indicated. It is important to recognize that cancer cells may be able to tolerate inhibition of mitochondrial function by upregulation of glycolysis and other survival mechanisms. As such, a combination of mitochondria-targeted agents with glycolytic inhibitors and other chemotherapeutic drugs may be required to achieve maximum efficacy. However, caution must be exercised to prevent potential increase in toxic side effects. Understanding mitochondrial biology in cancer cells and the interaction between cellular metabolism and drug action is important to developing mitochondrial-targeted agents for cancer treatment. The in vitro mitochondrial potential resistance bioassay of the invention could be utilized to discover new antioxidants alone or in combination for cancer treatment, including, for example, cancer of the breast, bone, muscle, kidney, liver, pancreas, or brain. [0067] The bioassay described herein can be used for the discovery of anti-oxidants for the treatment of many diseases or for the treatment of a disease affecting only one individual. For example, after biopsy of a specific tissue affected by the disease, mitochondrial membrane potential resistance can be measured in cells isolated from such tissue in the presence of anti- oxidants. This technology can be used to identify specific anti-oxidants that can reverse disease in that particular individual as a means of personalized medicine. [0068] The bioassay described herein could be used for the discovery of new anti-oxidants to treat stroke, spinal cord injury, eye related disorders, rheumatoid arthritis, burn, neurological disorders including Alzheimer, Parkinson disease, multiple sclerosis, cerebral vascular attack, immune and autoimmune disorders including Huntington's disease, rheumatoid arthritis, lupus, diabetes type I and Crohn’s disease. The methods of the invention can also be used to screen for agents to treat: Stroke, spinal cord injury, eye related disorders, rheumatoid arthritis, burn; Neurological disorders including Alzheimer’s, Parkinson’s disease, multiple sclerosis, cerebral vascular attack; Immune and autoimmune disorders including Huntington's disease, rheumatoid arthritis, lupus, diabetes type I and Crohn’s disease; Anti-aging (e.g. cosmetic treatments ,via oral as cream products); Mitochondria transfer and generation of oocytes for in vitro fertilization for treatment in advanced maternal age; and screening for cancer treatment. [0069] Accordingly, the invention provides a method of optimizing anti-oxidant treatment for a patient. In one embodiment, the method comprises culturing in vitro a biopsy sample of cells obtained from the patient; exposing the sample of cells to a stressor in the presence of one or more candidate treatment agents; measuring the mitochondrial membrane potential of the sample cells in the presence of stressor relative to a reference level; and selecting an optimized anti-oxidant treatment agent for the patient when mitochondrial membrane potential of the sample cells is maintained at or significantly greater than the reference level upon exposure to the stressor and/or at a greater level than other candidate treatment agents. In one

embodiment, the patient suffers from cancer or diabetes, or from a disorder associated with apoptosis, cellular stress, and/or oxidation. Kits [0070] The invention provides kits comprising a set of reagents and/or dyes as described herein, and optionally, one or more suitable containers containing such materials for use with the invention. The kit can optionally include a buffer. In one embodiment, the kit includes materials to perform the assay in a single reaction tube or well. EXAMPLES [0071] The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.

Example 1: Bioassay for mitochondrial potential resistance to stress [0072] The methods described herein have been tested and the results, shown in the accompanying figures, provide proof of concept. Reproducibility of the results versus standard techniques, such as fluorescence microscope and flow cytometry, has been demonstrated (See figures 5-8). High sensitivity and specificity of the bioassay is demonstrated in Figures 9-13. High correlation between mitochondrial potential resistance to stress and clinical values related with oogenesis and embryogenesis is demonstrated in Figures 9-13. It is possible to miniaturize the detection system by using a low number of cumulus cells (e.g., 100 cells). The bioassay can be adapted for using different known antioxidants (CoQ10, resveratrol, different vitamins, etc), and other agents known in the art. For example, any cell line, including cancerous cell types, can be incubated with antioxidants either before, concurrently, or after exposure to the stressor and then processed with the bioassay to determine resistance to stress. The timing of the antioxidant incubation can determine if the antioxidant has protective qualities against the stressor, can compete with the stressor, or reverse the harm caused by the stressor. The concentration of the stressor is critical, because the antioxidant will not have an effect on cells that are completely damaged. Thus, the IC₅₀ of the cells must be determined beforehand.

