Modifications To Human Oocytes Vitrification Method-fundamental Study And Clinical Application

Partâ… The effect of volume changes on developmental competence of Metaphaseâ…¡stage porcine oocytesObjective:Mammalian oocytes cryopreservation is still a relative inefficient technique despite the high potential benefits of it.The oocytes,with some special delicate ultrastructures,are very sensitive to many physical changes.This study was aimed to evalued the porcine oocytes torlerance to gradual volume changes rather than osmotality changes.Methods:Porcine oocytes were purchased commercially(BOMED,Inc. Madison),and immature oocytes were incubated in maturation medium for 40-44 hours.According to Boyle-Van't Hoff relationship equation,different osmotic levels were calculated in relation to different cell volume ratio.In vitro matured oocytes were randomly divided into 13 groups and be exposed to different osmotic treatments for 10 min.Experiment 1:In the first several replicates,in vitro matured oocytes were exposure to hyper/hyposmotic and isosmotic solutions for 10min,after that oocytes would be incubated without fertiliazation and observed parthenogenesis development.Experiment 2:After Experiment 1,more replicates were carried out within the osmotality region without significant parthenogenesis development. Blastocyst formation of porcine oocytes after exposured to different isosmotic groups for 10min would be recorded after fertilized by freezing porcine sperms.Results:1) Parthenogenetic activation could be observed in extra hypo/hyperosmotic solutions.In present study,blastocysts were observed after oocytes treated in the volumes of 0.3V-0.6V and 1.4V-2.0V("V"represents original volume).2) More replicates were performed between treatments 0.6V and 1.4V,with additional treatments of 0.95V and 1.1V.Development potential decreased as oocytes expanding or shrinking in hypo/hyperosmotic solutions.Oocytes could maintain a more than 70%development potential between about 0.7 and 1.2 times of original volume(â…¤).After curve fitting,a binary quadratic formula was got, which could describe the relationship between the blastocyst formation and volume change of porcin oocytes:Y=-1.104+3.945X-1.946X²;R²=0.641.Conclusions:This study provided forcible evidence about the intimate relationship between oocytes volumes and their developmental competence,which is different from the previous studies about the effect of extracellular osmotic pressure to oocytes.Application of the result of present study could estimate the developmental competence of the oocytes according to certain volumes,which provide fundamental supports about designing vitrifying/thawing procedures in consideration of volume maintenance. Partâ…¡Modifications of human oocyte vitrification on sucrose concentration,equilibration steps and thawing temperatureObjective:Oocytes cryopreservation is very valuable in human assisted reproductive technologies in clinical settings.However,the technology itself is far beyond satisfactory towards clinical application.In present study, several modifications of human oocyte vitrification were carried out to investigate development potential of human oocytes using different protocols.Methods:Patients undergoing Intracytoplasmic sperm injection(ICSI) treatment volunteered for the study and donated their immature oocytes.In vitro matured oocytes were subjected to one of the following 3 experiments: vitrification solutions with different sucrose concentrations(0.35M vs.0.5M); different equilibration procedures during vitrification(four-step method vs. two-step method) and different warming temperatures(37℃vs.room temperature).Survival,fertilization,high-quality embryo and blastocyst formation among the four groups were recorded.Results:1.There were no statistical differences between 0.5 M(control group)and 0.35 M sucrose concentration in vitrification solutions.2.The four-step protocol got higher survival and fertilization rate,while a relative lower high-quality embryo proportion.But there were no statistical differences between the outcomes of two groups.3.There were no statistical differences between the 37℃thawing group and room temperature(20～22℃) group in the survival,fertilization,and high quality embryo rates.4.There was a statistical difference of high quality embryo rates between control group and 37℃thawing group(Fisher exact test,P＜0.05).Conclusions:In conclusion,this experiment demonstrated that it is feasible to use a lower sucrose concentration in oocytes vitrification,and similarly,a lower concentration of sucrose in thawing solutions as well.. Therefore,a lower sucrose concentration in oocytes vitrification protocol is recommended here.More steps of CPAs addition and removal procedures in oocytes vitrification method did not show advantages in oocytes developmental potential after thawing,and prolonged equilibration time might be the main problem of this modification.Finally thawing procedures at 37℃didn't increase the development potential of vitrified oocytes,and additional researches are needed in order to find an optimal thawing temperature. Partâ…¢Primary application of human oocytes vitrification in clinic workObjective:Improvements in human oocyte cryopreservation and related techniques could offer many advantages for the use of human assisted reproductive technologies in clinical settings.Vitrification method has showed great advantages in human oocytes cryopreservation.This study aimed at estimating the effect of human oocytes vitrification method in application of clinic work.Methods:Human oocytes were vitrified and thawed with Medicult vitrification/thawing solutions after consented by the patients,who got more than 25 oocytes in fresh IVF/ICSI cycles.Some oocytes were thawed with a circumstances temperature of 37℃.Survived oocytes were underwent ICSI after 3h incubation in 37℃,5%CO₂ circumstances.Fertilization, embryos development and clinic pregnancy were recorded of each cycle.Results:1)11 patients,who didn't get pregnancy after transfering fresh embryos and cryopreserved embryos,used their vitrified oocytes and carried out ICSI-ET.Frozen cycles had a higher fertilization rates than fresh cycles,however,a lower high quality embryo rate was got in frozen cycles. 2) 11 patients accepted donation vitrified oocytes,There were statistically differences of both fertilization and high quality embryos rates between the two groups.3) Thirteen patients thawed oocytes under 37℃circumstance, and twelve patients thawed under 20℃.The survival,fertilization and implantation rates increased in 37℃group.There was a statistical difference of fertilization rate between the two groups.Conclusions:Human occytes cryopreservation would benefit infertile patients both through self-oocyte cryopreservation and donated-oocyte cyopreservation.Thawing at 37℃could preserve oocyte develpement competence than room temperature.The development and modification of human oocytes vitrification method would bring it into routine reproductive clinical work,and increases accumulative pregnancy rate.