玻璃化冷冻对小鼠种植前不同发育时期胚胎发育潜能的影响
|
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。
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 embryos
Objective
Vitrification 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.
Methods
Embryos 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.
Result
Vitrified 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.
Conclusion
Cryotop 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 method
Objective
We compared the effects of cryotop vitrification on embryonic developmental competence of 2-cell, 4-cell, 8-cell stage murine embryos in vitro.
Methods
In 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.
Result
Vitrified 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).
Conclusion
Cryotop 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 blastocysts
Objective
The purpose of this study was to apply the Cryotop vitrification to human blastocysts.
Methods
After 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.
Result
Of 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.
Conclusion
Both 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.
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。
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 embryos
Objective
Vitrification 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.
Methods
Embryos 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.
Result
Vitrified 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.
Conclusion
Cryotop 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 method
Objective
We compared the effects of cryotop vitrification on embryonic developmental competence of 2-cell, 4-cell, 8-cell stage murine embryos in vitro.
Methods
In 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.
Result
Vitrified 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).
Conclusion
Cryotop 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 blastocysts
Objective
The purpose of this study was to apply the Cryotop vitrification to human blastocysts.
Methods
After 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.
Result
Of 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.
Conclusion
Both 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.
引文
1. WhittinghamDG, LeiboSP, MazurP. Survival of mice embryos frozen to -196℃and - 269℃. Science, 1972, 178: 411一414.
2. Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature, 1983, 305 (5936): 707-709.
3. Takeda T, Elsden RP, Seidel GE. Cryopreservation of mouse embryos by direct plunging into liquid nitrogen. Theriogenology, 1984, 21: 266~268.
4. Rall WF, Fahy GM: Ice-free cryopreservation of mice embryos at -196℃by vitrification. Nature, 1985, 313: 573–575.
5. T. Mukaida, S. Wada, K. Takahashi, P.B. Pedro, T.Z. An and M. Kasai. Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mice embryos. Hum Reprod, 1998, 13 (10): 2874-2879.
6. Youssry M, Ozmen B, Zohni K, Diedrich K, Al-Hasani S. Current aspects of blastocyst cryopreservation. Reprod Biomed Online, 2008, 16 (2): 311-320.
7. Stehlik E, Stehlik J, Katayama KP, Kuwayama M, Jambor V, Brohammer R, Kato O. Vitrification demonstrates significant improvement versus slow freezing of human blastocysts. Reprod Biomed Online, 2005, 11 (1): 53- 57.
8. Balaban, B. Urman, B. Ata1, A. Isiklar, M.G. Larman, R. Hamilton and D.K. Gardner. A randomized controlled study of human Day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation. Human Reproduction, 2008, (9): 1976-1982.
9. Roser Morató, Dolors Izquierdo, Maria Teresa Paramio, Teresa Mogas.Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: Effects on spindle and chromosome configuration and embryo development. Cryobiology, 2008, 57: 137–141.
10. Shaw JM, Kola I, MacFarlane DR, Trounson AO. An association between chromosomal abnormalities in rapidly frozen 2-cell mice embryos and the ice forming properties of the cryoprotective solution.J Reprod Fertil, 1991, 91: 9–18.
11. Larman MG, Sheehan CB, Gardner DK. Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mice oocytes. Reproduction, 2006, 131: 53–61.
12. Iwatani M, Ikegami K, Kremenska Y, Hattori N, Tanaka S, Yagi S, Shiota K. Dimethyl sulfoxide has an impact on epigenetic profile in mice embryoid body. Stem Cells, 2006, 24: 2549–2556.
13. Kartberg AJ, Hambiliki F, Arvidsson T, Stavreus-Evers A, Svalander P. Vitrification with DMSO protects embryo membrane integrity better than solutions without DMSO. Reprod Biomed Online, 2008, 17(3): 378-384.
14. Kasai M, Komi JH, Takakama A etal. A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution without appreciable loss of viability. Reprod Fert, 1990, 89(1): 92.
15. Cuello, Sanchez-Osorio, Almina, Gil, Perals, Lucas, Roca, Vazquez. MartinezEffect of the cryoprotectant concentration on the in vitro embryo development and cell proliferation of OPS-vitrified porcine blastocysts. Cryobiology, 2008, 56: 189–194.
16. Kalliopi E, Loutradi, M.Med.Sc., Efstratios M. Kolibianakis, Christos A. Venetis, M.Sc., Evangelos G. Papanikolaou, George Pados, Ioannis Bontis, and Basil C. Tarlatzis. Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertility and Sterility, 2008, 90(1): 187-193.
17. E.Go′mez, A.Rodr?′guez, M.Mun?oz, J.N.Caaman?o, C.O.Hidalgo, E.Mora′n, N. Facal, C. Diez. Serum free embryo culture medium improves in vitro survival ofbovine blastocysts to vitrification. Theriogenology, 2008, 69: 1013–1021.
18. Chi-Gu Kim Hwanyul Yong, Gene LEE and Jaejin Cho. Effect of the Polyvinylpyrrolidone Concentration of Cryoprotectant on Mice Embryo Development and Production of Pups: 7.5% of PVP is Beneficial for In Vitro and In Vivo Development of Frozen-Thawed Mice Embryos. Journal of Reproduction and Development, 2008, 54 (4): 250-253.
19. Miyake T, Kasai M, Zhu SE, et al. Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycole based solution by a simple method. Theriogenology, 1993, 40: 121–134.
20. Han MS, Niwa K, Kasai MTheriogenology. Vitrification of rat embryos at various developmental stages. Theriogenology, 2003, 59 (8): 1851-1863.
21. Y. Yokota, S. Sato, M. Yokota, Y. Ishikawa, M. Makita, T. Asada and Y. Araki. Successful pregnancy following blastocyst vitrification: Case report. Human Reproduction, 2000, (8): 1802-1803.
22. Imam El-Danasouri, Helmy Selman Vitrification versus conventional cryopreservation technique. Middle East Fertility Society Journal, 2005, 10 (3): 205-206.
23. Valadimir Isachenko, Frank Nawroth, Salvatore Dessole and Hansvanderven developmental rate and ultrastructure of vitrified human pronuclear oocytes after step-wise versus direct rehydration. Human Reproduction, 2004, 19(3): 660-665.
24. Hyon-Jin Cho, Weon-Young Son, San-Hyun Yoon, Suk-Won Lee and Jin-Ho Lim. An improved protocol for dilution of cryoprotectants from vitrified human blastocysts. Hum Reprod, 2002, 17 (9): 2419-2422.
25. Papis K, Shimizu M and Izaike Y (2000) Factors affecting the survivability of bovine oocytes vitrified in droplets. Theriogenology, 15: 651–658.
26. Matsumoto H., Jiang JY, Tanaka T., Sasada H., and E. Sato. Vitrification oflarge quantities of immature bovine oocytes using nylon mesh. Cryobiology, 2001, 42 (2): 139-144.
27. Se-Pill Park, Eun Young Kim, Deok Im Kim, Noh Hyung Park, Yu Seok Won, San Hyun Yoon, Kil Saeng Chung and Jin Ho Lim.Simple , efficient and successful vitrification of bovine blastocysts using electron microscopic grid. Hum Reprod, 1999, 14 (11): 2838-2843.
28. Weon-Young Son, San-Hyun Yoon, Hye-Jin Yoon, Sang-Min Lee and Jin-Ho Lim. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum Reprod, 2003, 18 (1): 137-139.
29. P. Vanderzwalmen, G. Bertin, Ch. Debauche, V. Standaert, N. Bollen, E.van Roosendaal, M. Vandervorst, R. Schoysman and N. Zech. Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing. Human Reproduction, 2003, 18 (7): 1504-1511.
30. J. liebermann and M.J.Tucker. Effect of carrier system on the yield of human oocytes and embryos as assessed by survival and developmentqal potential after vitrifaction. Reproduction, 2002, 124: 483-489.
31. Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, Callesen H. Open pulled straw (OPS) vitrification: A new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998, 51: 53-58.
32. Shee-Uan Chen, Yih-Ron Lien, Hsin-Fu Chen, Kuang-Han Chao, Hong-Nerng Ho and Yu-Shih Yang. Open pulled straws for vitrification of mature mice oocytes preserve patterns of meiotic spindles and chromosomes better than conventional straws. Hum Reprod, 2000, 15 (12): 2598-2603.
33. Lane M, Schoolcraft WB, Gardner DK. Vitrification of mice and human blastocysts using a novel cryoloop container less technique. Fertil Steril, 1999,72 (6): 1073-1078.
