IN VITRO EMBRYO VIABILITY ASSESSMENT

我们已经建立了一个系统，允许非侵入性 单个卵母细胞和胚胎的生理测量 保持其随后的发展潜力。我们现在是 独一无二地准备采用非侵入性自参照电极 技术：1.识别来自卵母细胞的生理信号 可以诊断发育潜力的植入前胚胎。 2.确定卵母细胞的生理变化或 胚胎是与年龄相关的生殖衰退的原因。几个 我们开发的系统的特点值得一提； 加热台、生理介质、显微镜台板、 电极运动控制器和软件、放大器和数据 采集软件。我们发现有必要设计和建造一个 符合我们规格的加热台。我们的供暖舞台 将卵母细胞/胚胎维持在体温(37摄氏度)，同时 通过握住吸管，就可以充分接触到胚胎， ICSI移液管和生理探头。这个房间有一个盖子玻璃。 允许对样品进行高级显微镜观察的底部 在操作和生理诊断过程中。我们找到了它 有必要修改商业上可用的媒体的组成 以保持孵化器外的胚胎活力。这使得 超微结构的观察和生理研究 活卵母细胞/胚胎最多2小时，然后成功 调职。我们还发现有必要改装显微镜载物台 允许安装加热台的板，2 Narishige 微操作器--用于自参考的运动控制器 电极和接地电极座。除上述外， 更换现货蔡司Axiovert 100电视，我们已经安排了 显微镜允许DIC成像、荧光显微镜和 PolScope成像。这些修改加在一起，最大限度地 多功能性，同时允许稳定的非侵入性生理 来自活的卵母细胞/胚胎的测量。该委员会的成员 生物电流研究中心(NIH-NCRR)已经适应了 自参考电极运动控制器，包括X、Y、Z电机， 针对特定约束的运动控制器和软件接口 对卵母细胞/胚胎进行非侵入性记录。该中心还拥有 协助准备前置放大器主舞台、放大器 以及进行生理检查所需的数据采集软件 来自卵母细胞/胚胎的测量。生物流派的成员 研究中心对特里马奇博士进行了使用 自参比电极技术及其性能分析 结果数据。除了测量钙离子流量外， 自参比电极技术可以测量钾、质子 和氧气流量。我们已经开始描述这些运动的特点 穿过单个卵母细胞质膜的物种和 植入前胚胎。这些研究将大大提高我们的 了解卵母细胞和早期胚胎的生理学。 此外，更深入地了解 卵母细胞/胚胎的维护和利用应有利于这些物种 一种生理学诊断方法的鉴定 发展潜力。

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.