Study On The Effect Of Microinjecting Trehalose On Oocytes Vitrification

Objective (1)To explore a stable and high efficient control ovarian hyperstimulationmethod for rabbits to obtain sufficient high-quality rabbit oocytes；(2) To investigatewhether the application of trehalose can improve the effect of vitrification in rabbitoocytes or not;(3) To explore the effect of artificial oocyte activation on thedevelopment ability of frozen-thawing oocytes.Methods (1)30New Zealand white rabbits, randomly divided into3groups:HMG+HCG group; FSH+HCG group; PMSG+HCG group, compared the ovarianhyperstimulation effects of three treatment methods.(2)438mature oocytes whichcollected from HMG+HCG group and FSH+HCG group were chosen, and wererandomized divided into four groups: Intra-and extracellular trehalose group（n=143）:0.15mol/L intra-and0.5mol/L extracellular trehalose+15%ethylene glycol (EG)+15%1,2-propanediol(PROH), trehalose was introduced into oocytes by microinjection;Extracellular trehalose group(n=127):0.5mol/L trehalose+15%EG+15%PROH;Sucrose group(n=118):0.5mol/L sucrose+15%EG+15%PROH; Fresh controlgroup(n=50): without vitrification.(3) All the oocytes were thawed after1month, fertilized by ICSI after incubated for2hours. Then the thawed oocytes from Intra-andextracellular trehalose group, Extracellular trehalose group and Sucrose group wererandomly divided into two groups respectively: the controlled group and artificialoocyte activation group (AOA). The controlled groups’ oocytes were cultured incleavage medium after ICSI directly. The AOA groups’ oocytes were activated byexposure to7%anhydrous alcohol for6minutes, and then cultured in cleavagemedium. The survival rates, fertilization rates, cleavage rates, blastocyst rates werecompared.Results (1) The average number of follicles retrievaled from HMG+HCG group wassignificantly higher than which retrievaled from FSH+HCG group and PMSG+HCGgroup (P<0.05). And the average number of follicles retrievaled from FSH+HCGgroup was significantly higher than the PMSG+HCG group (P<0.05). The averagenumber of hyperaemia follicles and ovarian cysts on the ovary of PMSG+HCG groupwas significantly higher than which retrievaled from HMG+HCG group andFSH+HCG group (P<0.05). A part of the oocytes had abnormal morphology (such asfirst polar body fregment, egg peripheral gap, cytoplasm abnormal morphology) whichwas retrievaled form PMSG+HCG group.(2)The survival rate, fertilization rate,cleavage rate and blastocyst rate had no significant differences among the Intra-andextracellular trehalose controlled group, Extracellular trehalose controlled group andSucrose controlled group (P>0.05); The survival rates, fertilization rates, cleavagerates and blastocyst rates of all the three vitrification controlled groups weresignificantly lower than those of fresh control group (P<0.05);(3) The survival rates,fertilization rates, cleavage rates and blastocyst rates had no significant differencesamong the Intra-and extracellular trehalose AOA group, Extracellular trehalose AOAgroup and Sucrose AOA group (P>0.05); but the survival rates, fertilization rates,cleavage rates and blastocyst rates of all the three vitrification AOA groups were significantly lower than those of fresh control group (P<0.05);(4) The survival rate,cleavage rate and blastocyst formation rate had no significant difference between Intra-and extracellular trehalose controlled group and Intra-and extracellular trehalose AOAgroup(P>0.05), but the fertilization rate had significant difference (P<0.05); Thesurvival rate, cleavage rate and blastocyst formation rate had no significant differencebetween Extracellular trehalose controlled group and Extracellular trehalose AOAgroup(P>0.05), but the fertilization rate had significant difference (P<0.05); Thesurvival rate, cleavage rate and blastocyst formation rate had no significant differencebetween Sucrose controlled group and Extracellular trehalose AOA group(P>0.05), butthe fertilization rate had significant difference (P<0.05).Conclusions (1) HMG+HCG is a stable and effective method for rabbit ovarianhyperstimulation;(2) Intracellular trehalose seems could not improve the effect ofvitrification in rabbit oocytes, as well as post-thawed rabbit oocytes’ developmentability.(3) AOA followed by ICSI appears to improve the fertilization rates of therabbit oocytes.(4) The development ability of thawed rabbit oocytes after vitrificationdeclined significantly. Objectives (1) To explore whether the application of trehalose can improve the effect ofvitrification in human oocytes, as well as post-thawed oocytes’ development;(2) Toassess the technical safety based on the incidence of abnormal embryos. Methods Fresh human oocytes (n=189) obtained from IVF/ICSI patients wererandomly distributed into four groups:(1) Intra-and extracellular trehalose group(n=63):0.15mol/L intra-and0.5mol/L extracellular trehalose+15%ethylene glycol(EG)+15%1,2-propanediol(PROH), trehalose was introduced into oocytes bymicroinjection;(2) Extracellular trehalose group:0.5mol/L extracellular trehalose+15%EG+15%PROH;(3) Sucrose group:0.5mol/L sucrose+15%EG+15%PROH;(4)Fresh control group: without vitrification. The first three groups were chosen toundergo freezing and thawing. After oocytes were thawed, all survival oocytes werefertilized by ICSI. Survival rate, feritilization rate, cleavage rate, high-quality embryoformation rate, blastocyst rate, high-quality blastocyst formation rate were compared.When the embryo developed to blastocyst，we tested the chromosomal status of humanembryos by fluorescence in situ hybridization (FISH)（13,15,16,18,21,22,X,Y）, theabnormal embryos rates were compared.Results (1) The survival rate, high-quality embryo formation rate and blastocyst ratehad no significant differences among three vitrification groups (P>0.05); Thefertilization rate, cleavage rate and high-quality blastocyst formation rate weresignificantly higher in the Intra-and extracellular trehalose group than Extracellulartrehalose group and Sucrose group(P<0.05), the fertilization rate and cleavage ratewere significantly higher than Extracellular trehalose group(P<0.01), and the cleavagerate were significantly higher than Sucrose group(P<0.01); The fertilization rate,cleavage rate, high-quality embryo formation rate, blastocyst rate and high-qualityblastocyst formation rate had no significant differences between Extracellular trehalosegroup and Sucrose group(P>0.05); The high-quality embryo formation rate andhigh-quality blastocyst formation rate had significant differences between the Intra-and extracellular trehalose group and Fresh control group(P<0.05); The fertilizationrate, cleavage rate, high-quality embryo formation rate and high-quality blastocystformation rate were significantly higher in the Fresh control group than the Extracellular trehalose group and Sucrose group (P<0.01).(2) The percentage ofabnormal embryo of the intra-and extracellular trehalose group was higher than theother three groups, but there were no statistically significance among the four groups(P>0.05).Conclusions (1) Intracellular trehalose appears to improve the effect of vitrification inhuman oocytes, as well as post-thawed human oocytes’ development;(2)Microinjection of trehalose into oocytes followed by vitrification, ICSI and in-vitroembory culture seems not to induce more abnormal blastocysts.