卵母细胞玻璃化冷冻液配方的改良研究及临床应用
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摘要
第一部分改良玻璃化冷冻液对体外成熟卵母细胞冷冻复苏后发育潜能的影响
目的针对目前卵母细胞玻璃化冷冻效果不稳定的问题,参考常用的两种卵母细胞玻璃化冷冻液配方,设计一种保持渗透性不变而降低各种渗透性冷冻保护剂浓度的玻璃化冷冻液配方,利用ICSI患者捐赠的未成熟卵母细胞进行玻璃化冷冻方法的研究,验证新型改良卵母细胞玻璃化冷冻液对体外成熟卵母细胞玻璃化冷冻效果的影响。
方法收集常规ICSI患者捐赠的未成熟卵母细胞进行体外成熟培养,将18～24小时体外成熟的卵母细胞作为研究对象。随机分为4组,其中三组采用不同的方法进行冷冻保存,分别为B组(EG/DMSO法)、C组(EG/PROH法)和D组(EG/DMSO/PROH法),解冻后进行授精和胚胎培养,A组为对照组,培养成熟后直接进行授精和胚胎培养。以每解冻卵母细胞为分母,观察计算存活率、受精率、优胚率、囊胚形成率和优质囊胚率。
结果不同玻璃化冷冻配方对体外成熟卵母细胞的发育潜能均有较大的影响,其受精率、优胚率和囊胚形成率均显著低于新鲜组(P<0.01),尽管D组的存活率、2PN率、优胚率、囊胚形成率及优质囊胚率高于B组和C组,但统计学分析并无显著性差异(P>0.05)。D组的存活率最高(84.8%),与B(77.2%)、C组(80.0%)比较没有显著性差异(P>0.05)。D、B、C各组的2PN率分别为46.7%、42.4%和40.0%,优胚率分别为5.7%,2.2%和2.2%,囊胚形成率为7.6%,2.2%和3.3%(P>0.05),仅D组形成了优质囊胚,与A组比较没有显著性差异(1.9%和6.7%,P>0.05)。
结论改良卵母细胞玻璃化冷冻液可以改善体外成熟卵母细胞的发育潜能,有助于获得更多的优质囊胚。
第二部分改良玻璃化冷冻对体外成熟卵母细胞形态及超微结构的影响
目的卵母细胞作为人体最大的细胞,细胞结构特点决定了其对低温和高渗环境更加敏感,同慢速冷冻一样,玻璃化冷冻也可能导致卵母细胞超微结构的改变。针对卵母细胞骨架系统中的微丝分布和电镜下超微结构形态,进一步验证改良卵母细胞玻璃化冷冻液对体外成熟卵母细胞解冻后形态和超微结构的影响。
方法收集常规ICSI患者捐赠的未成熟卵母细胞进行体外成熟培养,将体外成熟的卵母细胞用两种冷冻方案冷冻解冻后进行形态学和超微结构评价。实验组分为传统组(EG/DMSO冷冻法)和改良组(EG/DMSO/PROH冷冻法),对照组为新鲜体外成熟卵母细胞,直接固定后行超微结构观察。每组卵母细胞均进行共聚焦显微镜下微丝分布观察和透射电镜下超微结构观察。
结果倒置镜下,冷冻两组均有部分卵母细胞存在周边空泡状结构。改良组的空泡发生率为18.7%,远远低于传统组(66.7%)(P=0.000)。改良组的微丝分布正常率(43.8%)略高于传统组(33.3%),两者比较没有显著性差异(P>0.05)。改良组和传统组的微丝分布正常率均低于对照组(71.1%)(分别是P<0.05和P<0.01),透射电镜结果显示,无论是传统组还是改良组,冷冻复苏后卵母细胞中的空泡状结构略多于新鲜卵母细胞。而线粒体和空泡的形态结构与对照组比较没有明显差异,冷冻两组中卵母细胞皮质颗粒的数量略有减少,且更加绕周边分布,未观察到明显的皮质颗粒外排现象。结论改良玻璃化冷冻方案较传统玻璃化冷冻方案可以更加有效的改善卵母细胞解冻后的光镜下形态,两种玻璃化冷冻方案都会影响体外成熟卵母细胞的超微结构。
第三部分不同玻璃化冷冻液对卵母细胞发育潜能的影响及其临床结局评价
目的评价一种卵母细胞玻璃化冷冻液配方的优劣,最直接的证据是临床妊娠结果,本研究回顾性分析了使用不同玻璃化冷冻液玻璃化冷冻卵母细胞的实验室培养数据及临床结局,为改良冷冻液的推广应用找到临床证据。
