IN VITRO EMBRYO VIABILITY ASSESSMENT

我们已经建立了一个非侵入性的系统

We have established a system that allows non-invasive physiological measurements from individual oocytes and embryos while maintaining their subsequent development potential. We are now uniquely poised to employ non-invasive self-referencing electrode techniques to: 1. Identify physiological signals from oocytes and preimplantation embryos thatare diagnostic of developmental potential. 2. Determine whether changes in the physiology of the oocyte or embryo underlie age-dependent decline in reproduction. Several features of the system we have developed are worthy of remark; the heating stage, the physiological media, the microscope stage plate, the electrode motion controller and software, the amplifier and data acquisition software. We found it necessary to design and construct a heating stage that meets our specifications. Our heating stage maintains the oocytes/embryos at body temperature (37 0C) while allowing sufficient access to the embryos by holding pipettes, ICSIpipettes, and physiological probes. The chamber has a cover-glass bottom that permits superior microscopic observation of specimens during manipulation and physiological diagnosis. We found it necessary to modify the composition of commercially available media in order to maintain embryo viability outside the incubator. This allows extended microscopic observations and physiological investigations of living oocytes/embryos for up to 2 hours followed by successful transfer. We also found it necessary to modify the microscope stage plate to permit mounting the heating stage, 2 Narishige micromanipulators, the motion controller for the self-referencing electrode and the ground electrode holder. In addition to the above changes to the stock Zeiss Axiovert 100 TV, we have arranged the microscope to permit DIC imaging, fluorescence microscopy, and PolScope imaging. Together these modifications allow maximal versatility while permitting stable non-invasive physiological measurements from living oocytes/embryos. The members of the BioCurrents Research Center (NIH-NCRR) have adapted the self-referencing electrode motion controller including X,Y,Z motors, motion controller and software interface to the particular constraints of non-invasively recording from oocytes/embryos. The Center has also been instrumental in preparing the preamplifier headstage, amplifier and data acquisition software necessary for conducting physiological measurements from oocytes/embryos. The members of the BioCurrents Research Center have trained Dr. Trimarchi in the use of self-referencing electrode techniques and the analysis of the resulting data. In addition to measuring calcium flux, self-referencing electrode techniques can measure potassium, proton and oxygen flux. We have begun characterizing the movement of these species across the plasma membrane of individual oocytes and preimplantation embryos. These studies will greatly enhance our understanding of the physiology of oocytes and early embryos. Moreover, a deeper understanding of mechanisms by which oocytes/embryos maintains and utilize these species should facilitate the identification of a physiological diagnostic for determining developmental potential.