卵母细胞玻璃化冷冻液配方的改良研究及临床应用
发布时间：2018-09-08 06:07
【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(P0.01),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(P0.05)。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(P0.05)。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%(P0.05),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%,P0.05)。 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(P0.05)。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P0.05和P0.01),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%)(P0.05)。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(P0.05)。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(P0.05),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)(P0.05)。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(P0.01)。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(P0.05)。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(P0.05),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。
[Abstract]:Part 1 the effect of modified vitrified fluid on the developmental potential of mature oocytes after cryopreservation.
Objective To study the unstable effect of vitrified oocytes cryopreservation and to design a vitrified cryoprotectant formula which can keep the osmotic property unchanged and reduce the concentration of various osmotic cryoprotectants. The immature oocytes donated by ICSI patients were used for vitrification. The freezing method was studied to verify the effect of the new modified vitrification solution on the vitrification of mature oocytes in vitro.
Methods Immature oocytes from ICSI patients were collected and cultured in vitro. The oocytes matured in vitro for 18-24 hours were divided into four groups randomly. Three groups were cryopreserved by different methods, namely, group B (EG/DMSO), group C (EG/PROH) and group D (EG/DMSO/PROH). Sperm and embryo culture, group A as control group, fertilization and embryo culture were carried out directly after maturation. The survival rate, fertilization rate, good embryo rate, blastocyst formation rate and high quality blastocyst rate were observed and calculated with each thawed oocyte as denominator.
Results Different vitrification refrigeration formulas had significant effects on the development potential of oocytes matured in vitro. The fertilization rate, the rate of superior embryo and blastocyst formation were significantly lower in group D than in group C (P 0.01). Although the survival rate, the rate of 2PN, the rate of superior embryo, the rate of blastocyst formation and the rate of superior blastocyst formation in group D were higher than those in group B and group C, there was no significant difference in statistical analysis. (P 0.05). The survival rate of group D was the highest (84.8%) and there was no significant difference between group B (77.2%) and group C (80.0%) (P 0.05). The 2PN rates of group D, B and C were 46.7%, 42.4% and 40.0%, respectively. The excellent embryo rates were 5.7%, 2.2% and 2.2%, and blastocyst formation rates were 7.6%, 2.2% and 3.3% (P 0.05). 0.05).
Conclusion Modified vitrification solution can improve the development potential of oocytes matured in vitro and help to obtain more high quality blastocysts.
The second part is the effect of modified vitrification on the morphology and ultrastructure of in vitro maturation oocytes.
Objective As the largest cell in human body, the characteristics of cell structure determine that oocytes are more sensitive to hypothermia and hyperosmotic environment. Like slow freezing, vitrification may also lead to ultrastructural changes of oocytes. Effect of oocyte cryopreservation on morphology and ultrastructure of mature oocytes in vitro after thawing.
Methods Immature oocytes from ICSI patients were collected and cultured in vitro. The oocytes were frozen and thawed in two freezing methods. The experimental group was divided into traditional group (EG / DMSO freezing method) and improved group (EG / DMSO / PROH freezing method). The control group was fresh oocytes matured in vitro. Ultrastructure of oocytes was observed after direct immobilization. Microfilament distribution and ultrastructure of oocytes were observed under confocal microscope and transmission electron microscope.
Results Under inverted microscope, some oocytes in both groups had peripheral vacuoles. The incidence of vacuoles in the improved group was 18.7%, which was much lower than that in the traditional group (66.7%) (P = 0.000). The normal distribution of microfilaments in the modified group (43.8%) was slightly higher than that in the traditional group (33.3%). There was no significant difference between the two groups (P 0.05). The normal cloth rate was lower than that of the control group (71.1%) (P 0.05 and P 0.01). Transmission electron microscopy (TEM) showed that there were slightly more vacuoles in oocytes after cryopreservation than fresh oocytes in both traditional and modified groups. Conclusion Modified vitrification is more effective than traditional vitrification in improving the morphology of oocytes after thawing. Both vitrification schemes can affect the in vitro maturation of oocytes. Ultrastructure.
The third part is the effect of different vitrification refrigerants on the developmental potential of oocytes and the evaluation of their clinical outcomes.