34. Mukaida T, Nakamura S, Tomiyama T, Wada S, Kasai, Takahashi K. Successful birth after transfer of vitrified human blastocyst using a cryoloop container-less technique. Fertil Steril, 2001, (76): 618-620.
35. T. Mukaida, S. Nakamura, T. Tomiyama, S. Wada, C. Oka, M. Kasai and K. Takahashi. Vitrification of human boastocysts using cryoloops: clinical outcomes of 223 cycles. Hum Reprod, 2003, 18 (2): 384-391.
36. Kenichiro Hiraoka1, Kaori Hiraoka, Masayuki Kinutani and Kazuo Kinutani. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Human Reproduction, 2004, 19(12): 2884–2888.
37. Hochi S, Terao T, Kamei M, Kato M, Hirabayashi M, Hirao M .Successful vitrification of pronuclear-stage rabbit zygotes by minimum volume cooling procedure Theriogenology, 2004, 15, 61 (2-3): 267-275.
38. MoratóR, Izquierdo D, Paramio MT, Mogas T. Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on spindle and chromosome configuration and embryo development. Cryobiology, 2008, 57 (2): 137-141.
39. El-Gayar M, Holtz, W. Technical note: vitrification of goat embryos by the open pulled-straw method [J] J. Anim Sci, 2001, 79: 2436-2438.
40. Kuwayama M, Vajta G, Ieda S, Kato O. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reprod Biomed Online, 2005, 11(5): 608-614.
41. Prudenico B.Fedro, Elji Yokoyama, Shi EnZhu. Permeability of mice oocytes and embryos at various developmental stages to five cryoprotectants. Jouranl of Reproduction and Development, 2005, 51: 235-246.
42. Zhao XH, Yue TF, Sui XL. Cryopreservation of mice morulas and blastocysts by vitrification. Zhonghua Fu Chan Ke Za Zhi, 2004, 39 (8): 526-528.
43. Zhou GB, Hou YP, Jin F, Yang QE, Yang ZQ, Quan GB, Tan HM, Zhu SE. Vitrification of mice embryos at various stages by open-pulled staws (OPS) method. Anim Biotechnol, 2005, 16 (2): 153-163.
44. Haibin Wang and Sudhansu K. Dey, Roadmap to embryo implantation: clues from mice models (review). Nature, 2006, 7: 185-199.
45. Hardy K, Handyside AH, Winston RML. The human blastocyst: cell number, death and alloation during late preimplantation development invitro. Development, 1989, 107: 597-604.
46. Chun-Chia Huang, Tsung-Hsien Lee, Shee-Uan Chen, Hsiu-Hui Chen, Tzu-Chun Cheng, Chung-Hsien Liu, Yu-Shih Yang and Maw-Sheng Lee Successful pregnancy following blastocyst cryopreservation using super-cooling ultra-rapid vitrification. Human Reproduction, 2005, 20(1): 122-128.
47. Jun Tao, Robert Tamis and Katharine Fink. Animal Experimentation: Cryopreservation of Mice Embryos at Morula/Compact StageJournal of Assisted. Reproduction and Genetics, 2001, 18(4): 237-245.
48. Vanderzwalmen P, Bertin G, Debauche Ch, Standaert V, van Roosendaal E, Vandervorst M, Bollen N, Zech H, Mukaida T, Takahashi K, Schoysman R. Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod, 2002, 17 (3): 744-751.
49. Chen SU, Lee TH, Lien YR, Tsai YY, Chang LJ, Yang YS. Microsuction of blastocoelic fluid before vitrification increased survival and pregnancy of mice expanded blastocysts, but pretreatment with the cytoskeletal stabilizer did not increase blastocyst survival. Fertil Steril, 2005, 84: 1156-1162.
50. SonWY, Yoon SH, YoonHJ, Lee SM, LimJH. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum Reprod, 2003, 18: 137–139.
51. Campos-Chillon LF, Walker DJ, de la Torre-Sanchez JF, Seidel GE Jr.In vitro assessment of a direct transfer vitrification procedure for bovine embryos. Theriogenology, 2006, 65(6): 1200-1214.
52. Cuello C, Gil MA, Parrilla I, Tornel J, Vazquez JM, Roca J, Berthelot F, Martinat-Botte F, Martinez EA.Vitrification of porcine embryos at various developmental stages using different ultra-rapid cooling procedures. Theriogenology, 2004, 62(1-2): 353-361.
53. Vajta G. Vitrification of oocytes and embryos of domestic animals. Anim Reprod Sci, 2000, 61: 357–364.
54. Hitoshi Ushijima,Hiroki Yoshioka,Ritsuko Esaki, keiji Takahashi, Masashige Kuwayama, Takashi Nakane and Hiroshi Nagashima. Improved Survival of Vitrified in Vivo-Derived Porcine Embryos. Jouranl of Reproduction and Development, 2004, 50(4): 481-486.
55. N.Cremades, M.Sousa, J.Silva, P.Viana, S.Sousa, C.Oliveira,J.Teixeira da Silva and A.Barros.Experimental vitrification of human compacted morula and early blastocysts using fine diameter plastic micropipettes 3. Human Reproduction, 2004, 19: 300-305.
56. Abe H, Yamashita S, Satoh T, Hoshi H. Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serumfree or serum-containing media. Mol Reprod Dev, 2002, 61: 57–66.
57. Garrisi GJ, Talansky BE, Sapira V, Gordon JW, Navot D. An intact zona pellucida is not necessary for successful mouse embryo cryopreservation. FertilSteril, 1992, 57(3): 677-681.
58. Dobrinsky J R.Cryopreservation of pig embryos:adaptation of vitrification technology for embryo transfer. Reprod Suppl, 2001, 58: 325-333.
59. Yildiz C, Ottaviani P, Law N, Ayearst R, Liu L, McKerlie C. Effects of cryopreservation on sperm quality, nuclear DNA integrity, in vitro fertilization, and in vitro embryo development in the mouse. Reproduction, 2007, 133(3): 585-595.
60. Coticchio G, Bonu MA, Bianchi V, Flamigni C, Borini A. Criteria to assess human oocyte quality after cryopreservation. Reprod Biomed Online, 2005, 11(4): 421-427.
61. Khalifa T A, Rekkas C A, Lymberopoulos A G. Factors affecting chromatin stability of bovine spermatozoa. Anim Reprod Sci, 2007, 11: 112-118.
62. Damario MA, Hammitt DG, Session DR, Dumesic DA. Embryo cryopreservation at the pronuclear stage and efficient embryo use optimizes the chance for a liveborn infant from single oocyte retrieval. Fertil.Steril, 2000, 73 (4): 767-773.
63. Tong Z B, Gold L, Pfeifer K E, Dorward H, Lee E, Bondy C A, Dean J, Nelson L M. Mater, a maternal effect gene required for early embryonic development in mice.Nat Genet, 2000, 26 (3): 267-268.
64. El-Danasouri I, Selman H Successful pregnancies and deliveries after a simple Vitrification protocol for day 3 human embryos. Fertil Steril, 2001, 76 (2): 400-402.
65. Rama Raju GA, Haranath GB, Krishna KM, Prakash GJ, Madan K. Vitrification of human 8-cell embryos, a modified protocol for better pregnancy rates. Reprod Biomed Online, 2005, 11: 434–437.
66. Desai N, Blackmon H, Szeptycki J, Goldfarb J. Cryoloop vitrification of human day 3 cleavage-stage embryos: post-vitrification development, pregnancyoutcomes and live births. Reprod Biomed Online, 2007, 14: 208–213.
67. Tada N, Sato M, Amann E, Ogawa S A simple and rapid method for cryopreservation of mouse 2-cell embryos by vitrification: Beneficial effect of sucrose and raffinose on their cryosurvival rate. Theriogenology, 1993, 40(2): 333-344.
68. Mojtaba rezazadeh valojerdi, mansoureh movahedin and Ahmad hosseini. Improvement of development of vitrified two-cell mice embryos by vero cell coculture. Journal of Assisted Reproduction and Genetics, 2002, 19(1): 31-38.
69. A.Dhali, V. M. Anchamparuthy, S.P.Butler, R.E.Pearson, I. K. Mullarky, F.C.Gwazdauskas. Gene expression and development of mice zygotes following droplet vitrification. Theriogenology, 2007, (68): 1292-1298.
