The Influence Of Different Buffer Media And Developmental Stage On Human Blastocyst Vitrification

BackgoundDuring in vitro fertilization/Intracytoplasmic sperm injection (IVF/ICSI) treatments, we usually select high-quality cleavage-stage embryos to transfer. It is difficult to predict The developmental potential of day 2 or day 3 embryos. However, blastocyst culture and transplantation may contribute to select the developmental potential embryos. Blastocyst stage conforms to the conditions of female reproductive physiology. For high embryo implantation rate, blastocyst transplantation can reduce the number of embryo for transplantation, enhance the pregnancy rate, and reduce the multiple pregnancy rates. Many benefits of blastocyst culture and transplantation promote the rapid development of blastocyst culture techniques. Especially with the advent of sequential media, human blastocyst formation rate and quality have been greatly improved, and blastocyst culture has become relatively easy. The wide spread of blastocyst culture and transplantation technology needs to improve the blastocyst freezing technology for protection. Because of the advantages of its fast, efficient, and easy to operate, vitrification technology is developing rapidly, and has become the main method of frozen blastocysts. In the profess of blastocyst vitrification, there are still some problem to affect the freezing effect in the frozen period, frozen solution preparation and other aspects, so they need further research and standardization.Objectives1. We try to establish a more stable cryopreservation system by comparison of effects of different buffer media on blastocyst vitrification;2. This study compares the effects of different developmental stages on human blastocyst vitrification and we try to find a more appropriate blastocyst cryopreservation period.MethodsFrom January 2011 to February 2012 in the Second Affiliated Hospital of Zhengzhou University, Center for Reproductive in IVF / ICSI-ET 1629 fresh discarded embryos from 542 couples were collected. These embryos were cultured by using a sequential microdrop culture and formed 422 blastocysts. The study had been approved by the hospital ethics committee. Each patient signed informed consent form, and agreed with the remaining embryos for scientific research.The experiment was divided into two parts:the first part compared the effects of PBS and Hepes two different buffer media prepared for the vitrification solution and recovery solution, the effects of vitrification and recovery of 78 early blastocysts by PBS buffer medium prepared for frozen solution and recovery solution and of 96 early blastocysts by Hepes buffer medium prepared frozen solution and recovery solution were compared; the second part compared the vitrification effects of the different developmental stages of blastocysts, after vitrification and recovery of 96 early blastocysts,86 blastocyst stage blastocysts and 82 expansion of blastocysts by Hepes buffer medium prepared for the frozen solution and recovery solution, the survival rates and the hatching rates of each stage of blastocysts were compared; in the same period,80 fresh blastocysts were in each control group.All data were dealt with SPSS 17.0 statistical package byχ2 test. a=0.05 is considered as test criterion for statistical analysis. P<0.05 were considered to be significant.Results1. The blastocyst culture and the blastocyst formation rates of embryos of different sources 1629 fresh cycles discarded embryos, using a sequential micro-drop culture, formed 422 blastocysts.389 2PN(2 pronucleus) embryos formed 76 blastocysts, and the blastocyst formation rate was 19.5%; 380 0PN(No pronucleus) embryos formed 108 blastocysts, and the blastocyst formation rate was 28.4%; 446 1PN(Single pronucleus) embryos formed 173 blastocysts, and the blastocyst formation rate was 38.8%; 414 3PN (3 pronucleus)embryos formed 65 blastocysts, and the blastocyst formation rate was 15.7%. The blastocyst formation rates of 1PN and OPN embryos were significantly higher than those of 3PN and 2PN low quality embryos. The blastocyst formation rate of 1PN embryos was higher than that of OPN embryos, and the differences were statistically significant (P<0.05). Embryos of different sources are randomly distributed in different blastocyst stage, and the difference was no statistically significant (P>0.05).2. Different buffer media on blastocyst survival rate and hatching rateThe survival rate after vitrification and recovery were 65.1% and 77.1%, respectively, for early blastocyst in Phosphate Buffered Saline (PBS Buffer) solution and HEPES buffer solution. The difference was statistically significant (P<0.05). After vitrification and cryopreservation, the hatching rate of the survived blastocyst in PBS group was significantly lower than those in Hepes group andcontrol group, and the differences were statistically significant(54.2%vs72.9%; 54.2%vs81.3%, P<0.05). However, there was no significant difference between Hepes group and control group in hatching rates of blastocysts (P>0.05).3. Comparison of the survival rates and hatching rates of blastocysts after cryopreservation in three different developmental stagesThe survival rates after vitrification and recovery were 77.1%,62.8% and 48.9 % respectively, for early blastocyst, blastocyst stage blastocysts and expansion blastocysts. The blastocyst survival rates of early blastocysts and blastocyst stage blastocysts were significantly higher than that of the expansion blastocysts, and the differences were statistically significant (P<0.05). However, there was no statistically significant difference between early blastocyst compared with blastocyst stage blastocysts in survival rates.After recovery continued to foster the survival blastocys and observed the hatching rate. The survival rates after vitrification and recovery were 12.9%,60.0% and 81.3% respectively, for early blastocyst, blastocyst stage blastocysts, expansion blastocysts, and control group, and there were no statistically significant differences (P>0.05) between blastocyst stage blastocysts group, and early blastocyst group or expansion blastocysts. The hatching rate of the early blastocysts was significantly higher than that of the expansion blastocysts, and the difference was statistically significant (P<0.05). There was no significant difference in hatching rate between early blastocysts group and control group. But the hatching rates of the blastocyst stage and expansion blastocysts were significantly lower than that of control group, and the differences were statistically significant (P<0.05)Conclusions1 Low D2-D3 morphology score 2PN embryos should be cultured to D6, and then decided to transplantation, cryopreservation, or abandon.2 OPN and 1PN embryos have good development potential, and you can consider using them in condition without normal fertilization embryos.3 Hepes buffer may be more suitable than PBS buffer for the preparation of vitrification and recovery solution.4 Early blastocysts might be more suitable for vitrification.