Studies On The In Vitro Maturation Of Oocytes And Vitrification Of Human Embryos

1. The in vitro meiotic process and developmental competence of buffalo oocytes from different size follicles was investigated. There was a significant difference in the meiotic progress of buffalo oocytes from different size follicles, and the meiotic speed of oocytes increased as their follicle size increased. The developmental competence of oocytes from follicles at diameter of 2-4 and 4-6mm was higher than oocytes from follicles at other diameter.2. Effects of EGF concentration (0,10 ng/ml, 25 ng/ml, 50 ng/ml, 100 ng/ml) on the in vitro maturation of buffalo oocytes was examined. Addition of EGF to the maturation medium did not affect the cumulus expansion of oocytes but significantly increased the proportion of oocytes extruded the first polar body (PB1) and hatch rate of blastocysts derived from these oocytes following parthenogenetic activation. The appropriate concentration of EGF was proved to be 25 ng/mL3. Effects of MEM Vitamins (0, 0.2%, 0.5%, 1.0%, 1.5%) on in vitro maturation of buffalo oocytes was explored. Addition of 0.2% or 0.5% MEM vitamins to the maturation medium did not affect the cumulus expansion of buffalo oocytes, but significantly increased the percentage of oocytes developing to blastocysts after parthenogenetic activation, blastocyst hatch rate and cell number. However, When the MEM Vitamins concentration was increased to either 1.0% or 1.5%, the cumulus expansion degree and proportion of oocytes extruded PB1 decreased.4. Effects of HCG (0.75 IU/ml, 2.5 IU/ml, 5 IU/ml, 7.5IU/ml) on in vitro maturation of buffalo oocytes was examined. Addition of HCG to the maturation medium increased the cumulus expansion degree and proportion of oocytes extruded PB1, but did not affect the embryo development and blastocyst cell number of oocytes after parthenogenetic activation. 5. Feasibility of in vitro maturation of cumulus-free buffalo and human oocytes were explored. Surrounding cumulus-free oocytes with cumulus pieces (M3) and supporting cumulus-free oocytes with expanding cumulus clumps (M4) could rebuild the gap junction between oocytes and cumulus cells, and then improved the in vitro maturation of buffalo cumulus-free oocytes. Co-culture with monolayer cumulus cells (M2) did not improve the maturation of buffalo cumulus-free oocytes. However, surrounding denuded oocytes with ovarian tissues inhibited the maturation of buffalo oocytes due to the secretion of some factors that may not be favorable to the oocyte maturation. Moreover, M4 was also proved to improve the in vitro maturation of human denuded oocytes in the presence of follicular fluid.6. The feasibility and clinical value of vitrification of human 4-8 cell embryos were explored. The apparatus of loading embryos and freezing sample volume had a significant influence on the vitrification of human 4-8 cell embryos. Glass capillary was proved to be efficient in vitrification of human 4-8 cell embryos and the freezing sample volume should be controlled as small as possible.7. Feasibility and clinical value of rescuing human day-3 (D3) low grade embryos by in vitro culture and vitrification were investigated. Human D3 low grade embryos were proved to be unsuitable for freezing, but could develop into blastocysts after in vitro culture of 2-3 days. These blastocysts from D3 low grade embryos could be frozen by vitrification, and result in healthy baby birth after thawed and transferred.