方法收集2012年～2013年的卵母细胞解冻的所有患者的数据,包括自卵使用患者和接受供卵的患者。卵母细胞来源于2008年～2013年不同时间冷冻的卵母细胞。卵母细胞冷冻动机以获卵数超过22枚者为多,部分因为取卵日取精失败。解冻周期患者均采用替代周期方案,卵母细胞胞解冻后行黄体支持。记录冷冻卵母细胞所用的试剂类型(MC组、KT组和改良组)、解冻卵母细胞使用者的年龄、冷冻存活率、受精率、优胚率、囊胚形成率、冷冻和移植胚胎数、临床妊娠率及着床率。同时计算每解冻卵母细胞获得的胚胎利用率。
结果自卵使用数据中,卵母细胞的冷冻复苏率以改良组最高(92.0%),显著高于其他两组(MC和KT组分别是88.2%和77.3%)(P<0.05)。改良组受精卵分裂后获得的优胚率最高(35.8%),与MC和KT组(29.0%和28.3%)相比有显著性差异(P<0.05)。胚胎移植后的临床妊娠率与着床率各组之间没有显著性差异(P>0.05),MC、KT和改良配方组临床妊娠率分别是37.2%,30.2%和39.6%,着床率分别是21.9%、18.8%和27.4%。但改良组每周期胚胎移植个数是1.89±0.59,显著低于MC组(2.28±0.83)(P=0.000),也少于KT组(2.02±0.93)(P>0.05)。按照每解冻卵母细胞的效率看,改良配方组的卵母细胞授精后获得的优胚率显著高于MC组和KT组(P<0.01)。每解冻卵母细胞获得的胚胎利用率高于另外两组,但没有显著性差异(P>0.05)。在供卵使用数据中仅用到了MC试剂和KT试剂,两组比较,存活率、受精率、优胚率、临床妊娠率及各项卵母细胞利用率,均没有显著性差异(P>0.05),供卵组的各项数据均比自卵组好,临床妊娠率可以分别达到50%和43.5%。
结论改良冷冻液配方获得了较商品化试剂更高的存活率和优胚率,在移植更少胚胎的同时,没有降低临床妊娠率和着床率。相对于价格昂贵保质期超长的商品化试剂,改良冷冻液获得了更多的可利用胚胎,今后将继续观察改良冷冻液的使用效果,同时追踪冷冻胚胎的使用结果,以获得更有价值的冷冻卵母细胞累积利用率指标。
Part1Effects of modified vitrification media on the development potential of human vitrification-warmed in vitro matured oocytes
Objective To certify the effects of modified vitrification media on the developmenta potential of human in-vitro matured oocytes.
Methods Immature oocytes were collected from controlled ovarian stimulation cycles for ICSI treatment. All these immature oocytes were cultured for18-24hrs for maturation. Only matured oocytes were enrolled in this study. Gathered human in-vitro matured oocytes were randomly divided into control and three vitrified group.Group A was control group without vitrification treatment. Group B was vitrified with EG and DMSO. Group C was vitrified with EG and PROH. Group D was vitrified with EG, DMSO and PROH. The developmental potential was evaluated with survival rate, fertilization rate,2PN rate, cleavage rate of2PN, high-quality embryo rate, balstocyst rate and high-quality blastocyst rate.