70. Keisuke Edashige, Satoshi Ohta, Mitsunobu Tanaka, Tatsunaga Kuwano, Delgado M. Valdez, Jr.,Takao Hara, Bo Jin,Sei-ichi Takahashi,Shinsuke Seki, Chihiro Koshimoto,and Magosaburo Kasai.The Role of Aquaporin 3 in the Movement of Water and Cryoprotectants in Mice Morulae. Biology of reproduction, 2007, 77: 365–375.
71. Keisuke Edashige, Mitsunobu Tanaka, Natsuko Ichimaru,Satoshi Ota, Ken-ichi Yazawa,Yuki Higashino, Megumi Sakamoto, Yohei Yamaji,Tatsunaga Kuwano, Delgado M. Valdez Jr., F.W. kleinhans, and magosaburo kasai. Channel-dependent permeation of water and glycerol in mice morulae. Biology of reproduction, 2006, 74: 625–632.
72. Diez C, Heyman Y, Le Bourhis D, Guyader-Joly C, Degrouard J,Renard JP. Delipidating in vitro-produced bovine zygotes: effect on further development and consequences for freezability. Theriogenology, 2001, 55: 923–936.
73. Nasr-esfahani M,Johnson M H,Aitken R J.The effect of iron and iron chelators on the in-vitro block to development of the mouse preimplantationembryo:BAT6 a new medium for improved culture of mouse embryos in vitro. Hum Reprod, 1990, 5 (8): 997-1003.
74. Pfaff RT, Liu J, Gao D, Peter AT, Li TK, Critser JK. Water and DMSO,membrane permeability characteristics of in-vivo and in-vitro derived and cultured murine oocytes and embryos. Mol Hum Reprod, 1998, 4: 51–59.
75. Barnett DK, Clayton MK, Kimura J, Bavister BD.Glucose and phosphate toxicity in hamster preimplantation embryos involves disruption of cellular organization, including distribution of active mitochondria. Mol Reprod Dev, 1997, 48 (2): 227-237.
76. Josiane Van der Elst, Youri Amerijckx1, and Andre′Van Steirteghem.Ultra-rapid freezing of mice oocytes lowers the cell number in the inner cell mass of 5 day old in-vitro cultured blastocysts. Human Reproduction, 1998, (13): 1595–1599.
77. Michelle Lane, Elizabeth A.Lyons and Barry D.Bavister.Cryopreservation reduces the ability of hamster 2-cell embryos to regulate intracellular pH. Human Reproduction, 2000, 15(2): 389-394.
78. Willingham M C.Cytochemical methods for the detection of apoptosis. J Histochem Cytochem, 1999, 47 (9): 1101-1110.
79. ReedML, LaneM, GardnerDK, Jensen NL, Thompson J. Vitrification of human blastocysts using the cryoloop method: successful clinical application and birth of offspring. J Assist Reprod Genet, 2002, 19: 304.
80. Yokota, Y. Sato S., Yokota M., Ishikawa Y., Makita M., Asada T.and Araki Y. Successful pregnancy following blastocyst vitrification: Case report Human Reproduction, 2000, 15(8):1802-1803.
81. Yokota Y, Sato S , Yokota M , et al . Birth of a healthy baby following vitrification of human blastocysts. Fertil Steril, 2001, 75 :1027-1029.
82. Takahashi K, Mukaida T, Goto T, Oka C. Perinatal outcome of blastocyst transferwith vit rification using cryoloop : a 4-year follow-up study. Fertil Steril , 2005 , 84: 88-92.
83. Mukaida T,Nakamura S, Tomiyama T, Wada S, Kasai M, Takahashi K.Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique. Fertil Steril 2001, 76(3): 618–623.
84. Reed ML, Lane M, Gardner DK, Jensen NL, Thompson J. Vitrification of human blastocysts using the cryoloop method: successful clinical application and birth of offspring. J Assist Reprod Genet, 2002, 19( 6):304-306.
85. M. Antinori, E. Licata, G. Dani, F. Cerusico, C. Versaci, S. Antinori, Cryotop vitrification of human oocytes results in high survival rate and healthy deliveries, Reprod. Biomed, 2007, 14: 72–79.
86. Kuwayama M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology, 2007, 67: 73-80.
87. Son WY, Yoon SH, Yoon HJ, Lee SM, Lim JH. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Human Reproduction, 2003, 18 (1): 137-139.
88. Motoishi. Cryopreservation of human blastocyst. Clin Embryol, 2000, 5: 6–14.
89. Hiraoka K, Kinutani M, Kinutani K. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Human Reprod, 2004, 19: 2884-2888.
90. Mukaida T, Oka C, Goto T, Takahashi K. Artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts. Hum Reprod, 2006, 21 (12): 3246-3252.
1. Rall WF, Fahy GM: Ice-free cryopreservation of mice embryos at -196℃by vitrification. Nature, 1985, 313: 573–575.
2. Mukaida, S. Wada, K. Takahashi, P.B. Pedro, T.Z. An and M. Kasai. Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mice embryos. Hum Reprod, 1998, 13(10) : 2874-2879.
3. Roser Morató, Dolors Izquierdo, Maria Teresa Paramio, Teresa Mogas. Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: Effects on spindle and chromosome configuration and embryo development. Cryobiology, 2008, 57: 137–141.
4. Shaw JM, Kola I, MacFarlane DR, Trounson AO. An association between chromosomal abnormalities in rapidly frozen 2-cell mice embryos and the ice forming properties of the cryoprotective solution. Reprod Fertil, 1991, 91: 9–18.
5. Kasai M, Komi JH, Takakamo A, Tsudera H, Sakurai T, Machida T, A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution without appreciable loss of viability. Reprod Fert, 1990, 89(1): 91-97.
6. Cho HJ, Son WY, Yoon SH, Lee SW, Lim JH. An improved protocolfor dilution of cryoprotectants from vitrified human blastocysts. HumReprod, 2002, 17 (9): 2419- 2422.
7. Ali J, Bongso A, Ratnam SS. Vitrification of human embryo. Med Sci Res, 1995, 23 (1): 539-540.
8. Kalliopi E. Loutradi, M.Med.Sc., Efstratios M. Kolibianakis, Christos A. Venetis, M.Sc., Evangelos G. Papanikolaou, George Pados, Ioannis Bontis, and Basil C. Tarlatzis.Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertility and Sterility, 2008, 90(1): 187-193.
9. Y. Yokota, S. Sato, M. Yokota, Y. Ishikawa, M. Makita, T. Asada and Y. Araki. Successful pregnancy following blastocyst vitrification: Case report Human Reproduction, 2000, 15(8): 1802-1803.
10. Imam DV, Helmy S. Successful pregnancies and deliveries after a simple vit rification protocol for 3 human embryos. Fertil Steril, 2001, 76 (2): 400-402.
11. Vanderzwalmen P, Bertin G, Debauche C, Standaert V, van Roosendaal E, Vandervorst M, Bollen N,Zech H, Mukaida T, Takahashi K, Schoysman R. Births after vitrification at morula and blastocyst stages: Effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod, 2002, 17: 744-571.
12. Shimizu M and Izaike Y Factors affecting the survivability of bovine oocytes vitrified in droplets. Theriogenology, 2002, 15: 651–658.
13. Matsumoto H., Jiang JY, Tanaka T., Sasada H., and E. Sato. Vitrification of large quantities of immature bovine oocytes using nylon mesh. Cryobiology, 2001, 42 (2): 139-144.
14. Se-Pill Park, Eun Young Kim, Deok Im Kim, Noh Hyung Park, Yu Seok Won, San Hyun Yoon, Kil Saeng Chung and Jin Ho Lim. Simple, efficient and successful vitrification of bovine blastocysts using electron microscopic grid. Hum Reprod, 1999, 14 (11): 2838-2843.
15. Weon-Young Son, San-Hyun Yoon, Hye-Jin Yoon, Sang-Min Lee and Jin-Ho Lim. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum Reprod, 2003, 18 (1): 137-139.
16. P. Vanderzwalmen, G Bertin, Ch. Debauche, V. Standaert, N. Bollen, E.van Roosendaal, M. Vandervorst, R. Schoysman and N. Zech. Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatchingafter thawing. Human Reproduction, 2003, 18 (7): 1504-1511.
17. J.liebermann and M. J. Tucker. Effect of carrier system on the yield of human oocytes and embryos as assessed by survival and developmentqal potential after vitrifaction Reproduction, 2002, 124: 483-489.
18. Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, Callesen H. Open pulled straw (ops) vitrification: A new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev, 1998, 51: 53-8.