Example 2: Resistance of cumulus cell (CC) mitochondria to stress in vitro as a predictor of oocyte competence during ovarian stimulation for in vitro fertilization (IVF) [0073] This example establishes a CC mitochondria in vitro bioassay based upon mitochondrial resistance to stress to predict oocyte competence during ovarian stimulation for IVF. See also Dumesic et al., 2016, J. Clin. Endocrinol. Metab.101(5):2235-45. Methods [0074] Twenty-five women (ages, 34.5±1.1 years; body mass index [BMI], 23.2±0.9 kg/m² [mean±^SEM]) undergoing ovarian stimulation for IVF were studied. Pooled CC from each woman were collected at oocyte retrieval and immediately analyzed. A CC mitochondrial resistance bioassay was developed by measuring mitochondrial potential to the membrane- permeable JC-1 dye in the presence of the mitochondrial membrane potential disrupter, carbonyl cyanide 3-chlorophenylhydrazone (CCCP). For bioassay optimization, human T Lymphocyte Jurkat cells were incubated with varying CCCP doses (0.02 - 2.0 μM) and then exposed to JC-1 (1.0 μM). Exposure of CC to 1.0 μM CCCP reduced mitochondrial potential to 50% of untreated cells; thus 1.0 μM CCCP was used in all CC studies. Regression analysis compared 1) CC mitochondrial resistance to CCCP and amount of follicle-stimulating hormone (FSH) administered with total and mature oocyte numbers, and also 2) correlated CC mitochondrial resistance to CCCP with numbers of 2-pronuclear (2PN), 6-8 cell cleavage and blastocyst embryos, before and after adjusting for mature oocyte number. Results [0075] CC mitochondrial resistance to CCCP positively correlated with numbers of total (r=0.78, P<0.0001), and mature (r=0.83, P<0.00001) oocytes, while negatively correlating with amount of FSH administered (r=-0.67, P<0.0022). Amount of FSH administered, in turn, negatively correlated with numbers of total (r=-0.75, P<0.0005) and mature (r=-0.83, P<0.0001) oocytes. Adjusting for FSH administered, CC mitochondrial resistance to CCCP remained a positive predictor of mature oocyte number (r=0.60, P<0.025). During pre-implantation embryogenesis, CC mitochondrial resistance to CCCP positively correlated with numbers of 2 PN (r = 0.79, P<0.0002), 6-8 cell cleavage (r = 0.84, P<0.0001) and blastocyst (r = 0.63, P<0.01) embryos. However, mature oocyte number also positively correlated with numbers of 2 PN (r=0.94, P<0.0001), 6-8 cell cleavage (r=0.87, P<0.0001) and blastocyst (r=0.73, P<0.001) embryos. Adjusting for mature oocyte number (mitochondrial resistance X mature oocytes interaction), CC mitochondrial resistance to CCCP remained a positive predictor of 6-8 cell cleavage embryo number (P<0.05), with higher values of mitochondrial bioassay-predicted cleaving embryos accompanying higher numbers of mature oocytes retrieved. [0076] This Example has shown that resistance of CC mitochondria to stress in vitro predicts oocyte competence based upon ovarian responsiveness to FSH administration during IVF. Example 3: Resistance of individual oocyte cumulus cell complex mitochondria to stress in vitro as a predictor of oocyte competence and embryogenesis during ovarian stimulation for in vitro fertilization (IVF) [0077] This example establishes an individual oocyte CC complex mitochondria in vitro bioassay based upon mitochondrial resistance to stress to predict oocyte competence and embryogenesis during ovarian stimulation for IVF. Methods [0078] At the time of retrieval, cumulus cell complexes of individual oocytes from each woman were collected and analyzed separately. The cells were processed in the same manner as described in Example 2. This technique will be developed to serve as a predictor of oocyte competence and embryogenesis, which could be even more effective than Example 2.