Result The vitrification process affected the development potential of these in-vitro matured oocytes. The fertilization rates, high-quality embryo rates, and balstocyst rates in the three vitrification groups were significantly lower than the control group (P<0.01).Though the survival rate (84.8%),2PN rate (46.7%), high-quality embryo rate (5.7%), balstocyst rate (7.6%) in Group D were higher than those of in Group B (77.2%,42.4%,2.2%,2.2%, respectively) and Group C (80.0%,40.0%,2.2%,3.3%, respectively), there was no significant difference (P>0.05). In three vitrifcaiton goups, high-quality blastocysts were got only in Group D (1.9%). Though high-quality blastocysts in Group D was lower than that of in Group A (6.7%), there was no significant difference (P>0.05).
Conclusion The vitrification process reduced the development potential of the in-vitro matured oocytes. The modified vitrification media is effective for in-vitro matured oocytes vitrification through increasing the high-quality blastocyst rate.
Part2Effects of modified vitrification media on the morphorlogy and
ultrastructure of human vitrification-warmed in vitro matured oocytes.
Objective To study the effects of modified vitrification media on the morphorlogy and ultrastructure of human in vitro matured oocytes.
Methods Immature oocytes were collected from controlled ovarian stimulation cycles for ICSI treatment. All these immature oocytes were cultured for18-24hrs for maturation. Only matured oocytes were enrolled in this study. Gathered human in-vitro matured oocytes were randomly divided into control and two vitrified group. Matured oocytes in conventinonal vitrification group were vitrified with EG and DMSO. Modified vitrification group was vitrified with EG, DMSO and PROH. All the vitrified oocytes were warmed in1to5months. Control group was not given vitrification. The oocytes in control group and two vitrification groups were stained by Rhodamine-phalloidine and observed by laser confocal microscopy (LSCM). Some of the oocytes in the three groups were fixed and observed under transmission electron microscopy (TEM).
Result There were a lot of vesicles in part of the warmed oocytes in the two vitrification groups, distributed under the membrane of the oocytes. The vesicles enclosed oocytes rate was18.7%in the modified vitrification group, which was lower significantly than the conventional group (66.7%)(P=0.000). There was no significant difference between modified vitrification group and conventional group in the rate of microfilament normal distribution (43.8%vs33.3%, P>0.05), but lower significantly than the control group (71.1%)(P<0.05). TEM results revealed that there were more vesicles in the cytoplasm in the two vitrification groups comparing with the control group. The morphology of mitochondria and vesicles were similar between the vitrification groups and control group. The distribution of the cortical granules in the vitrification-warmed oocytes was nearer to the periphery of the oocytes. Though the cortical granules extrusion was not observed, the number of the cortical granules was less in the vitrificaiton-warmed oocytes
Conclusion The modified vitrification protocol is useful for the recovery of morphorlogy and ultrastructure of the vitrification-warmed human in vitro matured oocytes compared with conventional vitrification protocol. Vitrificaiton process affected the number and distribution of cortical granules.
Part3Effects of different vitrification media on the development potential of vitrification-warmed in vivo matured oocytes and clinical results
Objective To study the effects of different vitrification media on the development potential of vitrification-warmed in vivo matured oocytes and clinical results.
Methods This retrospective study was involved in all of the vitrification-warmed oocytes between2012January and2013December. All of the oocytes were vitrified in the last five years. Different vitrification media were used (MC media, KT media and modified media). After warming, the rates of oocyte survival, fertilization, high-quality embryo, blastulation, clinical pregnancy and implantation were recorded and analyzed. Also, the usage rate of every warmed oocyte was calculated. Data from self-oocytes warming and donor-oocytes warming were calculated separately.
