不同冷冻载体对小鼠成熟卵母细胞玻璃化冷冻的影响

目的:探讨玻璃化冷冻小鼠成熟减数第二次分裂中期(MⅡ期)卵母细胞的最佳冷冻载体及冷冻方法。方法:用乙二醇和蔗糖为玻璃化冷冻保护剂,将小鼠成熟MⅡ期卵母细胞分为冻融、暴露和对照组。冻融组分别采用普通麦管、封闭式拉细麦管(CPS)和开放式拉细麦管(OPS)作载体,采用玻璃化快速冷冻和快速复温的方法冻存小鼠成熟卵母细胞;暴露组卵母细胞仅暴露在冷冻和复苏液中,不进行冷冻;对照组卵母细胞不接触冷冻和复苏液。观察各组处理后卵子回收、受精、卵裂等情况。结果:冻融组普通麦管、CPS和OPS处理的卵母细胞回收率分别为92.0%、92.3%和72.3%,OPS处理卵母细胞的回收率显著低于普通麦管和CPS处理的卵母细胞(P<0.001);普通麦管、CPS和OPS处理的卵母细胞复苏存活率分别为43.5%、72.2%和76.5%,CPS和OPS处理卵母细胞的复苏存活率显著高于普通麦管组处理的卵母细胞(P<0.001)。冻融、暴露和对照组的卵裂率分别为38.6%、69.2%和73.5%,冻融组显著低于暴露和对照组(P<0.001)。结论:CPS法在玻璃化冷冻保存成熟的卵母细胞方面有价值。     

颗粒细胞在玻璃化冷冻中对卵母细胞的影响

目的 采用开放式拉细麦管(open pulled straws，OPS)法玻璃化冷冻技术，探讨卵丘颗粒细胞对未成熟卵母细胞冻融后存活率及其发育能力的影响。方法 小鼠生殖泡期卵丘-卵母细胞复合物(COC)或完全去颗粒细胞后的裸卵(DO)用OPS法玻璃化冻存，复苏后行体外成熟培养(IVM)；COC直接行IVM作为对照组。结果 COC及裸卵冻融后存活率(分别为69．49％、77．90％)、成熟率(分别为56．10％、56．74％)无显著性差异，但成熟率均显著低于对照组(82．28％)。结论 在未成熟卵的玻璃化冷冻中颗粒细胞对卵母细胞冻存无保护作用。     

OPS法及自制冷冻叶法玻璃化冷冻小鼠MⅡ期卵母细胞的研究

目的:以小鼠为研究模型,建立优化的玻璃化冷冻卵母细胞方法以及研究超排卵对卵母细胞质量的影响,为人类卵母细胞玻璃化冷冻技术的改善提供基础研究依据.方法: 建立小鼠超排卵周期模型,比较ops(open pulled straw,开放式拉细麦管)和(自制)冷冻叶两种玻璃化冷冻方法冷冻小鼠MⅡ期卵母细胞的复苏率及体外受精率.结果:超排卵周期组中(自制)冷冻叶法玻璃化冷冻卵母细胞复苏率为66.5%,高于OPS法的冷冻复苏率(45.2%),2者比较差异有统计学意义(P<0.05).(自制)冷冻叶法玻璃化冷冻卵母细胞冷冻后体外受精率为47.4%,高于OPS法的冷冻后体外受精率(32.9%),2者比较差异有统计学意义(P<0.05).结论:(自制)冷冻叶玻璃化法冷冻是小鼠MⅡ期卵母细胞玻璃化冷冻的较优方法.     

猪卵母细胞OPS玻璃化冷冻保存的研究

猪卵母细胞由于脂肪含量高,对低温敏感,相较其他动物卵母细胞的冷冻保存更加困难,因此在种质的保护保存、胚胎工程研究产生极大的阻碍。本试验研究了猪卵母细胞的OPS玻璃化冷冻保存方法,研究乙二醇、甘油和聚乙烯吡咯烷酮(PVP)作为冷冻保护剂,对比不同的浓度梯度和两种冷冻保护剂联合使用对冷冻效果的影响。研究未经体外成熟培养和不同时间体外成熟培养的猪卵母细胞的冷冻保存效果。对比改良优化的OPS玻璃化冷冻方法和传统的程序化冷冻方法的保存效果。实验表明,体外成熟12h(GV期)的猪卵母细胞用乙二醇添加聚乙烯吡咯烷酮(PVP)进行玻璃化冷冻,解冻后体外成熟率、细胞的形态正常率与未经体外成熟的的卵母细胞相比差异显著,与成熟48h(MII期)卵母细胞相比差异不显著。但卵裂率和桑椹胚囊胚率体外成熟12h(GV期)的猪卵母细胞用乙二醇添加聚乙烯吡咯烷酮(PVP)作为冷的保护剂组要好于其他组。     