19. Shee-Uan Chen, Yih-Ron Lien, Hsin-Fu Chen, Kuang-Han Chao, Hong-Nerng Ho and Yu-Shih Yang. Open pulled straws for vitrification of mature mice oocytes preserve patterns of meiotic spindles and chromosomes better than conventional straws. Hum Reprod, 2000, 15 (12): 2598-2603.
20. Lane M, choolcraft WB, Gardner DK. Vitrification of mice and human blastocysts using a novel cryoloop container less technique. Fertil Steril, 1999, 72 (6): 1073-1078.
21. Mukaida T, Nakamura S, Tomiyama T, Wada S, Kasai, Takahashi K Successful birth after transfer of vitrified human blastocyst using a cryoloop container-less technique. Fertil Steril, 2001, (76): 618-620.
22. T. Mukaida, S. Nakamura, T. Tomiyama, S. Wada, C. Oka, M. Kasai and K. Takahashi. Vitrification of human boastocysts using cryoloops: clinical outcomes of 223 cycles. Hum Reprod, 2003, 18 (2): 384-391.
23. Kenichiro Hiraoka1, Kaori Hiraoka, Masayuki Kinutani and Kazuo Kinutani. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Human Reproduction, 2004, 19 (12): 2884–2888.
24. MoratóR, Izquierdo D, Paramio MT, Mogas T. Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on0indle and chromosome configuration and embryo development. Cryobiology, 2008, 57 (2): 137-141.
25. Vladimir Isachenko, Markus Montag, Eugenia Isachenko, Frank Nawroth, Salvatore Dessole and Hans van der Ven. Developmental rate and ultrastructure of vitrified human pronuclear oocytes after step wise versue direct rehydration. HumRep rod, 2004, 19 (3): 660-665.
26. Jelinkova L, Selman HA, Arav A, Strehler E, Reeka N, Sterzik K. Twin pregnancy after vitrification of 2-pronuclear human embryos. Fertil Steril, 2002, 77 (2): 412-414.
27. Cremades N, Sousa M,Silva J, Viana P, Sousa S, Oliveira C, Teixeira da Silva J, Barros A. Experimental vitrification of human compacted morulae and early blastocysts using fine diameter plastic micropipettes. Hum Reprod, 2004, 19(2): 300-305.
28. Desai N, Blackmon H, Szeptycki J, Goldfarb J. Cryoloop vitrification of human day 3 cleavage-stage embryos: post-vitrification development, pregnancy outcomes and live births. Reprod Biomed Online, 2007, 14: 208–213.
29. Takahashi K, Mukaida T, Goto T, Oka C. Perinatal outcome of blastocyst transfer with vit rification using cryoloop: a 4year follow-up study. Fertil Steril, 2005, 84: 88-92.
30. Kuwayamam, Teramoto S, Hata K. Pregnancies resulting rom transfer of human blastocysts vitrified by the minimum- volume- cooling method. Ferti Steri, 2004, 82 (Suppl 2): 114-115.
31. Nowshari MA, Brem G1 Effect of freezing rate and exposure time to cryop rotectant on the development of mouse pronuclear stage embryos. Hum Rep rod, 2001, 16 (11): 2368-2373.
32. Keynezhad P, J enabi M, Aram R. Cryopervation and frozen embryo t ransfer.Fertil Steril, 2004, 82 (l2): 2368-2373.
33. Liebermann J, Tucker MJ, Graham JR, Han T, Davis A, Levy MJ. Blastocyst development after vitrification of multipronuclear zygotes using the Flexipet denuding pipette. Rep roductive Bio Medicine Online, 2002, 4 (2): 146-150.
34. Selman HA, E-Danasouri I.Pregnancies derived from vitrified human zygotes. Fertil steril, 2002, 77 (2): 422-423.
35. Al-Hasani S, Ozmen B, Koutlaki N, Schoepper B, Diedrich K, Schultze-Mosgau A.Three years of routine vitrification of human zygotes: is it still fair to advocate slow-rate freezing?Reprod Biomed Online, 2007, 14(3): 288-293.
36. El-Danasouri I, Selman H Successful pregnancies and deliveries after a simple Vitrification protocol for day 3 human embryos. Fertil Steril, 2001, 76(2): 400-402.
37. B. Balaban, B. Urman B. Ata1, A. Isiklar1, M.G. Larman, R. Hamilton andD. K. Gardner. A randomized controlled study of human Day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation. Human Reproduction, 2008, (23): 1976–1982.
38.王俊霞,朱桂金,魏玉兰.人卵裂期胚胎玻璃化冷冻成功妊娠一例生殖医学杂志2003年06期:143.
39.童先宏,刘雨生,金仁桃,吴丽敏,骆丽华,栾红兵,周桂香,季静娟,郭通航封闭式拉细麦管法玻璃化冷冻保存人类早期胚胎的临床结果观察。现代妇产科进展,2008年,第17卷第8期:613-619.
40. Y. Yokota, S. Sato,M. Yokota, Y. Ishikawa, M. Makita, T. Asada and Y. Araki Successful pregnancy following blastocyst vitrification: Case report Human Reproduction, 2000, 15 (8) : 1802-1803.
41. Son WY, Yoon SH, Yoon HJ, Lee SM, Lim JH. Pregnancy outcome followingtransfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Human Reproduction, 2003, 18 (1): 137-139.
42. Hiraoka K, Kinutani M, Kinutani K. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Human Reprod, 2004, 19: 2884-2888.
43. Mukaida T, Oka C, Goto T, Takahashi K. Artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts. Hum Reprod, 2006, 21 (12): 3246-3252.
44. Edmond Edi-Osagie, Lee Hooper and Mourad W. Seif The impact of assisted hatching on livebieth rates and outcome of assested conception:a systematic review. Hum Reprod, 2003, 18 (9): 1828-1835.
45. ASRM Practice Committee. The role of assisted hatching in in vitro fertilization: a review of the literature. A Committee opinion. Fertil Steril, 2008, 90 (5): 196-198.
46. Basak Balaban, Bulent Urman1, Kayhan Yakin and Aycan Isiklar. Laser-assisted hatching increases pregnancy and implantation rates in cryopreserved embryos that were allowed to cleave in vitro after thawing: a prospective randomized study. Human Reproduction, 2006, (8): 2136-2140.
47. Valojerdi MR, Eftekhari-Yazdi P, Karimian L, Ashtiani SK. Effect of laser zona pellucida opening on clinical outcome of assisted reproduction technology in patients with advanced female age, recurrent implantation failure, or frozen-thawed embryos. Fertil Steril, 2008, 90 (1): 84-91.
48. Feng HL, Hershlag A, Scholl GM, Cohen MA A retroprospective study comparing three different assisted hatching techniques. Fertil Steril. 2008 Epubahead of print.
49. Elliott TA, Colturato LF, Taylor TH, Wright G, Kort HI, Nagy ZP Elliott TA, Colturato LF, Taylor TH, Wright G, Kort HI, Nagy ZP. Lysed cell removal promotes frozen-thawed embryo development. Fertil Steril, 2007, 87 (6): 1444-1449.
50. Nagy ZP, Taylor T, Elliott T, Massey JB, Kort HI, Shapiro DB.Removal of lysed blastomeres from frozen-thawed embryos improves implantation and pregnancy rates in frozen embryotransfer cycles. Fertil Steril, 2005, 84 (6): 1606-1612.
51. Rienzi L, Nagy ZP, Ubaldi F, Iacobelli M, Anniballo R, Tesarik J, Greco E. Laser assisted removal of necrotic blastomeres from cryopreserved embryos that were partially damaged. Fertil.Steril, 2002, 77 (6): 1196-1201.
52. MORRIS G J. The origin, ultrastructure and microbiology of the sediment accumulating in Dewars used for the storage of cryopreserved samples. Ferti Steri, 2004, 82 (2): 114.
53. Bielanski A, Bergeron H, Lau PC, Devenish J. Microbial contamination of embryos and semen during long term banking in liquid nitrogen. Cryobiology, 2003, 46 (2): 146-152.
54. Liebermann J, Tucker MJ. Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. Fertil Steril, 2006, 86 (1): 20- 26.
55. Zech N. Vitrification of hatching and hatched human blastocysts: Effect of an opening in the zona pellucida before vitrification. Reprod Biomed Online, 2005, 11: 355-361.
56. Kuleshova LL, MacFarlane DR, Trounson AO, Shaw JM. Sugars exert a major influence on the vitrification properties of ethylene glycol-based solutions andhave low toxicity to embryos and oocytes. Cryobiology, 1999, 38: 119-130.