Objective To evaluate the advantages and disadvantages of a vitrified oocyte cryopreservation solution, the most direct evidence is the clinical pregnancy results. This study retrospectively analyzed the laboratory culture data and clinical outcomes of vitrified oocytes frozen with different vitrified cryopreservation solutions.
Methods The data of all patients with oocyte thawing from 2012 to 2013 were collected, including those who used the oocytes and those who received the donated oocytes. Patients were treated with alternative cycles and oocytes were thawed for luteal support. The types of reagents used in cryopreservation (MC, KT and modified groups), the age of the users of thawed oocytes, cryopreservation survival rate, fertilization rate, excellent embryo rate, blastocyst formation rate, number of frozen and transplanted embryos, clinical pregnancy rate and implantation rate were recorded. The utilization rate of embryos obtained from each thawed oocyte.
Results The frozen recovery rate of oocytes in the improved group was the highest (92.0%) and significantly higher than that in the other two groups (88.2% and 77.3% in the MC and KT groups, respectively) (P 0.05). The improved group had the highest rate of excellent embryos (35.8%) after cleavage, which was significantly different from that in the MC and KT groups (29.0% and 28.3%). The clinical pregnancy rates of MC, KT and modified formula groups were 37.2%, 30.2% and 39.6%, 21.9%, 18.8% and 27.4% respectively. However, the number of embryo transfer per week in the modified group was 1.89 (+ 0.59), significantly lower than that in the MC group (2.28 (+ 0.83) (P = 0.000) and less than that in the KT group (2.02 (+ 0.93) (P 0.05)). The efficiency of frozen oocytes was significantly higher in the improved formula group than in the MC and KT groups (P 0.01). The utilization rate of frozen oocytes was higher than that in the other two groups, but there was no significant difference (P 0.05). There was no significant difference (P 0.05) in the rate of good embryo, clinical pregnancy and utilization rate of oocytes. The data of donor group were better than that of self-ovulated group. The clinical pregnancy rate could reach 50% and 43.5% respectively.
Conclusion The improved cryogenic liquid formula has higher survival rate and better embryo rate than commercial reagent, and it does not reduce clinical pregnancy rate and implantation rate while transplanting fewer embryos. The use effect and the use result of frozen embryos were tracked to obtain more valuable index of cumulative utilization of frozen oocytes.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R714.8
本文编号：2229602
[Abstract]:Part 1 the effect of modified vitrified fluid on the developmental potential of mature oocytes after cryopreservation.
Objective To study the unstable effect of vitrified oocytes cryopreservation and to design a vitrified cryoprotectant formula which can keep the osmotic property unchanged and reduce the concentration of various osmotic cryoprotectants. The immature oocytes donated by ICSI patients were used for vitrification. The freezing method was studied to verify the effect of the new modified vitrification solution on the vitrification of mature oocytes in vitro.
Methods Immature oocytes from ICSI patients were collected and cultured in vitro. The oocytes matured in vitro for 18-24 hours were divided into four groups randomly. Three groups were cryopreserved by different methods, namely, group B (EG/DMSO), group C (EG/PROH) and group D (EG/DMSO/PROH). Sperm and embryo culture, group A as control group, fertilization and embryo culture were carried out directly after maturation. The survival rate, fertilization rate, good embryo rate, blastocyst formation rate and high quality blastocyst rate were observed and calculated with each thawed oocyte as denominator.
Results Different vitrification refrigeration formulas had significant effects on the development potential of oocytes matured in vitro. The fertilization rate, the rate of superior embryo and blastocyst formation were significantly lower in group D than in group C (P 0.01). Although the survival rate, the rate of 2PN, the rate of superior embryo, the rate of blastocyst formation and the rate of superior blastocyst formation in group D were higher than those in group B and group C, there was no significant difference in statistical analysis. (P 0.05). The survival rate of group D was the highest (84.8%) and there was no significant difference between group B (77.2%) and group C (80.0%) (P 0.05). The 2PN rates of group D, B and C were 46.7%, 42.4% and 40.0%, respectively. The excellent embryo rates were 5.7%, 2.2% and 2.2%, and blastocyst formation rates were 7.6%, 2.2% and 3.3% (P 0.05). 0.05).