Result For the self-oocytes warming group, the oocytes survival rate of modified media (92.0%) was significantly higher than the MC media (88,2%) and KT media (77.3%)(P<0.05). High-quality embryo rate in modified media group (35.8%) was significantly higher than the other groups (29.0%vs28.3%)(P<0.05). There was no significant difference in the clinical pregnancy rate and implantation rate between MC, KT and modified media groups (37.2%vs30.2%vs39.6%and21.9%vs18.8%vs27.4%)(P>0.05). In spite of that, the number of embryo transfered in every cycle (1.89±0.59) was significantly lower in modified group compared with MC media group (2.28±0.83)(P=0.000). When it comes to oocytes usage efficiency, the high-quality embryo rate for every warmed oocyte was higher significantly than other two groups (P<0.01). Though the utilization of the oocyte was higher than the other two groups, there was no significant difference (P>0.05). Only MC media and KT media were used in the donor-oocyte cycles. There was no statistical difference in the rates of survival, fertilization, high-quality embryo, clinical pregnancy and implantation (P>0.05). Compared with data in the self-oocytes group for MC media and KT media, the clinical pregnancy rates were perfectly higher (50%and43.5%, respectively).
Conclusion This clinical study showed that the modified vitrification media could get more high-quality embryos and higher clinical pregnancy rate. This modified media would be used effectively in clinical application since it is an efficient vitrification protocol. More subtle study should be carried out for a reliable conclusion.
目的针对目前卵母细胞玻璃化冷冻效果不稳定的问题,参考常用的两种卵母细胞玻璃化冷冻液配方,设计一种保持渗透性不变而降低各种渗透性冷冻保护剂浓度的玻璃化冷冻液配方,利用ICSI患者捐赠的未成熟卵母细胞进行玻璃化冷冻方法的研究,验证新型改良卵母细胞玻璃化冷冻液对体外成熟卵母细胞玻璃化冷冻效果的影响。
方法收集常规ICSI患者捐赠的未成熟卵母细胞进行体外成熟培养,将18～24小时体外成熟的卵母细胞作为研究对象。随机分为4组,其中三组采用不同的方法进行冷冻保存,分别为B组(EG/DMSO法)、C组(EG/PROH法)和D组(EG/DMSO/PROH法),解冻后进行授精和胚胎培养,A组为对照组,培养成熟后直接进行授精和胚胎培养。以每解冻卵母细胞为分母,观察计算存活率、受精率、优胚率、囊胚形成率和优质囊胚率。
结果不同玻璃化冷冻配方对体外成熟卵母细胞的发育潜能均有较大的影响,其受精率、优胚率和囊胚形成率均显著低于新鲜组(P<0.01),尽管D组的存活率、2PN率、优胚率、囊胚形成率及优质囊胚率高于B组和C组,但统计学分析并无显著性差异(P>0.05)。D组的存活率最高(84.8%),与B(77.2%)、C组(80.0%)比较没有显著性差异(P>0.05)。D、B、C各组的2PN率分别为46.7%、42.4%和40.0%,优胚率分别为5.7%,2.2%和2.2%,囊胚形成率为7.6%,2.2%和3.3%(P>0.05),仅D组形成了优质囊胚,与A组比较没有显著性差异(1.9%和6.7%,P>0.05)。
