【作者】 凌秀凤；

【导师】 王心如；

【作者基本信息】 南京医科大学 ， 卫生毒理学， 2010， 博士

【摘要】 随着辅助生育技术(assisted reproductive technology,ART)的发展,胚胎冷冻技术已经成为其发展不可分割的一部分。玻璃化冷冻是一种比较新的冷冻保存方法,是在高浓度的冷冻保护剂的存在下,使细胞快速冷冻,形成玻璃态,避免了细胞内外冰晶形成对细胞的破坏作用。由于其大大简化了冷冻程序,缩短了冷冻时间,不需要昂贵的冷冻设备,提高了冷冻效果,具有广阔的应用前景。影响玻璃化冷冻的因素很多,首先冷冻保护剂的毒性是玻璃化冷冻的最大障碍,为了降低其毒性作用,人们不断改进冷冻技术和冷冻载体,提高冷冻速率。其次胚胎本身的原因也逐渐被人们所重视,理论上讲,玻璃化冷冻可以用于种植前胚胎的任何阶段,但由于胚胎从1细胞到囊胚,细胞体表面积、细胞膜成分及通透性不断发生改变,冷冻效果有明显差异。目前研究主要集中在囊胚冷冻,但在许多生殖中心,通常选择卵裂期胚胎移植,而卵裂期胚胎的冷冻是玻璃化冷冻的薄弱环节。所以尽管玻璃化冷冻已获得多例临床妊娠和分娩,但因其冷冻效果并不稳定,各项技术不成熟,现仍处于实验阶段,各家报道的胚胎复苏率差异很大。提高玻璃化冷冻后胚胎复苏率的稳定性,建立公认的玻璃化冷冻方案,并研究玻璃化冷冻对各个发育阶段胚胎的影响,是当前玻璃化冷冻急需了解的问题。第一部分Cryotop玻璃化冷冻对小鼠桑椹胚和囊胚发育潜能的影响目的用Cryotop玻璃化冷冻桑椹胚和囊胚发育影响的研究尚属空白,我们以小鼠的桑椹胚和囊胚作为研究对象,探讨该方法对冻融后桑椹胚和囊胚存活率及胚胎继续发育潜能的影响,为临床医学建立可靠的实验室依据。方法1. ICR小鼠常规促排卵,体内受精,HCG后75~80小时和85~96小时分别取桑椹胚和囊胚进行玻璃化冷冻。2.玻璃化冷冻与复苏:采用两步法,预平衡液为7.5% (v/v)乙二醇(EG)、7.5%(v/v)二甲基亚砜(DMSO),胚胎平衡10分钟;玻璃化冷冻液为:15% (v/v) EG、15%(v/v)DMSO、0.5M蔗糖(sucrose)溶液,胚胎作用控制在1分钟内。胚胎复苏采用1.0M、0.5M、0M sucrose复苏液,梯度稀释逐步洗脱冷冻保护剂。3.冻融后,首先是根据胚胎解冻后的形态完整性,判断胚胎存活情况,用存活率表示;其次是胚胎在体外的继续发育潜能,用囊胚形成率和孵出率表示。最后通过双重荧光染色评价囊胚各个胚层的细胞数,间接反映囊胚的质量。结果1.玻璃化冷冻对桑椹胚和囊胚组胚胎形态学无影响,两组存活率无差异(95.4% vs 96.5%,P>0.05)。胚胎体外继续培养显示冻融后的桑椹胚组囊胚形成率为90.3%,低于未冷冻组囊胚形成率98.4%(P<0.05),冻融后的桑椹期胚胎和囊胚期胚胎的囊胚孵出率无差异(79.6% vs 81.0%,P>0.05)。2.第五天孵出囊胚细胞进行荧光染色,冻融后的桑椹胚组TCN(total cell numbers)、TE(trophectoderm)和ICM(inner cell mass)分别为:111.53±25.12,73.00±19.72,38.53±11.29,ICM/TE比值:0.56±0.19;冻融囊胚组TCN、TE和ICM分别为87.23±13.50、54.13±10.60、33.10±5.00,ICM/TE比值:0.63±0.12,与对照组和冻融后桑椹胚组相比,囊胚组的TCN、TE和ICM明显下降,有统计学差异(P<0.05),玻璃化冷冻的两组中ICM/TE值,在冻融后桑椹胚组更接近未冷冻组(0.58±0.17)。结论用Cryotop法玻璃化冷冻桑椹胚和囊胚,冻融后两组存活率、囊胚孵出率无差异。Cryotop玻璃化冷冻对桑椹期胚胎的TCN、TE、ICM无影响,而在囊胚组的TCN、TE、ICM显著减少。两组中ICM/TE值,桑椹胚组更接近未冷冻组。说明在本实验条件下,桑椹胚期胚胎冷冻能获得更好的冷冻效果。第二部分小鼠2细胞、4细胞、8细胞胚胎玻璃化冷冻研究目的在玻璃化冷冻桑椹胚和囊胚的基础上,首次将Cryotop应用到卵裂期胚胎。