57. Shaw JM, Kuleshova LL, MacFarlane DR, Trounson AO. Vitrification properties of solutions of ethylene glycol in saline containing PVP, Ficoll, or dextran. Cryobiology, 1997, 35: 219-229.
58. Chi-Gu KIM, Hwanyul YONG3, Gene LEE) and Jaejin CHO. Effect of the Polyvinylpyrrolidone Concentration of Cryoprotectant on Mouse Embryo Development and Production of Pups: 7.5% of PVP is Beneficial for In Vitro and In Vivo Development of Frozen-Thawed Mouse Embryos. Reproduction and Development, 2008, 54(4): 250-253.
2. Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature, 1983, 305 (5936): 707-709.
3. Takeda T, Elsden RP, Seidel GE. Cryopreservation of mouse embryos by direct plunging into liquid nitrogen. Theriogenology, 1984, 21: 266~268.
4. Rall WF, Fahy GM: Ice-free cryopreservation of mice embryos at -196℃by vitrification. Nature, 1985, 313: 573–575.
5. T. Mukaida, S. Wada, K. Takahashi, P.B. Pedro, T.Z. An and M. Kasai. Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mice embryos. Hum Reprod, 1998, 13 (10): 2874-2879.
6. Youssry M, Ozmen B, Zohni K, Diedrich K, Al-Hasani S. Current aspects of blastocyst cryopreservation. Reprod Biomed Online, 2008, 16 (2): 311-320.
7. Stehlik E, Stehlik J, Katayama KP, Kuwayama M, Jambor V, Brohammer R, Kato O. Vitrification demonstrates significant improvement versus slow freezing of human blastocysts. Reprod Biomed Online, 2005, 11 (1): 53- 57.
8. Balaban, B. Urman, B. Ata1, A. Isiklar, M.G. Larman, R. Hamilton and D.K. Gardner. A randomized controlled study of human Day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation. Human Reproduction, 2008, (9): 1976-1982.
9. Roser Morató, Dolors Izquierdo, Maria Teresa Paramio, Teresa Mogas.Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: Effects on spindle and chromosome configuration and embryo development. Cryobiology, 2008, 57: 137–141.
10. Shaw JM, Kola I, MacFarlane DR, Trounson AO. An association between chromosomal abnormalities in rapidly frozen 2-cell mice embryos and the ice forming properties of the cryoprotective solution.J Reprod Fertil, 1991, 91: 9–18.
11. Larman MG, Sheehan CB, Gardner DK. Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mice oocytes. Reproduction, 2006, 131: 53–61.
12. Iwatani M, Ikegami K, Kremenska Y, Hattori N, Tanaka S, Yagi S, Shiota K. Dimethyl sulfoxide has an impact on epigenetic profile in mice embryoid body. Stem Cells, 2006, 24: 2549–2556.
13. Kartberg AJ, Hambiliki F, Arvidsson T, Stavreus-Evers A, Svalander P. Vitrification with DMSO protects embryo membrane integrity better than solutions without DMSO. Reprod Biomed Online, 2008, 17(3): 378-384.
14. Kasai M, Komi JH, Takakama A etal. A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution without appreciable loss of viability. Reprod Fert, 1990, 89(1): 92.
15. Cuello, Sanchez-Osorio, Almina, Gil, Perals, Lucas, Roca, Vazquez. MartinezEffect of the cryoprotectant concentration on the in vitro embryo development and cell proliferation of OPS-vitrified porcine blastocysts. Cryobiology, 2008, 56: 189–194.
16. Kalliopi E, Loutradi, M.Med.Sc., Efstratios M. Kolibianakis, Christos A. Venetis, M.Sc., Evangelos G. Papanikolaou, George Pados, Ioannis Bontis, and Basil C. Tarlatzis. Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertility and Sterility, 2008, 90(1): 187-193.
17. E.Go′mez, A.Rodr?′guez, M.Mun?oz, J.N.Caaman?o, C.O.Hidalgo, E.Mora′n, N. Facal, C. Diez. Serum free embryo culture medium improves in vitro survival ofbovine blastocysts to vitrification. Theriogenology, 2008, 69: 1013–1021.
18. Chi-Gu Kim Hwanyul Yong, Gene LEE and Jaejin Cho. Effect of the Polyvinylpyrrolidone Concentration of Cryoprotectant on Mice Embryo Development and Production of Pups: 7.5% of PVP is Beneficial for In Vitro and In Vivo Development of Frozen-Thawed Mice Embryos. Journal of Reproduction and Development, 2008, 54 (4): 250-253.
19. Miyake T, Kasai M, Zhu SE, et al. Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycole based solution by a simple method. Theriogenology, 1993, 40: 121–134.
20. Han MS, Niwa K, Kasai MTheriogenology. Vitrification of rat embryos at various developmental stages. Theriogenology, 2003, 59 (8): 1851-1863.
21. Y. Yokota, S. Sato, M. Yokota, Y. Ishikawa, M. Makita, T. Asada and Y. Araki. Successful pregnancy following blastocyst vitrification: Case report. Human Reproduction, 2000, (8): 1802-1803.
22. Imam El-Danasouri, Helmy Selman Vitrification versus conventional cryopreservation technique. Middle East Fertility Society Journal, 2005, 10 (3): 205-206.
23. Valadimir Isachenko, Frank Nawroth, Salvatore Dessole and Hansvanderven developmental rate and ultrastructure of vitrified human pronuclear oocytes after step-wise versus direct rehydration. Human Reproduction, 2004, 19(3): 660-665.
24. Hyon-Jin Cho, Weon-Young Son, San-Hyun Yoon, Suk-Won Lee and Jin-Ho Lim. An improved protocol for dilution of cryoprotectants from vitrified human blastocysts. Hum Reprod, 2002, 17 (9): 2419-2422.
25. Papis K, Shimizu M and Izaike Y (2000) Factors affecting the survivability of bovine oocytes vitrified in droplets. Theriogenology, 15: 651–658.
26. Matsumoto H., Jiang JY, Tanaka T., Sasada H., and E. Sato. Vitrification oflarge quantities of immature bovine oocytes using nylon mesh. Cryobiology, 2001, 42 (2): 139-144.
27. Se-Pill Park, Eun Young Kim, Deok Im Kim, Noh Hyung Park, Yu Seok Won, San Hyun Yoon, Kil Saeng Chung and Jin Ho Lim.Simple , efficient and successful vitrification of bovine blastocysts using electron microscopic grid. Hum Reprod, 1999, 14 (11): 2838-2843.
28. Weon-Young Son, San-Hyun Yoon, Hye-Jin Yoon, Sang-Min Lee and Jin-Ho Lim. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum Reprod, 2003, 18 (1): 137-139.
29. P. Vanderzwalmen, G. Bertin, Ch. Debauche, V. Standaert, N. Bollen, E.van Roosendaal, M. Vandervorst, R. Schoysman and N. Zech. Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing. Human Reproduction, 2003, 18 (7): 1504-1511.
30. J. liebermann and M.J.Tucker. Effect of carrier system on the yield of human oocytes and embryos as assessed by survival and developmentqal potential after vitrifaction. Reproduction, 2002, 124: 483-489.
31. Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, Callesen H. Open pulled straw (OPS) vitrification: A new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998, 51: 53-58.
32. Shee-Uan Chen, Yih-Ron Lien, Hsin-Fu Chen, Kuang-Han Chao, Hong-Nerng Ho and Yu-Shih Yang. Open pulled straws for vitrification of mature mice oocytes preserve patterns of meiotic spindles and chromosomes better than conventional straws. Hum Reprod, 2000, 15 (12): 2598-2603.
33. Lane M, Schoolcraft WB, Gardner DK. Vitrification of mice and human blastocysts using a novel cryoloop container less technique. Fertil Steril, 1999,72 (6): 1073-1078.
34. Mukaida T, Nakamura S, Tomiyama T, Wada S, Kasai, Takahashi K. Successful birth after transfer of vitrified human blastocyst using a cryoloop container-less technique. Fertil Steril, 2001, (76): 618-620.
35. T. Mukaida, S. Nakamura, T. Tomiyama, S. Wada, C. Oka, M. Kasai and K. Takahashi. Vitrification of human boastocysts using cryoloops: clinical outcomes of 223 cycles. Hum Reprod, 2003, 18 (2): 384-391.
36. Kenichiro Hiraoka1, Kaori Hiraoka, Masayuki Kinutani and Kazuo Kinutani. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Human Reproduction, 2004, 19(12): 2884–2888.