结论改良卵母细胞玻璃化冷冻液可以改善体外成熟卵母细胞的发育潜能,有助于获得更多的优质囊胚。
第二部分改良玻璃化冷冻对体外成熟卵母细胞形态及超微结构的影响
目的卵母细胞作为人体最大的细胞,细胞结构特点决定了其对低温和高渗环境更加敏感,同慢速冷冻一样,玻璃化冷冻也可能导致卵母细胞超微结构的改变。针对卵母细胞骨架系统中的微丝分布和电镜下超微结构形态,进一步验证改良卵母细胞玻璃化冷冻液对体外成熟卵母细胞解冻后形态和超微结构的影响。
方法收集常规ICSI患者捐赠的未成熟卵母细胞进行体外成熟培养,将体外成熟的卵母细胞用两种冷冻方案冷冻解冻后进行形态学和超微结构评价。实验组分为传统组(EG/DMSO冷冻法)和改良组(EG/DMSO/PROH冷冻法),对照组为新鲜体外成熟卵母细胞,直接固定后行超微结构观察。每组卵母细胞均进行共聚焦显微镜下微丝分布观察和透射电镜下超微结构观察。
结果倒置镜下,冷冻两组均有部分卵母细胞存在周边空泡状结构。改良组的空泡发生率为18.7%,远远低于传统组(66.7%)(P=0.000)。改良组的微丝分布正常率(43.8%)略高于传统组(33.3%),两者比较没有显著性差异(P>0.05)。改良组和传统组的微丝分布正常率均低于对照组(71.1%)(分别是P<0.05和P<0.01),透射电镜结果显示,无论是传统组还是改良组,冷冻复苏后卵母细胞中的空泡状结构略多于新鲜卵母细胞。而线粒体和空泡的形态结构与对照组比较没有明显差异,冷冻两组中卵母细胞皮质颗粒的数量略有减少,且更加绕周边分布,未观察到明显的皮质颗粒外排现象。结论改良玻璃化冷冻方案较传统玻璃化冷冻方案可以更加有效的改善卵母细胞解冻后的光镜下形态,两种玻璃化冷冻方案都会影响体外成熟卵母细胞的超微结构。
第三部分不同玻璃化冷冻液对卵母细胞发育潜能的影响及其临床结局评价
目的评价一种卵母细胞玻璃化冷冻液配方的优劣,最直接的证据是临床妊娠结果,本研究回顾性分析了使用不同玻璃化冷冻液玻璃化冷冻卵母细胞的实验室培养数据及临床结局,为改良冷冻液的推广应用找到临床证据。
方法收集2012年～2013年的卵母细胞解冻的所有患者的数据,包括自卵使用患者和接受供卵的患者。卵母细胞来源于2008年～2013年不同时间冷冻的卵母细胞。卵母细胞冷冻动机以获卵数超过22枚者为多,部分因为取卵日取精失败。解冻周期患者均采用替代周期方案,卵母细胞胞解冻后行黄体支持。记录冷冻卵母细胞所用的试剂类型(MC组、KT组和改良组)、解冻卵母细胞使用者的年龄、冷冻存活率、受精率、优胚率、囊胚形成率、冷冻和移植胚胎数、临床妊娠率及着床率。同时计算每解冻卵母细胞获得的胚胎利用率。
结果自卵使用数据中,卵母细胞的冷冻复苏率以改良组最高(92.0%),显著高于其他两组(MC和KT组分别是88.2%和77.3%)(P<0.05)。改良组受精卵分裂后获得的优胚率最高(35.8%),与MC和KT组(29.0%和28.3%)相比有显著性差异(P<0.05)。胚胎移植后的临床妊娠率与着床率各组之间没有显著性差异(P>0.05),MC、KT和改良配方组临床妊娠率分别是37.2%,30.2%和39.6%,着床率分别是21.9%、18.8%和27.4%。但改良组每周期胚胎移植个数是1.89±0.59,显著低于MC组(2.28±0.83)(P=0.000),也少于KT组(2.02±0.93)(P>0.05)。按照每解冻卵母细胞的效率看,改良配方组的卵母细胞授精后获得的优胚率显著高于MC组和KT组(P<0.01)。每解冻卵母细胞获得的胚胎利用率高于另外两组,但没有显著性差异(P>0.05)。在供卵使用数据中仅用到了MC试剂和KT试剂,两组比较,存活率、受精率、优胚率、临床妊娠率及各项卵母细胞利用率,均没有显著性差异(P>0.05),供卵组的各项数据均比自卵组好,临床妊娠率可以分别达到50%和43.5%。
结论改良冷冻液配方获得了较商品化试剂更高的存活率和优胚率,在移植更少胚胎的同时,没有降低临床妊娠率和着床率。相对于价格昂贵保质期超长的商品化试剂,改良冷冻液获得了更多的可利用胚胎,今后将继续观察改良冷冻液的使用效果,同时追踪冷冻胚胎的使用结果,以获得更有价值的冷冻卵母细胞累积利用率指标。
Part1Effects of modified vitrification media on the development potential of human vitrification-warmed in vitro matured oocytes
Objective To certify the effects of modified vitrification media on the developmenta potential of human in-vitro matured oocytes.
