Basic study in cryopreservation and chromosomal analysis of single blastomeres.

植入前胚胎活检作为一种研究植入前胚胎发育潜能的工具，为产前诊断提供了可能，具有广泛的应用前景。本研究的目的是首先检测小鼠四细胞和八细胞胚胎单卵裂球的发育潜力，其次探讨单卵裂球成功冷冻保存和核型分析的可能性。(1)通过挤压技术从4-和8-细胞小鼠胚胎中去除单个卵裂球并不影响体外发育到囊胚期（对照组的80%，活检胚胎的75%和78.3%分别处于4-和8-细胞期）。然而，移植到假孕受体小鼠后，活检胚胎的植入率显著低于完整对照组（4-细胞组为24.3%，8-细胞组为34.8%，对照组为50%）。(2)大多数单个卵裂球在体外经历了多次细胞分裂。经过短期培养的单卵裂球，随后暴露于秋水仙胺16小时，27.3%的4-细胞和52.2%的8-细胞活检进行核型分析。然而，20%至30%的活组织检查的成纤维细胞球由于细胞死亡和技术问题而丢失。(3)用含1.5M DMSO+0.25M蔗糖的冷冻液，采用5步稀释法，不能成功地冷冻活检的单卵裂球。本研究所建立的技术为产前诊断的研究提供了有用的工具。

Biopsy of the preimplantation embryo has wide application as a tool to investigate the developmental potential of the preimplantation embryo and also offered a possibility for prenatal diagnosis. The purpose of this study were first, to examine the developmental potential of single blastomeres of four-and eight-cell mouse embryos, and second to investigate the possibility of successful cryopreservation and karyotyping of single blastomeres.(1) Removal of a single blastomere from 4- and 8-cell mouse embryos by extrusion technique did not affect development to the blastocyst stage in vitro (80% of control,75% and 78.3% of biopsied embryo at 4- and 8-cell stage, respectively). However,after transfer to pseudopregnant recipient mice, the implantation rates of biopsied embryos were significantly lower than intact-controls (24.3% at 4-cell, 34.8% at 8-cell and 50% at control, respectively).(2) Most single blastomeres had undergone several cell divisions in vitro. After a short culture of single blastomeres with subsequent exposure to colcemid for 16 hours, 27.3% of 4-cell and 52.2% of 8-cell biopsies were karyotyped. However, 20 to 30% of biopsied blstomeres were lost through cell death and technical problems.(3) Biopsied single blastomeres could not be successfully cryopreserved when the freezing medium contained 1.5M DMSO+0.25M sucrose and 5-step dilution method was used. The techniques developed in this study may provide the useful tool for the study of prenatal diagnosis.