Study On Effect Of Vitrification On Preimplantation Developmental Competence Of Murine Embryos

Assisted Reproductive Technology (ART) has been one of the important subfertility treatments. Embryos cryopreservation technology has become a necessary part of ART, allowing long term storage of valuable human embryos. Vitrification is a new method which was first described by Rall and Fahy (Rall and Fahy, 1985). This technology is drawing greater attention as it is simpler, faster and less expensive. Vitrification applies high concentration of cryoprotectants and rapid cooling rates as the embryos are plunged directly into liquid nitrogen, thus eliminating both intracellular and extracellular ice crystal formation which provides the embryo with greater protection from cryoinjury. Successful vitrification requires the combination of very rapid cooling rates and a high concentration of cryoprotective agents, which may cause osmotic and cytotoxic damage to the embryos. Therefore, much care must be taken to limit the exposure time to cryoprotectants and to establish their lowest effective concentration.Although this technology has been in existence for over two decades, it still yields variable results. The"universal"vitrification protocol has yet to be defined for many variables including embryo stage and quality, cooling and warming rates and culture conditions. Theoretically, vitrification could be applied to embryos at all preimplantation stages. But it is yet unknown which particular embryonic developmental stage would tolerate the vitrification process better and yield the highest survival rate and best developmental competence after thawing. It must be noted that different embryonic stages are differently affected by the same cryopreservation procedure, due to differences in blastomere volume and embryonic metabolism. The main research focuses on the blastocyst and oocyte vitrification at present mainly. Blastocyst stage embryos are frozen more successfully than early cleavage or pronuclear stage embryos, possibly since they have already surpassed embryonic genome activation. The high concentration of cryoprotectants used may compromise the later development of embryos, including their ability to implant, due to injuries or anomalies that may only be detected at an ultrastructural level. In light of this, it is important for researchers to achieve more consistent results from existing protocols and, thereby, to establish a standardized vitrification protocol that can be applied for cryopreservation of different developmental stages. Vitrification could eventually replace conventional slow freezing methods, due to its relative ease and superior results for preventing cryoinjury. Part I Effect of cryotop vitrification on preimplantation developmental competence of murine morula and blastocyst stage embryosObjectiveVitrification is an effective cryopreservation technique for mammalian embryos. Nevertheless, it is unclear which embryonic stage is most suited for vitrification. This study compared the effects of cryotop vitrification on the developmental competence of murine morula and blastocyst stage embryos.MethodsEmbryos at morula or at blastocyst were vitrified using two-step method. Embryos were first pretreated with 7.5% EG + 7.5% DMSO for 10 min, and then placed into 15% EG + 15% DMSO + 0.5 mol/L sucrose for 30 to 60 sec. They were plunged into liquid nitrogen. Embryos were warmed and diluted using 1.0mol/L, 0.5mol/L sucrose and 0mol/L sucrose. After vitrification and warming, the mouse embryonic developmental capacity was morphologically evaluated till to hatched blastocyst stage. Furthermore, trophectoderm (TE) and inner cell mass (ICM) cell numbers were compared in two hatched blastocyst groups derived from vitrified morula and blastocyst, respectively.ResultVitrified embryos at morula or at blastocyst were morphologically normal after thawing. The post-vitrification survival rate for morula was 95.4% (186/195) and 96.5% (195/202) for blastocyst, which was not significantly different (P>0.05). The blastocyst formation rate was significantly lower for the vitrified morula (90.3%) than non-vitrified control group (98.4%) ( P<0.05). The hatching rate was similar between vitrified morula (79.6%) and the vitrified blastocyst (81.0%) groups. When further development to fully hatched blastocyst stage was compared, fully hatched blastocyst derived from vitrified morula had significantly higher cell counts for both ICM and TE lineage than hatched blastocyst derived from vitrified blastocyst (P<0.001). Typically, TE cell counts in hatched blastocysts derived from vitrified blastocysts are substantially lower than vitrified morula group. Hence, it appears that the TE lineage is retarded upon vitrification at the blastocyst stage.ConclusionCryotop vitrification of mouse embryos at the morula stage rather than blastocyst stage would thus ensure a higher degree of post-thaw developmental competence.Part II Vitrification of murine embryos at 2-cell, 4-cell, 8-cell stage by Cryotop methodObjectiveWe compared the effects of cryotop vitrification on embryonic developmental competence of 2-cell, 4-cell, 8-cell stage murine embryos in vitro.MethodsIn this experiment, 2-cell, 4-cell, 8-cell stage embryos were cryopreserved by vitrification with two-step methods. After vitrification and thawing, the survival of mouse embryos was assessed by their morphology, their ability to develop to blastocysts and their ability to leave the zone pellucida (hatching) in vitro culture. Additionally, TE and ICM cell numbers were compared in hatched blastocyst from vitrified three groups, respectively. ResultVitrified embryos at 2-cell, 4-cell and 8-cell stage were morphologically normal after thawing. The post-vitrification survival rate for cleavage embryos was 96.7% (610/631) and there were no difference among 2-cell stage (96.1%), 4-cell stage (96.8%) and 8-cell stage (97.1%) respectively (P>0.05). The developmental rate of vitrified 2-cell embryos to blastocyst (69.4%) and hatched blastocyst (52.6%) was significantly lower than that from control group and 8-cell stage (P<0.05). There were some embryos appeared development block even their cellular integrity and morphology normal. These block embryos continued to culture 72 hours later, the embryonic blastomere morphology were still intact and remained in the 2-cell stage. In vitrified 4-cell group, the blastocyst formation rate (93.3%) was similar to 8-cell group (91.2%), but the hatching rate (60.0%) was significantly lower than that of non-vitrified control group (80.3%) and 8-cell stage embryo (78.4%)(P<0.05). When further development to the fully hatched blastocyst stage was compared, hatched blastocysts derived from vitrified 2-cell, 4-cell, 8-cell embryos had significantly lower cell counts both in ICM and TE than those from fresh blastocysts (P<0.05). Among vitrified 2-cell, 4-cell, 8-cell groups, there were no significant difference between ICM and TE (P>0.05).ConclusionCryotop vitrification is suitable for the cryopreservation of mice embryos from 2-cell stage, 4-cell stage, 8-cell stage embryos without a significant loss of survival. Vitrification influenced adversely the rate of cleavage and preimplantion development of 2-cell embryos. Embryos at the 8-cell stage had the best tolerance for cryopreservation in the present study. Part III Study on Cryotop vitrification of human blastocystsObjectiveThe purpose of this study was to apply the Cryotop vitrification to human blastocysts.MethodsAfter embryo transfer in IVF cycles, surplus embryos were cultured to D5 or D6 till expanded blastocyst stage, and the blastocysts were cryopreserved by vitrification using Cryotop tools. Before vitrification, the artificial shrinkage was induced in expanded blastocysts. Primary outcome measures after thawing were the following: blastocyst recovery and survival; cryotransfer cancellation; and the implantation, pregnancy (PR), and ongoing-pregnancy rates.ResultOf 142 blastocysts vitrified from 79 patients, 116 survived (81.7%) after thawing. 57 of them were hatched (49.1%) at the time of transfer. Nine patients cancelled trsnsfer for blastocyst degenerated. During the 70 patients, the pregnancy rate was 35.7% (25/70), and the implantation rate was 29.3% (34/116). Three patients delivered 3 health infants, 18 pregnancies are ongoing and four ended in miscarriage.ConclusionBoth vitrification using Cryotop and artifical shrinkage are useful technique for cryopreservation of human blastocysts because of high survival rate, clinical pregnant rate and implantation rate.