Methods Immature oocytes were collected from controlled ovarian stimulation cycles for ICSI treatment. All these immature oocytes were cultured for18-24hrs for maturation. Only matured oocytes were enrolled in this study. Gathered human in-vitro matured oocytes were randomly divided into control and three vitrified group.Group A was control group without vitrification treatment. Group B was vitrified with EG and DMSO. Group C was vitrified with EG and PROH. Group D was vitrified with EG, DMSO and PROH. The developmental potential was evaluated with survival rate, fertilization rate,2PN rate, cleavage rate of2PN, high-quality embryo rate, balstocyst rate and high-quality blastocyst rate.
Result The vitrification process affected the development potential of these in-vitro matured oocytes. The fertilization rates, high-quality embryo rates, and balstocyst rates in the three vitrification groups were significantly lower than the control group (P<0.01).Though the survival rate (84.8%),2PN rate (46.7%), high-quality embryo rate (5.7%), balstocyst rate (7.6%) in Group D were higher than those of in Group B (77.2%,42.4%,2.2%,2.2%, respectively) and Group C (80.0%,40.0%,2.2%,3.3%, respectively), there was no significant difference (P>0.05). In three vitrifcaiton goups, high-quality blastocysts were got only in Group D (1.9%). Though high-quality blastocysts in Group D was lower than that of in Group A (6.7%), there was no significant difference (P>0.05).
Conclusion The vitrification process reduced the development potential of the in-vitro matured oocytes. The modified vitrification media is effective for in-vitro matured oocytes vitrification through increasing the high-quality blastocyst rate.
Part2Effects of modified vitrification media on the morphorlogy and
ultrastructure of human vitrification-warmed in vitro matured oocytes.
Objective To study the effects of modified vitrification media on the morphorlogy and ultrastructure of human in vitro matured oocytes.
Methods Immature oocytes were collected from controlled ovarian stimulation cycles for ICSI treatment. All these immature oocytes were cultured for18-24hrs for maturation. Only matured oocytes were enrolled in this study. Gathered human in-vitro matured oocytes were randomly divided into control and two vitrified group. Matured oocytes in conventinonal vitrification group were vitrified with EG and DMSO. Modified vitrification group was vitrified with EG, DMSO and PROH. All the vitrified oocytes were warmed in1to5months. Control group was not given vitrification. The oocytes in control group and two vitrification groups were stained by Rhodamine-phalloidine and observed by laser confocal microscopy (LSCM). Some of the oocytes in the three groups were fixed and observed under transmission electron microscopy (TEM).
Result There were a lot of vesicles in part of the warmed oocytes in the two vitrification groups, distributed under the membrane of the oocytes. The vesicles enclosed oocytes rate was18.7%in the modified vitrification group, which was lower significantly than the conventional group (66.7%)(P=0.000). There was no significant difference between modified vitrification group and conventional group in the rate of microfilament normal distribution (43.8%vs33.3%, P>0.05), but lower significantly than the control group (71.1%)(P<0.05). TEM results revealed that there were more vesicles in the cytoplasm in the two vitrification groups comparing with the control group. The morphology of mitochondria and vesicles were similar between the vitrification groups and control group. The distribution of the cortical granules in the vitrification-warmed oocytes was nearer to the periphery of the oocytes. Though the cortical granules extrusion was not observed, the number of the cortical granules was less in the vitrificaiton-warmed oocytes
Conclusion The modified vitrification protocol is useful for the recovery of morphorlogy and ultrastructure of the vitrification-warmed human in vitro matured oocytes compared with conventional vitrification protocol. Vitrificaiton process affected the number and distribution of cortical granules.
Part3Effects of different vitrification media on the development potential of vitrification-warmed in vivo matured oocytes and clinical results
Objective To study the effects of different vitrification media on the development potential of vitrification-warmed in vivo matured oocytes and clinical results.