37. Hochi S, Terao T, Kamei M, Kato M, Hirabayashi M, Hirao M .Successful vitrification of pronuclear-stage rabbit zygotes by minimum volume cooling procedure Theriogenology, 2004, 15, 61 (2-3): 267-275.
38. MoratóR, Izquierdo D, Paramio MT, Mogas T. Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on spindle and chromosome configuration and embryo development. Cryobiology, 2008, 57 (2): 137-141.
39. El-Gayar M, Holtz, W. Technical note: vitrification of goat embryos by the open pulled-straw method [J] J. Anim Sci, 2001, 79: 2436-2438.
40. Kuwayama M, Vajta G, Ieda S, Kato O. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reprod Biomed Online, 2005, 11(5): 608-614.
41. Prudenico B.Fedro, Elji Yokoyama, Shi EnZhu. Permeability of mice oocytes and embryos at various developmental stages to five cryoprotectants. Jouranl of Reproduction and Development, 2005, 51: 235-246.
42. Zhao XH, Yue TF, Sui XL. Cryopreservation of mice morulas and blastocysts by vitrification. Zhonghua Fu Chan Ke Za Zhi, 2004, 39 (8): 526-528.
43. Zhou GB, Hou YP, Jin F, Yang QE, Yang ZQ, Quan GB, Tan HM, Zhu SE. Vitrification of mice embryos at various stages by open-pulled staws (OPS) method. Anim Biotechnol, 2005, 16 (2): 153-163.
44. Haibin Wang and Sudhansu K. Dey, Roadmap to embryo implantation: clues from mice models (review). Nature, 2006, 7: 185-199.
45. Hardy K, Handyside AH, Winston RML. The human blastocyst: cell number, death and alloation during late preimplantation development invitro. Development, 1989, 107: 597-604.
46. Chun-Chia Huang, Tsung-Hsien Lee, Shee-Uan Chen, Hsiu-Hui Chen, Tzu-Chun Cheng, Chung-Hsien Liu, Yu-Shih Yang and Maw-Sheng Lee Successful pregnancy following blastocyst cryopreservation using super-cooling ultra-rapid vitrification. Human Reproduction, 2005, 20(1): 122-128.
47. Jun Tao, Robert Tamis and Katharine Fink. Animal Experimentation: Cryopreservation of Mice Embryos at Morula/Compact StageJournal of Assisted. Reproduction and Genetics, 2001, 18(4): 237-245.
48. Vanderzwalmen P, Bertin G, Debauche Ch, Standaert V, van Roosendaal E, Vandervorst M, Bollen N, Zech H, Mukaida T, Takahashi K, Schoysman R. Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod, 2002, 17 (3): 744-751.
49. Chen SU, Lee TH, Lien YR, Tsai YY, Chang LJ, Yang YS. Microsuction of blastocoelic fluid before vitrification increased survival and pregnancy of mice expanded blastocysts, but pretreatment with the cytoskeletal stabilizer did not increase blastocyst survival. Fertil Steril, 2005, 84: 1156-1162.
50. SonWY, Yoon SH, YoonHJ, Lee SM, LimJH. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum Reprod, 2003, 18: 137–139.
51. Campos-Chillon LF, Walker DJ, de la Torre-Sanchez JF, Seidel GE Jr.In vitro assessment of a direct transfer vitrification procedure for bovine embryos. Theriogenology, 2006, 65(6): 1200-1214.
52. Cuello C, Gil MA, Parrilla I, Tornel J, Vazquez JM, Roca J, Berthelot F, Martinat-Botte F, Martinez EA.Vitrification of porcine embryos at various developmental stages using different ultra-rapid cooling procedures. Theriogenology, 2004, 62(1-2): 353-361.
53. Vajta G. Vitrification of oocytes and embryos of domestic animals. Anim Reprod Sci, 2000, 61: 357–364.
54. Hitoshi Ushijima,Hiroki Yoshioka,Ritsuko Esaki, keiji Takahashi, Masashige Kuwayama, Takashi Nakane and Hiroshi Nagashima. Improved Survival of Vitrified in Vivo-Derived Porcine Embryos. Jouranl of Reproduction and Development, 2004, 50(4): 481-486.
55. N.Cremades, M.Sousa, J.Silva, P.Viana, S.Sousa, C.Oliveira,J.Teixeira da Silva and A.Barros.Experimental vitrification of human compacted morula and early blastocysts using fine diameter plastic micropipettes 3. Human Reproduction, 2004, 19: 300-305.
56. Abe H, Yamashita S, Satoh T, Hoshi H. Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serumfree or serum-containing media. Mol Reprod Dev, 2002, 61: 57–66.
57. Garrisi GJ, Talansky BE, Sapira V, Gordon JW, Navot D. An intact zona pellucida is not necessary for successful mouse embryo cryopreservation. FertilSteril, 1992, 57(3): 677-681.
58. Dobrinsky J R.Cryopreservation of pig embryos:adaptation of vitrification technology for embryo transfer. Reprod Suppl, 2001, 58: 325-333.
59. Yildiz C, Ottaviani P, Law N, Ayearst R, Liu L, McKerlie C. Effects of cryopreservation on sperm quality, nuclear DNA integrity, in vitro fertilization, and in vitro embryo development in the mouse. Reproduction, 2007, 133(3): 585-595.
60. Coticchio G, Bonu MA, Bianchi V, Flamigni C, Borini A. Criteria to assess human oocyte quality after cryopreservation. Reprod Biomed Online, 2005, 11(4): 421-427.
61. Khalifa T A, Rekkas C A, Lymberopoulos A G. Factors affecting chromatin stability of bovine spermatozoa. Anim Reprod Sci, 2007, 11: 112-118.
62. Damario MA, Hammitt DG, Session DR, Dumesic DA. Embryo cryopreservation at the pronuclear stage and efficient embryo use optimizes the chance for a liveborn infant from single oocyte retrieval. Fertil.Steril, 2000, 73 (4): 767-773.
63. Tong Z B, Gold L, Pfeifer K E, Dorward H, Lee E, Bondy C A, Dean J, Nelson L M. Mater, a maternal effect gene required for early embryonic development in mice.Nat Genet, 2000, 26 (3): 267-268.
64. El-Danasouri I, Selman H Successful pregnancies and deliveries after a simple Vitrification protocol for day 3 human embryos. Fertil Steril, 2001, 76 (2): 400-402.
65. Rama Raju GA, Haranath GB, Krishna KM, Prakash GJ, Madan K. Vitrification of human 8-cell embryos, a modified protocol for better pregnancy rates. Reprod Biomed Online, 2005, 11: 434–437.
66. Desai N, Blackmon H, Szeptycki J, Goldfarb J. Cryoloop vitrification of human day 3 cleavage-stage embryos: post-vitrification development, pregnancyoutcomes and live births. Reprod Biomed Online, 2007, 14: 208–213.
67. Tada N, Sato M, Amann E, Ogawa S A simple and rapid method for cryopreservation of mouse 2-cell embryos by vitrification: Beneficial effect of sucrose and raffinose on their cryosurvival rate. Theriogenology, 1993, 40(2): 333-344.
68. Mojtaba rezazadeh valojerdi, mansoureh movahedin and Ahmad hosseini. Improvement of development of vitrified two-cell mice embryos by vero cell coculture. Journal of Assisted Reproduction and Genetics, 2002, 19(1): 31-38.
69. A.Dhali, V. M. Anchamparuthy, S.P.Butler, R.E.Pearson, I. K. Mullarky, F.C.Gwazdauskas. Gene expression and development of mice zygotes following droplet vitrification. Theriogenology, 2007, (68): 1292-1298.
70. Keisuke Edashige, Satoshi Ohta, Mitsunobu Tanaka, Tatsunaga Kuwano, Delgado M. Valdez, Jr.,Takao Hara, Bo Jin,Sei-ichi Takahashi,Shinsuke Seki, Chihiro Koshimoto,and Magosaburo Kasai.The Role of Aquaporin 3 in the Movement of Water and Cryoprotectants in Mice Morulae. Biology of reproduction, 2007, 77: 365–375.
71. Keisuke Edashige, Mitsunobu Tanaka, Natsuko Ichimaru,Satoshi Ota, Ken-ichi Yazawa,Yuki Higashino, Megumi Sakamoto, Yohei Yamaji,Tatsunaga Kuwano, Delgado M. Valdez Jr., F.W. kleinhans, and magosaburo kasai. Channel-dependent permeation of water and glycerol in mice morulae. Biology of reproduction, 2006, 74: 625–632.
