Confocal measurements of mitochondrial activity during human preimplantation embryo development.

Abstract

Objective: We have used ratiometric confocal microscopy in conjunction with the membrane potential-sensitive fluorescent dye JC-1 to study the activity of mitochondria in living human oocytes and preimplantation embryos. Our aim is to test whether the activity of mitochondria in diverse populations of oocytes differs, and to correlate differences in activity with oocyte maturation, embryo development and the reproductive characteristics of individual patients attending for in vitro fertilisation protocols.Design: Confocal analysis of mitochondrial activity in human oocytes and embryos donated for research.Materials/Methods: Patients donated spare oocytes and embryos after informed consent. Material was loaded with a final concentration of 0.5 μm dye for 30 minutes in embryo culture medium, prior to washing and transfer into fresh medium for confocal microscopy. Images were taken on an Olympus Fluoview confocal microscope and processed using the confocal software or Adobe Photoshop.Results: We analysed mitochondrial activity in a total of 22 fresh metaphase II oocytes obtained on the day of oocyte retrieval and 31 embryos obtained after transfer into the patients uterus. In both fresh human oocytes and preimplantation embryos, the mitochondrial membrane potential was inversely related to maternal age. Interestingly, we noticed a positive correlation between the rate of embryo development and mitochondrial membrane potential in individual blastomeres. No significant differences were observed when blastomere mitochondrial activity was plotted against either sperm count or sperm motility.Conclusions: We have shown that the inherent mitochondrial activity of human oocytes and embryos decreases in relation to maternal age. Furthermore, the mitochondrial activity of human embryos is related to rate of development. These data suggest that the inherent mitochondrial activity in the human oocyte is related to maternal age and can determine the potential of the oocyte to form a top quality embryo. The data underlies the observation that pregnancy rates are decreased in older couples through a decrease in the rate of development of embryos from these patients.Supported by: Serono Pharma, Italy and Fondazione Nuovi Orizzonti, Italy. Objective: We have used ratiometric confocal microscopy in conjunction with the membrane potential-sensitive fluorescent dye JC-1 to study the activity of mitochondria in living human oocytes and preimplantation embryos. Our aim is to test whether the activity of mitochondria in diverse populations of oocytes differs, and to correlate differences in activity with oocyte maturation, embryo development and the reproductive characteristics of individual patients attending for in vitro fertilisation protocols. Design: Confocal analysis of mitochondrial activity in human oocytes and embryos donated for research. Materials/Methods: Patients donated spare oocytes and embryos after informed consent. Material was loaded with a final concentration of 0.5 μm dye for 30 minutes in embryo culture medium, prior to washing and transfer into fresh medium for confocal microscopy. Images were taken on an Olympus Fluoview confocal microscope and processed using the confocal software or Adobe Photoshop. Results: We analysed mitochondrial activity in a total of 22 fresh metaphase II oocytes obtained on the day of oocyte retrieval and 31 embryos obtained after transfer into the patients uterus. In both fresh human oocytes and preimplantation embryos, the mitochondrial membrane potential was inversely related to maternal age. Interestingly, we noticed a positive correlation between the rate of embryo development and mitochondrial membrane potential in individual blastomeres. No significant differences were observed when blastomere mitochondrial activity was plotted against either sperm count or sperm motility. Conclusions: We have shown that the inherent mitochondrial activity of human oocytes and embryos decreases in relation to maternal age. Furthermore, the mitochondrial activity of human embryos is related to rate of development. These data suggest that the inherent mitochondrial activity in the human oocyte is related to maternal age and can determine the potential of the oocyte to form a top quality embryo. The data underlies the observation that pregnancy rates are decreased in older couples through a decrease in the rate of development of embryos from these patients. Supported by: Serono Pharma, Italy and Fondazione Nuovi Orizzonti, Italy.