Methods This retrospective study was involved in all of the vitrification-warmed oocytes between2012January and2013December. All of the oocytes were vitrified in the last five years. Different vitrification media were used (MC media, KT media and modified media). After warming, the rates of oocyte survival, fertilization, high-quality embryo, blastulation, clinical pregnancy and implantation were recorded and analyzed. Also, the usage rate of every warmed oocyte was calculated. Data from self-oocytes warming and donor-oocytes warming were calculated separately.
Result For the self-oocytes warming group, the oocytes survival rate of modified media (92.0%) was significantly higher than the MC media (88,2%) and KT media (77.3%)(P<0.05). High-quality embryo rate in modified media group (35.8%) was significantly higher than the other groups (29.0%vs28.3%)(P<0.05). There was no significant difference in the clinical pregnancy rate and implantation rate between MC, KT and modified media groups (37.2%vs30.2%vs39.6%and21.9%vs18.8%vs27.4%)(P>0.05). In spite of that, the number of embryo transfered in every cycle (1.89±0.59) was significantly lower in modified group compared with MC media group (2.28±0.83)(P=0.000). When it comes to oocytes usage efficiency, the high-quality embryo rate for every warmed oocyte was higher significantly than other two groups (P<0.01). Though the utilization of the oocyte was higher than the other two groups, there was no significant difference (P>0.05). Only MC media and KT media were used in the donor-oocyte cycles. There was no statistical difference in the rates of survival, fertilization, high-quality embryo, clinical pregnancy and implantation (P>0.05). Compared with data in the self-oocytes group for MC media and KT media, the clinical pregnancy rates were perfectly higher (50%and43.5%, respectively).
Conclusion This clinical study showed that the modified vitrification media could get more high-quality embryos and higher clinical pregnancy rate. This modified media would be used effectively in clinical application since it is an efficient vitrification protocol. More subtle study should be carried out for a reliable conclusion.
引文
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[2]Gook DA, Schiewe MC, Osborn SM, et al. Intracytoplasmic sperm injection and embryo development of human oocytes cryopreserved using 1,2-propanediol. Hum Reprod,1995,10(10):2637-41.
[3]Porcu E, Fabbri R, Seracchioli R, et al. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril,1997,68(4):724-6.
[4]Kuleshova L, Gianaroli L, Magli C, et al. Birth following vitrification of a small number of human oocytes:case report. Hum Reprod, 1999,14(12):3077-9.
[5]Practice Committees of American Society for Reproductive Medicine; Society for Assisted Reproductive Technology. Mature oocyte cryopreservation: a guideline. Fertil Steril.2013 Jan;99(1):37-43.
[6]Coticchio G, Borini A, Distratis V, Maione M, Scaravelli G, Bianchi V, Macchiarelli G, Nottola SA. Qualitative and morphometric analysis of the ultrastructure of human oocytes cryopreserved by two alternative slow cooling protocols. J Assist Reprod Genet.2010; 27(4):131-40.
[7]Aye M, Di Giorgio C, De Mo M, et al. Assessment of the genotoxicity of three cryoprotectants used for human oocyte vitrification:dimethyl sulfoxide, ethylene glycol and propylene glycol. Food Chem Toxicol,2010, 48(7):1905-12.
[8]Martinez-Burgos M, Herrero L, Megias D, et al. Vitrification versus slow freezing of oocytes:effects on morphologic appearance, meiotic spindle configuration, and DNA damage. Fertil Steril,2011,95(1):374-7.
[9]Oktay K, Cil A, Bang H. Efficiency of oocyte cryopreservation:a meta-analysis. Fertil Steril 2006;86:70-80.
[10]Noyes N, Porcu E, Borini A. Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies. Reprod Biomed Online, 2009,18(6):769-76.
[11]Scaravelli G, Vigiliano V, Mayorga JM, et al. Analysis of oocyte cryopreservation in assisted reproduction:the Italian National Register data from 2005 to 2007. Reprod Biomed Online,2010,21(4):496-500.