Following the development of the embryo in complement with the single oocyte’s mitochondrial resistance, the optimal embryo for implantation will be chosen. This saves time and money involved in genetic testing of the embryo and gives immediate security to the mom. [0079] Understanding the heterogeneity within a cumulus cell population remains a major impediment to understanding clinically effective mitochondrial therapies. [0080] Pooled cumulus cells collected from a single patient emit a wide range of red

fluorescence from 10^3-10^5 (Figs.14A, 15A). The data demonstrate that multiple

subpopulations exist within pooled cumulus cells from a single patient, defined by nonrandom peaks and can predict function properties of these cells. The peak of from 10^3 to 10^4 represent a population of cells less resistant to cells, while the peak from 10^4 to 10^5 represent a population of cells more resistant to stress. This represents a powerful new tool to elucidate the relationship between mitochondrial health and phenotypic variation within a cell population. [0081] Cumulus cells from individual oocytes emit a much narrower range of red fluorescence from 10^4 to 10^5 (Figs 14B, 15B). This indicates a single homogenous population within individual complexes. [0082] For example, the red/green ratio of the pooled cumulus cells of patient 1 is 8.6 (Fig.14A) while an individual cumulus complex (Fig.14B) from the same patient is 4.3 This indicates that this individual complex is less resistant to stress than the CC complexes combined in the pooled sample from the same patient. In contrast, the red/green ratio of the pooled cumulus cells of patient is 5.5 (Fig.15A) while an individual cumulus complex (Fig.15B) from the same patient is 7.1. This indicates that this individual complex is more resistant to stress than the CC

complexes combined in the pooled sample from the same patient. [0083] The importance of this bioassay is simple. Individual cumulus cell complexes with a greater resistance to mitochondrial stress indicate that the oocyte from which the cells originate have a higher chance of successful implantation. Example 4: Detailed Steps of Mitochondrial Membrane Potential Resistance Assay [0084] The Example provides a detailed description of the methodology used for detecting and measuring mitochondria potential resistance to CCCP in human cumulus cells obtained in patients undergoing IVF treatment:

[0085] 1- Pooled cumulus cells were isolated by mechanical stripping after oocyte retrieval when cumulus-oocyte complexes. [0086] 2- Cells were transferred to culture dishes and were washed several times in 5 mL of MOPS (4-morpholinepropanesulfonic acid) buffered medium (G-MOPS^TM, VitroLife, Englewood, CO), containing 10% serum substitute supplement (Irvine Scientific, Santa Ana, CA).

[0087] 3- Cells were then were resuspended in 100 μL of recombinant human hyaluronidase (40-120 U/ml) (ICSI Cumulase®, Malov, Denmark), and pipetted up and down for 1 minute before being placed in the MOPS buffered medium.

[0088] 4- Pooled cumulus cells were initially centrifuged at 1600 rpm for 5 minutes at 24°C in the IVF laboratory.

[0089] 5- Pooled cumulus cells were initially centrifuged at 1,600 rpm for 5 minutes at 24C in the IVF laboratory.

[0090] 6- Cell samples were then transported on ice to the research laboratory.

[0091] 7- Cells were incubated in the absence and presence of the disrupted mitochondrial membrane reagent (see above) at different concentrations (0.1-2 μM concentration) on the dark.

[0092] 8- 1 mL of full culture medium (Dulbecco’s Modified Eagle Medium 1× (DMEM; CellGro, MediatechInc, Manassas, VA) comprised of 10% fetal bovine serum (FBS; Thermo Scientific Hyclone, Logan, UT) and 5% antibiotic-antimycotic solution (CellGro, Mediatech Inc, Manassas, VA)] was added to 15 ml sterile conical tubes.

[0093] 9- Different amounts of CCCP (0.1-2 μM concentration) (125 μL of 50 mM CCCP in dimethyl sulfoxide, cat # M34152, MitoProbe JC-1 Assay Kit, Life Technologies, Grand Island, NY) were added to the tubes containing 1 ml of full culture.

[0094] 10- 50 μL of polyvinylpyrrolidone (PVP) (Irvine Scientific, Santa Ana, CA) was added to 450 μL full culture medium to prepare (10% PVP).

[0095] 11- Remaining MOPS buffered medium was removed r from the cumulus cell pellet.

[0096] 12- Cells were then resuspended in 10% PVP full medium.

[0097] 13- 50 μL of resuspended cells were added to 15 ml sterile conical tubes without and with CCCP at different concentrations.

[0098] 14- Cells were mixed and incubated for 5 minutes at 37°C/5% CO₂.

