The Experimental Research Of Vitrification Technique

Objective: To investigate the influences on the morphology and function of vitrified embryos by open pulled straw, close pulled straw, Cryoloop and straw.Methods:1. 300 embryos of 6-10 cell cleavage stage developed from three pronuclei zygotes of human were randomly separated into five groups, 60 embryos in control group and 60 embryos in open pulled straw (OPS), close pulled straw (CPS), Cryoloop and Straw respectively. The survival rates of the embryos after warming were compared by the observation of the embryo morphology.2. The culture medium where the warmed embryos were incubated for 24 hours was collected to survey the activity of lactate dehydrogenase (LDH) in it by the automatic biochemistry analyzer.Results:1. The survival rate of the Cryoloop ultra-rapid vitrification refrigeration group is highest and significant higher than the CPS, OPS and Straw groups (P<0.05). Compared with the OPS and the Straw groups, the survival rate of the CPS group is significant different (P<0.05).2. The activity of LDH of the Cryoloop group is lower than the other three groups and the difference is significant (P<0.05). The activity of LDH of the CPS and OPS was both lower than the Straw group (P<0.05). There are no differences were found between the CPS and OPS in the activity of LDH (P>0.05).Conclusion: Cryoloop displayed to have minimal influence on the morphology and function of the embryos suffered from the vitrification refrigeration compared to the other three groups. The CPS is second only to Cryoloop. Though no higher morphology survival rate were gained in the OPS compared with the Straw, the function loss was reduced. Objective: To investigate the influences on vitrified embryos of the low Na+ phosphate buffer and non-permeable protective Trehalose.Methods:1.The low Na+ phosphate buffer (mPBS) was made by replacing sodium chloride (NaCl) in the phosphate buffer with the choline chloride. The embryos were cooled and warmed by the vitrification solution contained the mPBS and phosphate buffer respectively, and then the survival rate was detailed. The culture medium where the warmed embryos were incubated for 24 hours was collected to survey the activity of lactate dehydrogenase (LDH) in it by the automatic biochemistry analyzer and compare.2.The cryoprotectants of vitrification were made by sucrose and D-(+) Trehalose. The survival rate and the development ability of embryos cooled with those solutions were detailed. The culture medium where the warmed embryos were incubated for 24 hours was collected to survey the activity of lactate dehydrogenase (LDH) in it by the automatic biochemistry analyzer and compare.Results:1.The survival rate of the mPBS group is a little higher than the phosphate buffer group but the difference is not significant (P>0.05). The activity of LDH of the mPBS group is lower than the phosphate buffer group (P<0.05).2.Both the survival rate and the activity of LDH were found no significant differences between the D-(+) Trehalose group and the sucrose group(P>0.05).Conclusion:1. Since the low Na+ phosphate buffer less influenced the function of the embryos warmed than the phosphate buffer, it is probably more suit for the vitrifying embryos.2. The vitrification solution with D-(+) Trehalose neither improves the survival rate nor lightens the influence on the function of vitrified embryos. Objective: To discuss the degeneration mechanism of the vitrified embryosMethods:1. 150 embryos in 6-10 cell cleavage stage developed from human three pronuclei zygotes were separated into five groups randomly, 30 embryos in control group, open pulled straw (OPS), close pulled straw (CPS), Cryoloop and Straw respectively. The survived embryos after thawing were stained with the rhodamine123, then photographed and analyzed under the laser scanning confocal microscopy to calculate their mitochondrial membrane potential (△Ψm).2. 158 embryos in 6-10 cell cleavage stage developed from human three pronuclei zygotes were separated into three groups randomly, 30 embryos in positive control group, 30 embryos in normal group and 98 embryos in CPS group respectively. The survived embryos after warming were fixed in the 4% paraformaldehyde PBS solution. The fixed embryos were labelled under the manual of the TUNEL kit to detect the DNA fragment in situ and photographed under the laser scanning confocal microscopy.Results:1. High fluorescent intensity was found at the normal embryos of the control group which△Ψm is stable at 22.2622±0.67067. The warmed embryos showed lower fluorescent intensity and decreased△Ψm than the normal embryo. There was a discrepancy on the fluorescent intensity in the four experimental groups. The fluorescent intensity of Cryoloop is the highest among the four vitrified groups. The CPS and OPS group both have higher fluorescent intensity and△Ψm than the Straw group(P<0.05) while there are no differences were found between CPS and OPS(P>0.05). The△Ψm of CPS ,OPS and Straw were lower than the control group and the differences was significant(P<0.05).2. After stained by the TUNEL, only 6.9% normal embryos have the expression of the green fluorescence. The expression of the fluorescence was stronger in the CPS group than the normal group (P<0.05).Conclusion: The mitochondrial membrane potential decreased under the LSCM and the DNA fragment was detected by TUNEL technique in the vitrified embryos. These results and the morphology of the warmed embryos all conformed the vitrified embryos were degenerated from cell apoptosis pathway. Objective: To establish a method to observe the spindle and the chromosome in the mature oocyte (MII stage) and detect the changes of the spindle and chromosome in the mature oocyte vitrified with OPS.Methods:The technologies of optical section and 3D image reconstruction of laser scanning confocal microscope (LSCM) were combined with the immunochemistry technique of the suspended cells to examine the spindle and chromosome in the mice MII oocytes.The mice MII stage oocytes were collected and vitrified by OPS vitrification refrigeration. The survival oocytes after warming were fixed and stained by cell immuno-fluorescence sign method then observed under the LSCM to examine the spindle and chromosome.The mice MII stage oocytes were collected and vitrified by OPS vitrification refrigeration. The survival oocytes after warming were fertilized in vitro and further cultured to blastocyst stage.Results:1. The configurations of spindle and chromosome can be observed clearly by LSCM. The normal rates of spindle and chromosome were 82% and 86% respectively.2. The normal rates of spindle and chromosome of the thawed oocytes after OPS vitrification refrigeration were 46% and 52% respectively. The difference is significant between the OPS and control group (P<0.05). The survival rate was 58%. The fertilization rate and the blastocyst formation rate of the warmed oocytes after OPS vitrification refrigeration were 36% and 19.3%respectively.Conclusion:1. The configurations of spindle and chromosome can be observed clearly and effectively by the combination of the LSCM and cell immunochemistry techniques.2. The damage of the spindle and chromosome in the oocytes suffered the vitrificaton were detected by the combination of the LSCM and cell immunochemistry techniques so as that this method is a reliable means to observe the spindle and chromosome in the MII stage oocytes. The OPS vitrification refrigeration is a feasible method to cryopreserve the MII oocyte.