Vitrification On Oocytes Of Different Stage And Blastocyst From 3PN

PartⅠVitrification on Kunming Mouse Mature OocytesObjective To investigate the effect on the developmental capacity of mouse mature oocyte after vitrification using different vitrification methods. Try to establish the platform of vitrification, and to provide theoretical and technical reference for human oocytes cryopreservation of vitrification.Methods The study was using Kunming mice and vitrification technology to cryopreserve mouse mature oocytes. It was divided into vitrification group and control group, and then the vitrification group was divided into OPS group, artificial-straw group and cryoleaf group, and their survival rate, fertilization rate, cleavage rate and formed blastocyst rate were compared.Result 1. There was no significant difference on fertilization rate and cleavage rate between control group and vitrification group (P>0.05), but there was a significant difference on formed blastocyst rate (P<0.05). 2. The survival rate, fertilization rate, cleavage rate and blastocyst formed rate have no significant difference between OPS group and artificial group (P>0.05). 3. There were significant differences on survival rate between OPS group and cryoleaf group (P<0.05), as well as artificial group and cryoleaf group, while fertilization rate, cleavage rate, formed blastocyst rate have no significant difference between them (P>0.05).Conclusions 1. We initially established the vitrification technology platform of mouse mature oocytes. 2. The OPS and cryoleaf methods of vitrification can be used to cryopreserving mouse mature oocytes, furthermore, cryoleaf methods is much safer.PartⅡStudy On Vitrification of Human Immature OocytesObjective To analyze the fertilization and development ability after vitrification of different stage of human oocytes, and investigate the efficiency of vitrification of immature oocytes to expand the field of gamete cryopreservation.Methods The surplus GV oocytes from our ICSI programme were assigned to group with granule cells and group without granule cells. In addition, oocytes from IVM were as control group, GV vitrification group and MⅡvitrification group as test groups. We compared their survival rate, fertilization rate, cleavage rate and formed blastocyst rate.Result There was a significant difference of maturation rate between group with granule cells group and that without granule cells group(P<0.05), The survival rate, fertilization rate, cleavage rate(including 2 cells rate and more than 2 cells rate) had no significant difference (P>0.05), and the maturation rate, fertilization rate, cleavage rate were significantly different between IVM group and GV vitrification group, as well as IVM group and MⅡvitrification group(P<0.05). But there were no significant difference of survival rate, maturation rate, fertilization rate, cleavage rate between GV vitrification group and MⅡvitrification group(P>0.05).Conclusion 1. Vitrification of human immature oocytes must reserve with granule cells. 2. Granule cells play a great role in the in-vitro maturation after thawing. 3. It has already been established the platform of human immature oocytes vitrification technology which combine with in vitro maturation technology.PartⅢStudy on Vitrification of Human Mature OocytesObjective To study on the vitrification methods and cryopreservation solution of human mature oocytes, and to assess the feasibility and clinical value of cryopreservation of human oocytes.Methods Human oocytes were collected during stimulated cycle and then vitrificated with cryoleaf. Surviving oocytes were thawed and then inseminated by intracytoplasmic sperm injection (ICSI). The fertilized oocytes were further cultured in vitro for 48～72h. High-quality embryos were transferred to the patients of infertility with informed consent. The oocytes were divided into vitrification group and slow-freezing group, and then the vitrification group was divided into group with self-made up solution and that of commercial solution, and their survival rate, fertilization rate, cleavage rate and blastocyst rate were compared.Results The survival rate, cleavage rate, blastocyst rate had no significant difference between vitrification group and slow-freezing group (P>0.05), while the fertilization rate had a significant difference (P=0.05). There also had no significant difference of survival rate, fertilization rate, cleavage rate and blastocyst rate between slow-freezing group and group with self-made up solution, as well as slow-freezing group and vitrification group with commercial solution(P>0.05). In slow-freezing group, embryos were transferred to 4 patients, one of them achieved pregnancy and gave birth of one healthy boy. In vitrification group, embryos were transferred to 7 patients, and one of them got a biochemical pregnancy.Conclusions Both slow-freezing methods and vitrification methods can be used to human mature oocytes, and vitrification methods are more simple and convenient. Although vitrification of human mature oocytes is a best one to cryopreserve, the further investigation and improvement of technique is still needed until they are applied clinically.PartⅣStudy on Vitrification of Human Blastocysts from 3PNObjective To compare the vitrification using different methods with slow-freezing method for human blastocyst cryopreservation and to investigate the survival rate and development capacity after thawing. We try to lay a foundation of human blastocyst cryopreservation.Methods In IVF/ICSI cycles, supernumerary embryos were cultured to D5 or D6, and the formed blastocysts were cryopreserved using vitrification or slow-freezing method. And all blastocysts were divided into slow-freezing group and vitrification group. Then vitrification group was divided into artificial straw group and cryoleaf group. Their survival rate, expanded blastocyst rate and blastocyst-hatching rate were compared. Results The survival rate, expanded blastocyst rate and blastocyst-hatching rate had no significant difference between slow-freezing group and vitrification group, as well as artificial straw group and cryoleaf group.(P>0.05).Conclusions The platform of human blastocyst vitrification cryopreservation methods has already established. And it can be applied clinically.