【作者】 仝丽丽；

【导师】 谭丽；

【作者基本信息】 郑州大学 ， 妇产科学， 2012， 硕士

【摘要】 背景在体外受精/卵细胞浆内单精子注射(IVF/ICSI)治疗中,一般选择受精后第2天或第3天的卵裂期优质胚胎移植。这个时期进行移植很难预料胚胎的继续发育潜力,囊胚培养移植能筛选出发育潜能好的胚胎,且符合女性生殖生理条件,由于胚胎种植率高,可以减少胚胎移植数,因此,既可以提高妊娠率又可以降低多胎妊娠率。囊胚培养移植的诸多优势,推动了囊胚培养技术的迅速发展,尤其是序贯培养基的问世,使人类囊胚形成率及质量得到很大提升,囊胚培养变得相对容易。而囊胚培养移植技术的广泛普及还需要完善的囊胚冷冻技术作保障。玻璃化冷冻因其快捷、高效、操作方便等优点,已经成为冷冻囊胚的主要方法。囊胚玻璃化冷冻技术环节中,冷冻溶液、冷冻时期等方面仍存在一些影响冷冻效果的问题,还需要进一步的研究,进一步规范化。目的1.比较采用不同缓冲介质对囊胚玻璃化冷冻效果的影响,尝试建立一种更稳定的冷冻复苏体系。2.比较人类不同发育阶段囊胚的玻璃化冷冻效果,旨在寻求更适合冻存囊胚的时期。方法收集2011年1月～2012年2月在郑州大学第二附属医院生殖中心进行体外受精-胚胎移植/单精子胞浆内注射-胚胎移植(IVF/ICSI-ET)治疗的542对夫妇的新鲜废弃胚胎,总数为1629枚,将这些胚胎采用序贯法微滴培养,共形成422枚囊胚。本研究已获得本院伦理委员会批准,且每位患者均已签过剩余胚胎用于科学研究的知情同意书。本实验包括两部分：第一部分比较PBS和Hepes两种不同缓冲介质配制玻璃化冷冻液和复苏液的效果,比较用PBS缓冲介质配制的冷冻液和复苏液玻璃化冷冻和复苏78枚早期囊胚与用Hepes缓冲介质配制的冷冻液和复苏液玻璃化冷冻和复苏96枚早期囊胚的效果；第二部分比较不同发育阶段的囊胚对玻璃化冷冻效果的影响,采用Hepes缓冲介质配制冷冻液和复苏液玻璃化冷冻和复苏96枚早期囊胚、86枚囊胚期囊胚和82枚扩张期囊胚,比较各阶段囊胚复苏后的存活率和囊胚孵出率；同时以同期培养的80枚未冷冻囊胚为对照组。采用统计软件SPSS 17.0中的统计分析方法进行数据处理,率的比较采用χ2检验。检验标准取α=0.05,P<0.05为有显著性差异。结果1.不同来源胚胎的囊胚培养及囊胚形成率1629枚新鲜周期废弃胚胎,采用序贯法微滴培养,共形成422枚囊胚,其中389枚2PN(2原核)来源胚胎形成囊胚76枚,囊胚形成率为19.5%；380枚OPN(无原核)来源胚胎形成囊胚108枚,囊胚形成率为28.4%;446枚1PN(单原核)来源胚胎形成囊胚173枚,囊胚形成率为38.8%;414枚3PN(3原核)来源胚胎形成囊胚65枚,囊胚形成率为15.7%。1PN和OPN胚胎的囊胚形成率明显高于3PN和低质量2PN胚胎,1PN胚胎高于OPN胚胎,差异有统计学意义(P<0.05)；不同来源的胚胎随机分布于囊胚的不同阶段,差异无统计学意义(P>0.05)。2.不同缓冲介质对囊胚玻璃化冷冻效果的影响应用缓冲介质PBS配制冷冻溶液与复苏溶液,玻璃化冷冻早期囊胚复苏后的存活率为65.1%,低于Hepes组的77.1%,差异有统计学意义(P<0.05)。玻璃化冷冻复苏后,PBS组存活囊胚的孵出率显著低于Hepes组和对照组,差异有统计学意义(54.2%vs72.9%；54.2%vs81.3%,P<0.05),Hepes组与对照组的囊胚孵出率差异无统计学意义(P>0.05)。3.三组不同发育阶段囊胚玻璃化冷冻复苏后存活率与孵出率的比较玻璃化冷冻不同发育阶段囊胚,复苏后早期囊胚、囊胚期囊胚和扩张期囊胚的存活率分别为77.1%、62.8%和48.9%,早期囊胚、囊胚期囊胚的存活率明显高于扩张期囊胚,差异有统计学意义(P<0.05)；早期囊胚与囊胚期囊胚比较,差异无统计学意义(P>0.05)。囊胚复苏存活后继续培养观察,早期囊胚、囊胚期囊胚、扩张期囊胚和对照组的孵出率分别72.9%、60.0%、47.5%和81.3%,早期囊胚与囊胚期囊胚比较,囊胚期囊胚与扩张期囊胚比较,差异无统计学意义(P>0.05)；但早期囊胚明显高于扩张期囊胚,差异有统计学意义(P<0.05)。早期囊胚的孵出率与对照组比较,差异无统计学意义(P>0.05),而囊胚期囊胚和扩张期囊胚的孵出率明显低于对照组,差异有统计学意义(P<0.05)。结论1、D2-D3形态学上评分较低的2PN胚胎有一定的发育潜能,应该培养至D6,再决定移植、冷冻保存或废弃。2、OPN和1PN来源的胚胎有一定的发育潜能,无下常受精胚胎选择时,可以考虑利用。3、Hepes较PBS更适于配制玻璃化冷冻液与复苏液。4、应该选择早期囊胚进行玻璃化冷冻保存。更多还原

【Abstract】 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.更多还原