[0099] 15- 5 μL of JC-1 fluorescent dye (M34152, MitoProbe JC-1 Assay Kit, Life Technologies, Grand Island, NY) was added to each tube and incubated at 37°C/5% CO₂ for another for 15 minutes, mixing every 5 minutes.

[0100] 16- 2 mL of full culture medium was added to each tube to wash the cells. [0101] 17- Cells were centrifuged at 1000 RPM for 5 minutes at 20°C. Medium was aspirated and the cells were then resuspended with 60 μL of the 10% PVP Full Medium Solution.

[0102] 18- Cellometer setup instructions are indicated below.

[0103] 19- Transfer 17 μL of the control sample into the Nexcelom Cellometer cell counting chamber slide (Nexcelom CHT4-PD100-003).

[0104] 20- Insert chamber into the Cellometer.

[0105] 21- Press Preview B1, focus for sharp cell edges and bright centers, press stop B1.

[0106] 22- Press Preview F1, focus for sharp cell edges and bright centers,

[0107] 23- Stop F1.

[0108] 24- Press Count.

[0109] 25- Press Export.

[0110] 26- Choose the first option (already selected- Nexcelom Data Package)

[0111] 27- Press Next.

[0112] 28- Adjust gates on Mito Green Intensity histogram and Mito Red Intensity histogram based on the peaks.

[0113] 29- For the Mito Green Intensity, drag one edge of the green gate to between 10^3 and 10^4 and the other edge of the gate to 10^5.

[0114] 30- For the Mito Red Intensity, drag one edge of the red gate to the start of a clear peak around 10^4 and drag the other edge of the gate to 10^5.

[0115] 31- Record the value at the bottom of the spread sheet which states: Ratio Mito Red to Mito Green.

[0116] 32- Drag the edge of the gate which was at 10^5 to 10^2 and record the new value at the bottom of the speed sheet which states: Ratio Mito Red to Mito Green.

[0117] 33- The first value obtained from the ratio is the numerator and the second value obtained from the ratio is the denominator.

[0118] 34- Divide these values to obtain the final Red to Green Ratio.

[0119] 35- Each sample is determined by triplicate under identical conditions.

[0120] 36- Calculate the mean of the triplicate values to determine the final value of Red to Green Fluorescence ratio for each sample. [0121] Cellometer Setup (needs to be done only once while using the assay). [0122] 1- Power Cellometer Vision CBA on which is connected to a PC and has the Cellometer software installed.

[0123] 2- The filters in the back of the Cellometer should be set to: P/N: VB-535-402 in slot A; and P/N: VB-595-502 in slot B

[0124] 3- In the Cellometer software program, select Options on the top bar

[0125] 4- Select Instrument

[0126] 5- Select EDIT

[0127] 6- Password is: camera

[0128] 7- Select Login

[0129] 8- Select Modify Name

[0130] 9- Match the Fluorescence Optics Modules A and B with the numbers mentioned above [0131] 10- Select Done

[0132] 11- Select Save

[0133] 12- Select Save

[0134] 13- Select Assay Type on the top bar

[0135] 14- Select New Assay Type

[0136] 15- Under Assay Name enter CBA_JC-1

[0137] 16- Under Imaging Mode select Dual Fluoresce (F1,F2)

[0138] 17- Select Multimode FL Counting

[0139] 18- For the F1 Image:

[0140] 19- Under Cell Type select CBA_Mito Green FL

[0141] 20- Under Description Fluorophore type Mito Green and select VB-535-402

[0142] 21- Under Fluorescent Exp type 100.0 msec and select VLA-470

[0143] 22- For the F2 image:

[0144] 23- Under Cell Type select CBA_Mito Red FL

[0145] 24- Select Edit

[0146] 25- Set the Brightfield and Fluorescence Cell Diameter to 10.0 - 50.0 micron

[0147] 26- Select Save

[0148] 27- Under Fluorophore Type Mito Red and select VB-595-502 [0149] 28- Under Fluorescent Exp type 2500.0 msec and select VLA-470

[0150] 29- Unselect Show Data File Buttons

[0151] 30- Unselect Show Sample Adjustment Button

[0152] 31- Select Show Cell Size Distribution Button

[0153] 32- Select Set Dilution Factor for Assay to 1.000

[0154] 33- Select Show Percent F1, F2 Dual Expression

[0155] 34- Under Result Template select Browse and choose CBA_results Template.rlt_tm [0156] 35- Under Print Template select Browse and choose CBA_Results Template.rlt_tm [0157] 36- Under FCS Layout Style select Set FCS Layout and choose CBA_JC-1.fey