72. Diez C, Heyman Y, Le Bourhis D, Guyader-Joly C, Degrouard J,Renard JP. Delipidating in vitro-produced bovine zygotes: effect on further development and consequences for freezability. Theriogenology, 2001, 55: 923–936.
73. Nasr-esfahani M,Johnson M H,Aitken R J.The effect of iron and iron chelators on the in-vitro block to development of the mouse preimplantationembryo:BAT6 a new medium for improved culture of mouse embryos in vitro. Hum Reprod, 1990, 5 (8): 997-1003.
74. Pfaff RT, Liu J, Gao D, Peter AT, Li TK, Critser JK. Water and DMSO,membrane permeability characteristics of in-vivo and in-vitro derived and cultured murine oocytes and embryos. Mol Hum Reprod, 1998, 4: 51–59.
75. Barnett DK, Clayton MK, Kimura J, Bavister BD.Glucose and phosphate toxicity in hamster preimplantation embryos involves disruption of cellular organization, including distribution of active mitochondria. Mol Reprod Dev, 1997, 48 (2): 227-237.
76. Josiane Van der Elst, Youri Amerijckx1, and Andre′Van Steirteghem.Ultra-rapid freezing of mice oocytes lowers the cell number in the inner cell mass of 5 day old in-vitro cultured blastocysts. Human Reproduction, 1998, (13): 1595–1599.
77. Michelle Lane, Elizabeth A.Lyons and Barry D.Bavister.Cryopreservation reduces the ability of hamster 2-cell embryos to regulate intracellular pH. Human Reproduction, 2000, 15(2): 389-394.
78. Willingham M C.Cytochemical methods for the detection of apoptosis. J Histochem Cytochem, 1999, 47 (9): 1101-1110.
79. ReedML, LaneM, GardnerDK, Jensen NL, Thompson J. Vitrification of human blastocysts using the cryoloop method: successful clinical application and birth of offspring. J Assist Reprod Genet, 2002, 19: 304.
80. Yokota, Y. Sato S., Yokota M., Ishikawa Y., Makita M., Asada T.and Araki Y. Successful pregnancy following blastocyst vitrification: Case report Human Reproduction, 2000, 15(8):1802-1803.
81. Yokota Y, Sato S , Yokota M , et al . Birth of a healthy baby following vitrification of human blastocysts. Fertil Steril, 2001, 75 :1027-1029.
82. Takahashi K, Mukaida T, Goto T, Oka C. Perinatal outcome of blastocyst transferwith vit rification using cryoloop : a 4-year follow-up study. Fertil Steril , 2005 , 84: 88-92.
83. Mukaida T,Nakamura S, Tomiyama T, Wada S, Kasai M, Takahashi K.Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique. Fertil Steril 2001, 76(3): 618–623.
84. Reed ML, Lane M, Gardner DK, Jensen NL, Thompson J. Vitrification of human blastocysts using the cryoloop method: successful clinical application and birth of offspring. J Assist Reprod Genet, 2002, 19( 6):304-306.
85. M. Antinori, E. Licata, G. Dani, F. Cerusico, C. Versaci, S. Antinori, Cryotop vitrification of human oocytes results in high survival rate and healthy deliveries, Reprod. Biomed, 2007, 14: 72–79.
86. Kuwayama M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology, 2007, 67: 73-80.
87. Son WY, Yoon SH, Yoon HJ, Lee SM, Lim JH. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Human Reproduction, 2003, 18 (1): 137-139.
88. Motoishi. Cryopreservation of human blastocyst. Clin Embryol, 2000, 5: 6–14.
89. Hiraoka K, Kinutani M, Kinutani K. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Human Reprod, 2004, 19: 2884-2888.
90. Mukaida T, Oka C, Goto T, Takahashi K. Artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts. Hum Reprod, 2006, 21 (12): 3246-3252.
1. Rall WF, Fahy GM: Ice-free cryopreservation of mice embryos at -196℃by vitrification. Nature, 1985, 313: 573–575.
2. Mukaida, S. Wada, K. Takahashi, P.B. Pedro, T.Z. An and M. Kasai. Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mice embryos. Hum Reprod, 1998, 13(10) : 2874-2879.
3. Roser Morató, Dolors Izquierdo, Maria Teresa Paramio, Teresa Mogas. Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: Effects on spindle and chromosome configuration and embryo development. Cryobiology, 2008, 57: 137–141.
4. Shaw JM, Kola I, MacFarlane DR, Trounson AO. An association between chromosomal abnormalities in rapidly frozen 2-cell mice embryos and the ice forming properties of the cryoprotective solution. Reprod Fertil, 1991, 91: 9–18.
5. Kasai M, Komi JH, Takakamo A, Tsudera H, Sakurai T, Machida T, A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution without appreciable loss of viability. Reprod Fert, 1990, 89(1): 91-97.
6. Cho HJ, Son WY, Yoon SH, Lee SW, Lim JH. An improved protocolfor dilution of cryoprotectants from vitrified human blastocysts. HumReprod, 2002, 17 (9): 2419- 2422.
7. Ali J, Bongso A, Ratnam SS. Vitrification of human embryo. Med Sci Res, 1995, 23 (1): 539-540.
8. Kalliopi E. Loutradi, M.Med.Sc., Efstratios M. Kolibianakis, Christos A. Venetis, M.Sc., Evangelos G. Papanikolaou, George Pados, Ioannis Bontis, and Basil C. Tarlatzis.Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertility and Sterility, 2008, 90(1): 187-193.
9. Y. Yokota, S. Sato, M. Yokota, Y. Ishikawa, M. Makita, T. Asada and Y. Araki. Successful pregnancy following blastocyst vitrification: Case report Human Reproduction, 2000, 15(8): 1802-1803.
10. Imam DV, Helmy S. Successful pregnancies and deliveries after a simple vit rification protocol for 3 human embryos. Fertil Steril, 2001, 76 (2): 400-402.
11. Vanderzwalmen P, Bertin G, Debauche C, Standaert V, van Roosendaal E, Vandervorst M, Bollen N,Zech H, Mukaida T, Takahashi K, Schoysman R. Births after vitrification at morula and blastocyst stages: Effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod, 2002, 17: 744-571.
12. Shimizu M and Izaike Y Factors affecting the survivability of bovine oocytes vitrified in droplets. Theriogenology, 2002, 15: 651–658.
13. Matsumoto H., Jiang JY, Tanaka T., Sasada H., and E. Sato. Vitrification of large quantities of immature bovine oocytes using nylon mesh. Cryobiology, 2001, 42 (2): 139-144.
14. Se-Pill Park, Eun Young Kim, Deok Im Kim, Noh Hyung Park, Yu Seok Won, San Hyun Yoon, Kil Saeng Chung and Jin Ho Lim. Simple, efficient and successful vitrification of bovine blastocysts using electron microscopic grid. Hum Reprod, 1999, 14 (11): 2838-2843.
15. Weon-Young Son, San-Hyun Yoon, Hye-Jin Yoon, Sang-Min Lee and Jin-Ho Lim. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum Reprod, 2003, 18 (1): 137-139.
16. P. Vanderzwalmen, G Bertin, Ch. Debauche, V. Standaert, N. Bollen, E.van Roosendaal, M. Vandervorst, R. Schoysman and N. Zech. Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatchingafter thawing. Human Reproduction, 2003, 18 (7): 1504-1511.
17. J.liebermann and M. J. Tucker. Effect of carrier system on the yield of human oocytes and embryos as assessed by survival and developmentqal potential after vitrifaction Reproduction, 2002, 124: 483-489.
18. Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, Callesen H. Open pulled straw (ops) vitrification: A new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev, 1998, 51: 53-8.
19. Shee-Uan Chen, Yih-Ron Lien, Hsin-Fu Chen, Kuang-Han Chao, Hong-Nerng Ho and Yu-Shih Yang. Open pulled straws for vitrification of mature mice oocytes preserve patterns of meiotic spindles and chromosomes better than conventional straws. Hum Reprod, 2000, 15 (12): 2598-2603.
20. Lane M, choolcraft WB, Gardner DK. Vitrification of mice and human blastocysts using a novel cryoloop container less technique. Fertil Steril, 1999, 72 (6): 1073-1078.
21. Mukaida T, Nakamura S, Tomiyama T, Wada S, Kasai, Takahashi K Successful birth after transfer of vitrified human blastocyst using a cryoloop container-less technique. Fertil Steril, 2001, (76): 618-620.