Conclusion Modified vitrification solution can improve the development potential of oocytes matured in vitro and help to obtain more high quality blastocysts.
The second part is the effect of modified vitrification on the morphology and ultrastructure of in vitro maturation oocytes.
Objective As the largest cell in human body, the characteristics of cell structure determine that oocytes are more sensitive to hypothermia and hyperosmotic environment. Like slow freezing, vitrification may also lead to ultrastructural changes of oocytes. Effect of oocyte cryopreservation on morphology and ultrastructure of mature oocytes in vitro after thawing.
Methods Immature oocytes from ICSI patients were collected and cultured in vitro. The oocytes were frozen and thawed in two freezing methods. The experimental group was divided into traditional group (EG / DMSO freezing method) and improved group (EG / DMSO / PROH freezing method). The control group was fresh oocytes matured in vitro. Ultrastructure of oocytes was observed after direct immobilization. Microfilament distribution and ultrastructure of oocytes were observed under confocal microscope and transmission electron microscope.
Results Under inverted microscope, some oocytes in both groups had peripheral vacuoles. The incidence of vacuoles in the improved group was 18.7%, which was much lower than that in the traditional group (66.7%) (P = 0.000). The normal distribution of microfilaments in the modified group (43.8%) was slightly higher than that in the traditional group (33.3%). There was no significant difference between the two groups (P 0.05). The normal cloth rate was lower than that of the control group (71.1%) (P 0.05 and P 0.01). Transmission electron microscopy (TEM) showed that there were slightly more vacuoles in oocytes after cryopreservation than fresh oocytes in both traditional and modified groups. Conclusion Modified vitrification is more effective than traditional vitrification in improving the morphology of oocytes after thawing. Both vitrification schemes can affect the in vitro maturation of oocytes. Ultrastructure.
The third part is the effect of different vitrification refrigerants on the developmental potential of oocytes and the evaluation of their clinical outcomes.
Objective To evaluate the advantages and disadvantages of a vitrified oocyte cryopreservation solution, the most direct evidence is the clinical pregnancy results. This study retrospectively analyzed the laboratory culture data and clinical outcomes of vitrified oocytes frozen with different vitrified cryopreservation solutions.
Methods The data of all patients with oocyte thawing from 2012 to 2013 were collected, including those who used the oocytes and those who received the donated oocytes. Patients were treated with alternative cycles and oocytes were thawed for luteal support. The types of reagents used in cryopreservation (MC, KT and modified groups), the age of the users of thawed oocytes, cryopreservation survival rate, fertilization rate, excellent embryo rate, blastocyst formation rate, number of frozen and transplanted embryos, clinical pregnancy rate and implantation rate were recorded. The utilization rate of embryos obtained from each thawed oocyte.
Results The frozen recovery rate of oocytes in the improved group was the highest (92.0%) and significantly higher than that in the other two groups (88.2% and 77.3% in the MC and KT groups, respectively) (P 0.05). The improved group had the highest rate of excellent embryos (35.8%) after cleavage, which was significantly different from that in the MC and KT groups (29.0% and 28.3%). The clinical pregnancy rates of MC, KT and modified formula groups were 37.2%, 30.2% and 39.6%, 21.9%, 18.8% and 27.4% respectively. However, the number of embryo transfer per week in the modified group was 1.89 (+ 0.59), significantly lower than that in the MC group (2.28 (+ 0.83) (P = 0.000) and less than that in the KT group (2.02 (+ 0.93) (P 0.05)). The efficiency of frozen oocytes was significantly higher in the improved formula group than in the MC and KT groups (P 0.01). The utilization rate of frozen oocytes was higher than that in the other two groups, but there was no significant difference (P 0.05). There was no significant difference (P 0.05) in the rate of good embryo, clinical pregnancy and utilization rate of oocytes. The data of donor group were better than that of self-ovulated group. The clinical pregnancy rate could reach 50% and 43.5% respectively.
Conclusion The improved cryogenic liquid formula has higher survival rate and better embryo rate than commercial reagent, and it does not reduce clinical pregnancy rate and implantation rate while transplanting fewer embryos. The use effect and the use result of frozen embryos were tracked to obtain more valuable index of cumulative utilization of frozen oocytes.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R714.8
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