[0158] 37- Select Save

[0159] Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention pertains. [0160] Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

What is claimed is: 1. A method for selecting an oocyte that is competent for in vitro fertilization, the method comprising: (a) culturing in vitro a plurality of cumulus cells associated with an oocyte, wherein the cumulus cells have been isolated from the oocyte; (b) exposing the cumulus cells to a stressor; (c) measuring the amount of stressor required to inhibit mitochondrial membrane potential of cumulus cells by 50% relative to a reference level (IC₅₀); and (d) selecting the associated oocyte as competent for in vitro fertilization when the IC₅₀ after exposure to the stressor is maintained at or significantly higher than the reference level.

2. The method of claim 1, wherein the stressor is a mitochondrial membrane potential disruptor.

3. The method of claim 1, wherein the mitochondrial membrane potential disruptor is carbonyl cyanide 3-chlorophenylhydrazone (CCCP).

4. The method of claim 1, wherein the CCCP is about 1 μM.

5. The method of claim 1, wherein the culls are cultured in a medium that comprises a cationic carbocyanine dye (JC-1), and mitochondrial membrane potential is indicated by change in fluorescence emitted by JC-1.

6. The method of claim 1, wherein the reference level is obtained from the cumulus cells prior to the exposing of step (b).

7. The method of claim 1, further comprising transferring mitochondria harvested from autogeneic stem cells into the oocyte.

8. The method of claim 7, wherein the autogeneic stem cells are selected on the basis of mitochondrial membrane potential resistance to a stressor.

9. The method of claim 8, wherein the autogeneic stem cells are selected by: (i) culturing a plurality of autogeneic stem cells in vitro; (ii) exposing the stem cells to a stressor; and (iii) measuring the amount of stressor required to inhibit mitochondrial membrane potential of the stem cells by 50% relative to a reference level (IC₅₀); and (iv) selecting stem cells exhibiting the highest IC50 after exposure to the stressor.

10. A method of selecting an embryo isolated in vitro that is competent for subsequent implantation, the method comprising: (a) culturing in vitro a sample of cells obtained by biopsy from the embryo; (b) exposing the sample of cells to a stressor; and (c) measuring mitochondrial membrane potential of the sample cells relative to a reference level; (d) selecting the embryo as competent for implantation when mitochondrial membrane potential of the sample cells is maintained at or significantly greater than the reference level upon exposure to the stressor.

11. A method for optimizing the likelihood of a live birth after implantation of a human egg fertilized in vitro comprising performing the method of claim 10 on one or more embryos developed after in vitro fertilization and implanting an embryo selected according to step (d).

12. A method of optimizing anti-oxidant treatment for a patient, the method comprising: (a) culturing in vitro a biopsy sample of cells obtained from the patient; (b) exposing the sample of cells to a stressor in the presence of one or more candidate treatment agents; and (c) measuring the mitochondrial membrane potential of the sample cells in the presence of stressor relative to a reference level; (d) selecting an optimized anti-oxidant treatment agent for the patient when mitochondrial membrane potential of the sample cells is maintained at or significantly greater than the reference level upon exposure to the stressor and/or at a greater level than other candidate treatment agents.

13. The method of claim 12, wherein the patient suffers from cancer, viral infection, or diabetes.

14. The method of claim 12, wherein the patient suffers from a disorder associated with apoptosis, cellular stress, and/or oxidation.

15. A method for selecting a cell that is resistant to stress, the method comprising: (a) culturing in vitro a plurality of cells; (b) exposing the cells to a stressor selected from: CCCP, 2,4-dinitrophenol (DNP), and carbonyl cyanide p-triflouromethoxyphenylhydrazone (FCCP); (c) measuring the amount of CCCP, DNP, or FCCP required to inhibit mitochondrial membrane potential of cells by 50% relative to a reference level (IC₅₀); and (d) selecting a cell whose IC₅₀ after exposure to the stressor is maintained at or significantly higher than the reference level.

16. The method of any of the preceding claims, wherein the measuring is performed using an automated cell counting system.