Effect Of The Optimized Vitrified Cryopreservation Procedure On Human Oocytes

Part one Effect of FCS in the Vitrified Cryoprotective Agents on Frozen-thawed Human Oocytes of Different Stages at Different TemperatureObjectiveTo study the effect of 65% FCS which was used to optimized the vitrified cryoprotective agents (CPAs) on frozen-thawed human oocytes of different stages at different temperature.MethodsThe failed fertilization MII oocytes from the IVF-ET patients and discarded immature MI and GV stages oocytes from ICSI patients in our center were selected for this study.The MII oocytes were frozen-thawed by the following procedures:The vitrified CPAs consisted of 15% EG, 15%DMSO, 0.5 mol/l sucrose and 65%FCS.The study groups were divided in A,B,C by different operation conditions. Study A : the MII oocytes were placed into the balanced medium at 37℃,then placed them into the CPAs at room temperature(25±2℃), and when thawing ,the oocytes were placed into every warm medium at 37℃. Study B: all the frozen-thawed steps were operated at room temperature. Study C: all the frozen-thawed steps were operated at 37℃.The fresh oocytes were as the control group .The frozen-thawed procedures of GV stage oocytes were the same as the MII oocytes and the GV divided in group GA, GB, GC and GD. The frozen-thawed procedures of MI stage oocytes were also the same as the MII oocytes and the MI divided in group MA, MB, MC and MD. The control immature oocytes and the survival thawed immature oocytes were IVM .The matured MII oocytes,the survival thawed MII oocytes and the control MII oocytes were fixed and immunstaining, then the spindles and chromosomes of the oocytes were observed under Nikon CISI Confocal Laser Scanning Microscope.Results(1)The survival rates of studygroup A, B and C were 95.8%, 95.8% and 78.3%, respectively. There was no significant difference in the survival rate among the three groups (P﹥0.05). The rates with normal spindle and chromosome configurations of group A were 13%and 13%.There was no significant difference when compared with the control group which rates were 18.2% and 18.2%(P>0.05). The rates with absent spindle in study group A and control group were 65.3% and 39.4% respectively,and there was no significant difference between group A and control group(P﹥0.05). The rate with normal spindle configurations of groups B was 4.4%, and there was no significant difference between group B and control group(P﹥0.05). The rates with normal spindle and chromosome configurations of groups C both were 0% , and there was no significant difference between group C and control group(P﹥0.05).The rates with normal chromosome configurations of groups B was 0%,and there was significant difference between the groups B and control group(P﹤0.05). The rates with absent spindle in study group B,C were 78.2% and 88.9%,respectively, and there was statistic difference between the groups B,C and control group(P﹤0.01 and P﹤0.05).The rate of type III chromosome configurations of groups C was 100%,and there was significant difference between group C and group A(P﹤0.05). (2)The survival rates of GA,GB,GC group were 100%,83.3%and66.7%. There was no significant difference in the survival rate among the study groups(P﹥0.05) . The IVM rates of GA,GB and GC group were 40%,100% and 100%. there were no significant difference in the IVM rate among the three study groups and the study groups among the control group(P﹥0.05)).There was no significant difference in the normal spindle and chromosome configurations rate of IVM oocytes among the three study groups and the study groups among the control group(P﹥0.05). However, the normal spindle and chromosome configurations rate of GB group was the highest (40%).(3)The survival rates of MA,MB and MC group were 71.4%,100% and 83.3%. There was no significant difference in the survival rate among the three study groups (P﹥0.05). The IVM rates of MA,MB ,MC and control group were 0%, 66.6% ,80%.and57.1%. There was no significant difference in the IVM rate among the three study groups and the control group(P﹥0.05). However, there was significant difference in the IVM rate among the three study groups(MA vs MB or MC, P﹤0.05). But there was no significant difference in the IVM rate between MB and MC(P﹥0.05). No one IVM MII oocyte had the normal spindle and chromosome configurations .Conclusions(1)Adding FCS to the CPAs has positive effect to survival rate of the different stages of oocytes after thawing .(2)The condition of the study group A is better to freeze MII oocytes and that operation condition is less hamful to the spindle and chromosome configurations .(3)Placed into the high osmotic CPAs ,the oocytes should be choose to freeze at room temprature.This way make the oocytes dehydration more gently and reduce the harm that made by the volume change. (4)The better vitrification condition of MI stage oocytes is all frozen-thawed operation steps at 37°C.(5)Vitrification condition of GV stage oocytes which is operated at room temperature has the highest rate of the normal spindle and chromosome configurations .Part two Comparative Analysis of Two Vitrification Procedures in Mature Human OocytesObjectiveTo compare the effect of two vitrification procedures on the spindle and chromosome configurations of Mature (MII) human oocytes.MethodsThe failed fertilization MII oocytes from the IVF-ET patients in our center were selected for my study . The vitrified CPAs of study A consisted of 15% EG, 15%DMSO, 0.5 mol/l sucrose and 65%FCS . The MII oocytes of study A group were placed into the balanced medium at 37℃,then placed them into the vitrified CPAs at room temperature(25℃), and when thawing ,the oocytes were placed into every warm medium at 37℃. The vitrified CPAs of study B consisted of 15%EG, 15%DMSO,0.5mol/l sucrose . All the frozen-thawed steps of study B was the same as study A. The fresh oocytes were as the control group .After thawing , the survival oocytes of the study groups and the fresh oocytes which used as control group were stained by immunofluorescene , then the spindles and chromosomes of the oocytes were observed under Nikon CISI Confocal Laser Scanning Microscope.ResultsThe survival rates of group A,B were 95.8%,96.7% respectively,there was no significant difference between the study groups(P>0.05). The normal spindle and chromosome configurations rates in group A and control group were 13%,13%,18.2%,18.2%,respectively .There was no significantly difference between group A and control group( P>0.05 ) .But the rates of spindle and chromosome with normal configurations in group B were significantly lower than in control group (0%,0% VS 18.2%,18.2%,P<0.05). The rates with absent spindle in study group A and control group were 65.3% and 39.4% respectively,and there was no significant difference between group A and control group(P﹥0.05).The rate with absent spindle in study group B was 79.3% ,and there was very significant difference between the group B and control group(P﹤0.01).The rate with type III chromosome configurations of group B was 100%,and there was very significant difference between group B and control group(P﹤0.01).ConclusionThe Fetuin from the FCS may protect the microtubules of the frozen-thawed oocytes'spindles and can promote the normal rate of the spindle and chromosome configurations in right conditions by reducing water permeability of the oocytes'membrane.