22. T. Mukaida, S. Nakamura, T. Tomiyama, S. Wada, C. Oka, M. Kasai and K. Takahashi. Vitrification of human boastocysts using cryoloops: clinical outcomes of 223 cycles. Hum Reprod, 2003, 18 (2): 384-391.
23. Kenichiro Hiraoka1, Kaori Hiraoka, Masayuki Kinutani and Kazuo Kinutani. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Human Reproduction, 2004, 19 (12): 2884–2888.
24. MoratóR, Izquierdo D, Paramio MT, Mogas T. Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on0indle and chromosome configuration and embryo development. Cryobiology, 2008, 57 (2): 137-141.
25. Vladimir Isachenko, Markus Montag, Eugenia Isachenko, Frank Nawroth, Salvatore Dessole and Hans van der Ven. Developmental rate and ultrastructure of vitrified human pronuclear oocytes after step wise versue direct rehydration. HumRep rod, 2004, 19 (3): 660-665.
26. Jelinkova L, Selman HA, Arav A, Strehler E, Reeka N, Sterzik K. Twin pregnancy after vitrification of 2-pronuclear human embryos. Fertil Steril, 2002, 77 (2): 412-414.
27. Cremades N, Sousa M,Silva J, Viana P, Sousa S, Oliveira C, Teixeira da Silva J, Barros A. Experimental vitrification of human compacted morulae and early blastocysts using fine diameter plastic micropipettes. Hum Reprod, 2004, 19(2): 300-305.
28. Desai N, Blackmon H, Szeptycki J, Goldfarb J. Cryoloop vitrification of human day 3 cleavage-stage embryos: post-vitrification development, pregnancy outcomes and live births. Reprod Biomed Online, 2007, 14: 208–213.
29. Takahashi K, Mukaida T, Goto T, Oka C. Perinatal outcome of blastocyst transfer with vit rification using cryoloop: a 4year follow-up study. Fertil Steril, 2005, 84: 88-92.
30. Kuwayamam, Teramoto S, Hata K. Pregnancies resulting rom transfer of human blastocysts vitrified by the minimum- volume- cooling method. Ferti Steri, 2004, 82 (Suppl 2): 114-115.
31. Nowshari MA, Brem G1 Effect of freezing rate and exposure time to cryop rotectant on the development of mouse pronuclear stage embryos. Hum Rep rod, 2001, 16 (11): 2368-2373.
32. Keynezhad P, J enabi M, Aram R. Cryopervation and frozen embryo t ransfer.Fertil Steril, 2004, 82 (l2): 2368-2373.
33. Liebermann J, Tucker MJ, Graham JR, Han T, Davis A, Levy MJ. Blastocyst development after vitrification of multipronuclear zygotes using the Flexipet denuding pipette. Rep roductive Bio Medicine Online, 2002, 4 (2): 146-150.
34. Selman HA, E-Danasouri I.Pregnancies derived from vitrified human zygotes. Fertil steril, 2002, 77 (2): 422-423.
35. Al-Hasani S, Ozmen B, Koutlaki N, Schoepper B, Diedrich K, Schultze-Mosgau A.Three years of routine vitrification of human zygotes: is it still fair to advocate slow-rate freezing?Reprod Biomed Online, 2007, 14(3): 288-293.
36. El-Danasouri I, Selman H Successful pregnancies and deliveries after a simple Vitrification protocol for day 3 human embryos. Fertil Steril, 2001, 76(2): 400-402.
37. B. Balaban, B. Urman B. Ata1, A. Isiklar1, M.G. Larman, R. Hamilton andD. K. Gardner. A randomized controlled study of human Day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation. Human Reproduction, 2008, (23): 1976–1982.
38.王俊霞,朱桂金,魏玉兰.人卵裂期胚胎玻璃化冷冻成功妊娠一例生殖医学杂志2003年06期:143.
39.童先宏,刘雨生,金仁桃,吴丽敏,骆丽华,栾红兵,周桂香,季静娟,郭通航封闭式拉细麦管法玻璃化冷冻保存人类早期胚胎的临床结果观察。现代妇产科进展,2008年,第17卷第8期:613-619.
40. Y. Yokota, S. Sato,M. Yokota, Y. Ishikawa, M. Makita, T. Asada and Y. Araki Successful pregnancy following blastocyst vitrification: Case report Human Reproduction, 2000, 15 (8) : 1802-1803.
41. Son WY, Yoon SH, Yoon HJ, Lee SM, Lim JH. Pregnancy outcome followingtransfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Human Reproduction, 2003, 18 (1): 137-139.
42. Hiraoka K, Kinutani M, Kinutani K. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Human Reprod, 2004, 19: 2884-2888.
43. Mukaida T, Oka C, Goto T, Takahashi K. Artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts. Hum Reprod, 2006, 21 (12): 3246-3252.
44. Edmond Edi-Osagie, Lee Hooper and Mourad W. Seif The impact of assisted hatching on livebieth rates and outcome of assested conception:a systematic review. Hum Reprod, 2003, 18 (9): 1828-1835.
45. ASRM Practice Committee. The role of assisted hatching in in vitro fertilization: a review of the literature. A Committee opinion. Fertil Steril, 2008, 90 (5): 196-198.
46. Basak Balaban, Bulent Urman1, Kayhan Yakin and Aycan Isiklar. Laser-assisted hatching increases pregnancy and implantation rates in cryopreserved embryos that were allowed to cleave in vitro after thawing: a prospective randomized study. Human Reproduction, 2006, (8): 2136-2140.
47. Valojerdi MR, Eftekhari-Yazdi P, Karimian L, Ashtiani SK. Effect of laser zona pellucida opening on clinical outcome of assisted reproduction technology in patients with advanced female age, recurrent implantation failure, or frozen-thawed embryos. Fertil Steril, 2008, 90 (1): 84-91.
48. Feng HL, Hershlag A, Scholl GM, Cohen MA A retroprospective study comparing three different assisted hatching techniques. Fertil Steril. 2008 Epubahead of print.
49. Elliott TA, Colturato LF, Taylor TH, Wright G, Kort HI, Nagy ZP Elliott TA, Colturato LF, Taylor TH, Wright G, Kort HI, Nagy ZP. Lysed cell removal promotes frozen-thawed embryo development. Fertil Steril, 2007, 87 (6): 1444-1449.
50. Nagy ZP, Taylor T, Elliott T, Massey JB, Kort HI, Shapiro DB.Removal of lysed blastomeres from frozen-thawed embryos improves implantation and pregnancy rates in frozen embryotransfer cycles. Fertil Steril, 2005, 84 (6): 1606-1612.
51. Rienzi L, Nagy ZP, Ubaldi F, Iacobelli M, Anniballo R, Tesarik J, Greco E. Laser assisted removal of necrotic blastomeres from cryopreserved embryos that were partially damaged. Fertil.Steril, 2002, 77 (6): 1196-1201.
52. MORRIS G J. The origin, ultrastructure and microbiology of the sediment accumulating in Dewars used for the storage of cryopreserved samples. Ferti Steri, 2004, 82 (2): 114.
53. Bielanski A, Bergeron H, Lau PC, Devenish J. Microbial contamination of embryos and semen during long term banking in liquid nitrogen. Cryobiology, 2003, 46 (2): 146-152.
54. Liebermann J, Tucker MJ. Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. Fertil Steril, 2006, 86 (1): 20- 26.
55. Zech N. Vitrification of hatching and hatched human blastocysts: Effect of an opening in the zona pellucida before vitrification. Reprod Biomed Online, 2005, 11: 355-361.
56. Kuleshova LL, MacFarlane DR, Trounson AO, Shaw JM. Sugars exert a major influence on the vitrification properties of ethylene glycol-based solutions andhave low toxicity to embryos and oocytes. Cryobiology, 1999, 38: 119-130.
57. Shaw JM, Kuleshova LL, MacFarlane DR, Trounson AO. Vitrification properties of solutions of ethylene glycol in saline containing PVP, Ficoll, or dextran. Cryobiology, 1997, 35: 219-229.
58. Chi-Gu KIM, Hwanyul YONG3, Gene LEE) and Jaejin CHO. Effect of the Polyvinylpyrrolidone Concentration of Cryoprotectant on Mouse Embryo Development and Production of Pups: 7.5% of PVP is Beneficial for In Vitro and In Vivo Development of Frozen-Thawed Mouse Embryos. Reproduction and Development, 2008, 54(4): 250-253.