IN VITRO EMBRYO VIABILITY ASSESSMENT

体外胚胎活力评估

• 批准号: 6319680
• 负责人: DAVID KEEFE
• 金额: $ 1.06万
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6319680
• 关键词: VITRO; EMBRYO; VIABILITY; ASSESSMENT

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.

我们已经建立了一个系统，允许非侵入性 来自单个卵母细胞和胚胎的生理测量， 保持后续发展潜力。 我们现在 独特地准备采用非侵入性自参考电极 技术：1. 识别来自卵母细胞的生理信号， 具有发育潜力的植入前胚胎。 2. 确定卵母细胞的生理变化或 胚胎是年龄依赖性生殖下降的基础。 几 我们开发的系统的特点值得注意; 加热台，生理介质，显微镜载物台， 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 加热阶段符合我们的规格。 我们的加热阶段 将卵母细胞/胚胎维持在体温（37 ℃）， 允许通过保持移液管充分接近胚胎， ICSI移液器和生理探针。 腔室有一个盖玻片 允许对样品进行上级显微镜观察的底部 在操作和生理诊断过程中。 我们发现它 需要改变商业上可获得的介质的组成， 以保持胚胎在孵化器外的活力。 这允许 扩展的显微镜观察和生理学研究 活卵母细胞/胚胎长达2小时，然后成功 转移 我们还发现有必要修改显微镜载物台 板，以允许安装加热阶段，2成重 微操作器，运动控制器的自参考 电极和接地电极保持器。 除了上述 更改股票蔡司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。 这些修改一起允许最大的 多功能性，同时允许稳定的非侵入性生理 来自活卵母细胞/胚胎的测量。 的成员 BioCurrents研究中心（NIH-NCRR）已经调整了 自参考电极运动控制器，包括X、Y、Z电机， 运动控制器和软件接口的特定约束 卵母细胞/胚胎的非侵入性记录。 该中心还 我在准备舞台，放大器， 和数据采集软件， 卵母细胞/胚胎的测量。 BioCurrents的成员 研究中心已经培训了Trimarchi博士使用 自参比电极技术和分析 结果数据。 除了测量钙通量， 自参比电极技术可以测量钾、质子 和氧通量。 我们已经开始描述这些 穿过单个卵母细胞的质膜， 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/胚胎的维持和利用这些物种应有助于 用于确定的生理诊断的识别 发展潜力。