[12]Selman H, Angelini A, Barnocchi N, Brusco GF, Pacchiarotti A, Aragona C. Ongoing pregnancies after vitrification of human oocytes using a combined solution of ethylene glycol and dimethyl sulfoxide. Fertil Steril.2006 Oct;86(4):997-1000. Epub 2006 Sep 11.
[13]Lucena El, Bernal DP, Lucena C, Rojas A, Moran A, Lucena A.Successful ongoing pregnancies after vitrification of oocytes. Fertil Steril.2006 Jan;85(1):108-11.
[14]Noyes N, Knopman J, Labella P, et al. Oocyte cryopreservation outcomes including pre-cryopreservation and post-thaw meiotic spindle evaluation following slow cooling and vitrification of human oocytes. Fertil Steril,2010, 94(6):2078-82.
[15]Rudick B, Opper N, Paulson R, Bendikson K, Chung K.The status of oocyte cryopreservation in the United States. Fertil Steril.2010 Dec;94(7):2642-6. Epub 2010 Jun 18.
[16]Tong XH, Wu LM, Jin RT, Luo LH, Luan HB, Liu YS. Fertilization rates are improved after IVF if the corona radiata is left intact in vitrified-warmed human oocytes. Hum Reprod.2012 Nov;27(11):3208-14.
[17]Song WY,Sun YP,Jin HX,Xin ZM,Su YC,Guo YH,Chen ZJ.Effects of cryopreservation time and thawing method of human oocyte vitrification on the outcome of assisted reproduction[J].Chinese Journal of Obstetrics and Gynecology,2010,45(8):578-582.
[18]宋文妍,孙莹璞,金海霞,辛志敏,苏迎春,郭艺红,陈子江.卵母细胞玻璃化冷冻在睾丸取精失败周期中的临床应用(附8例报告)[J].中华男科学杂志,2010,16(4).
[19]严正杰,蔡令波,冯婷,马龙,陈娟,王媁,冒韵东,刘嘉茵.卵母细胞玻璃化冷冻对胚胎发育及妊娠结局的影响[J].生殖医学杂志,2009,18(1).
[20]滕晓明；李昆明；王羽；潘家坪；尹萍；梁珊珊；阮井玲；陈智勤。卵母细胞玻璃化冷冻在体外受精-胚胎移植中的初步应用。中华男科学杂志2012；18(6)：531-533
[21]孙贻娟；冯云；张爱军；陆小微；牛志宏；谷瑞环。人成熟卵母细胞玻璃化冷冻技术的临床应用。中国优生与遗传杂志2012；20(7)：110-111,113
[22]Coticchio G, Bromfield JJ, Sciajno R, et al. Vitrification may increase the rate of chromosome misalignment in the metaphase II spindle of human mature oocytes. Reprod Biomed Online,2009,19 Suppl 3:29-34.
[23]Forman EJ, Li X, Ferry KM, Scott K,Treff NR, Scott RT Jr. Oocyte vitrification does not increase the risk of embryonic aneuploidy or diminish the implantation potential of blastocysts created after intracytoplasmic sperm injection:a novel, paired randomized controlled trial using DNA fingerprinting. Fertil Steril.2012 Sep;98(3):644-9.
[24]Chamayou S, Bonaventura G, Alecci C, et al. Consequences of metaphase II oocyte cryopreservation on mRNA content. Cryobiology,2011,62(2):130-4.
[25]Di Pietro C, Vento M, Guglielmino MR, et al. Molecular profiling of human oocytes after vitrification strongly suggests that they are biologically comparable with freshly isolated gametes. Fertil Steril,2010,94(7):2804-7.
[26]Bielanski A, Bergeron H, Lau PC, et al. Microbial contamination of embryos and semen during long term banking in liquid nitrogen. Cryobiology,2003, 46(2):146-52.
[27]Cobo A, Romero JL, Perez S, et al. Storage of human oocytes in the vapor phase of nitrogen. Fertil Steril,2010,94(5):1903-7.
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