实验以小鼠2细胞、4细胞和8细胞胚胎作为实验对象,对卵裂期胚胎进行玻璃化冷冻研究,探讨临床卵裂期胚胎冷冻的可能。方法1. ICR小鼠常规促排卵,体内受精,分别于HCG后38~40小时、48~50小时和60~62小时取2细胞、4细胞和8细胞胚胎行玻璃化冷冻。2.玻璃化冷冻与复苏:胚胎首先用预平衡液7.5% (v/v) EG、7.5% (v/v) DMSO平衡10分钟;然后转入15% (v/v) EG、15% (v/v) DMSO、0.5M sucrose的玻璃化冷冻液中,时间控制在30~60秒内。胚胎复苏采用1.0M、0.5M、0M sucrose复苏液,梯度稀释逐步洗脱冷冻保护剂。3.冻融后,首先是根据胚胎解冻后的形态完整性来判断胚胎存活情况,用存活率表示;其次是在体外的继续发育能力,用囊胚形成率和孵出率表示。最后通过双重荧光染色评价囊胚各个胚层的细胞数,间接反映囊胚的质量。结果1.冻融后的2细胞、4细胞和8细胞胚胎的形态学无异常;共冷冻了卵裂期胚胎631枚,存活610枚,存活率为96.7%(610/631);三组胚胎冻融后的存活率之间没有差异(P>0.05)。冻融后的三组胚胎继续培养,囊胚形成率均低于相应对照组(P<0.05)。冻融后的2细胞组胚胎的囊胚形成率为69.4%,显著低于4细胞组胚胎(93.3%)和8细胞组胚胎(91.2%)(P<0.05);冻融后2细胞期和4细胞期的囊胚孵出率分别52.6%和60.0%,两组相比无统计学差异(P>0.05),但与冻融后8细胞期的囊胚孵出率(78.4%)相比,有显著下降(P<0.05)。2.第5天孵出囊胚细胞进行荧光染色,冻融后2细胞期的TCN、TE和ICM分别为:85.40±17.02,54.97±10.48,31.10±8.79,ICM/TE比值:0.57±0.13;4细胞组TCN、TE和ICM分别为81.00±13.02、51.40±10.06、29.60±7.06,ICM/TE比值:0.59±0.16,8细胞组的TCN、TE和ICM分别为84.94±13.75、53.44±10.18、31.50±7.23,ICM/TE比值:0.60±0.16,分别与相应对照组相比,三组TCN、TE和ICM均显著下降,有统计学差异(P<0.05),三组之间无统计学差异(P>0.05)。结论Cryotop法玻璃化冷冻三组卵裂期胚胎,冻融后的胚胎存活率三组之间无差异,但2细胞期胚胎冷冻导致冻融后胚胎出现明显的发育阻滞,之后的囊胚形成率、孵出率显著下降;冻融后4细胞组与8细胞组的胚胎相比,囊胚形成率没有下降,但囊胚孵出率显著下降;8细胞组囊胚孵出率与对照组相比,无显著差异。冻融后三组胚胎的孵出囊胚各个胚层细胞数均显著下降,说明玻璃化冷冻对胚胎发育潜能有不良影响。三组中,2细胞期胚胎对冷冻损伤最敏感,8细胞期胚胎更耐受玻璃化冷冻损伤,更适合玻璃化冷冻。第三部分玻璃化冷冻人类囊胚的临床应用探讨目的将前期玻璃化冷冻技术应用到临床,利用Cryotop法,结合人工辅助脱水技术,探讨玻璃化冷冻在临床生殖医学中人类囊胚冷冻的应用效果。方法1.将接受体外受精-胚胎移植患者剩余胚胎培养到扩张期囊胚。2.将扩张期囊胚进行针刺人工辅助脱水。3.将脱水后胚胎进行Cryotop玻璃化冷冻,根据临床需要,解冻囊胚,并分析囊胚复苏后的存活率、妊娠率及种植率等指标。结果玻璃化法冷冻囊胚共进行224个周期,冷冻了543枚囊胚;玻璃化冷冻复苏79例患者、142枚囊胚,存活116枚囊胚,存活率为81.7 %;有9例患者因冷冻后囊胚退化取消移植,其余70例患者均有囊胚移植,移植囊胚116枚,平均每人移植1.7枚囊胚;获得临床妊娠25例,其中单胎16例、双胎9例,着床了34枚囊胚;移植周期临床妊娠率为35.7%,种植率为29.3%。3例足月分娩,新生儿健康,18例继续妊娠,4例流产,流产率为16 %。结论使用Cryotop玻璃化冷冻法适于人类囊胚的冷冻保存,结合人工皱缩,获得存活率为81.7%、妊娠率为35.7%、种植率为29.3%,流产率没有明显提高,妊娠率达到国内外先进水平,在临床中值得应用和推广。更多还原

【Abstract】 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.更多还原