激光扫描共聚焦显微镜观察冻存对SHR卵母细胞的影响

目的 冻存未受精的成熟卵母细胞（MII期）比冻存同一品系的处于卵裂期的胚胎更困难。因为卵母细胞的某些内部结构对低温很敏感。本研究的目的是希望能够更有效地观察冻存对卵母细胞的影响。方法 我们运用激光扫描共聚焦显微术（CLSM）的光学切片和三维图像重建技术，研究冻存对自发性高血压大鼠（SHR）卵母细胞的结构，尤其是对纺锤体、染色体的影响，以及冻存对体外受精的影响。结果 我们不仅观察到有的卵母细胞冻存后仍维持着完整的桶状纺锤体以及紧密排列的染色体，而且能观察到卵母细胞内均匀分布着细胞质微管星体。冻存也造成对一些卵母细胞纺锤体和细胞质微管星体的破坏。结论 CLSM能够清晰有效地观察冻存对未受精卵纺锤体、染色体以及胞质微管星体的影响，我们推想冻存后卵母细胞的体外受精率下降与这些结构的受损有关。     

3种不同载体对卵母细胞玻璃化冷冻效果的影响

目的以小鼠成熟卵母细胞(MⅡ期)为试验对象,应用3种不同载体对其行玻璃化冷冻,旨在分析载体对冷冻效果的影响。方法采用玻璃化冷冻技术冻存昆明种小鼠成熟卵母细胞,设新鲜对照组和玻璃化冷冻组,玻璃化冷冻组又分为开放式脉管(OPS)组、自制麦管组和Cryoleaf组。比较冻融后鼠卵的存活率、受精率、卵裂率及囊胚形成率。结果新鲜对照组和玻璃化冷冻组的受精率、卵裂率差异无统计学意义,但囊胚形成率差异有统计学意义(P<0.05);OPS组与自制麦管组的鼠卵存活率、受精率、卵裂率、囊胚形成率差异均无统计学意义;OPS组与Cryoleaf组比较,存活率差异有统计学意义(P<0.05),受精率、卵裂率、囊胚形成率差异均无统计学意义;自制麦管组与Cryoleaf组比较,存活率差异有统计学意义(P<0.05),而受精率、卵裂率、囊胚形成率差异均无统计学意义。结论载体的不同会对卵母细胞玻璃化冷冻效果形成一定程度的影响。     

一种安全有效的卵母细胞封闭式玻璃化冷冻载体

目的 探讨封闭式玻璃化冷冻载体冻存小鼠卵母细胞的可行性.方法 以小鼠MII期卵母细胞为模型,以开放式玻璃微细管法(GMP)为对照组,比较两种玻璃化冷冻载体对小鼠卵母细胞冷冻后的存活率、受精率、卵裂率及囊胚率的影响.结果卵母细胞经冻融后,封闭式冷冻载体组和GMP组的存活率、受精率、卵裂率和囊胚率均没有明显差异(92.80%vs 93.11%,49.80%vs 51.67%,36.73%vs 35.83%,12.65%vs 14.17%%;P>0.05).结论 封闭式冷冻载体能安全、有效的冷冻保存小鼠卵母细胞.     

Effect of three different loadings on vitrification of oocyte

目的 以小鼠成熟卵母细胞(MⅡ期)为试验对象,应用3种不同载体对其行玻璃化冷冻,旨在分析载体对冷冻效果的影响.方法 采用玻璃化冷冻技术冻存昆明种小鼠成熟卵母细胞,设新鲜对照组和玻璃化冷冻组,玻璃化冷冻组又分为开放式脉管(OPS)组、自制麦管组和Cryoleaf组.比较冻融后鼠卵的存活率、受精率、卵裂率及囊胚形成率.结果 新鲜对照组和玻璃化冷冻组的受精率、卵裂率差异无统计学意义,但囊胚形成率差异有统计学意义(P<0.05);OPS组与自制麦管组的鼠卵存活率、受精率、卵裂率、囊胚形成率差异均无统计学意义;OPS组与Cryoleaf组比较,存活率差异有统计学意义(P<0.05),受精率、卵裂率、囊胚形成率差异均无统计学意义;自制麦管组与Cryoleaf组比较,存活率差异有统计学意义(P<0.05),而受精率、卵裂率、囊胚形成率差异均无统计学意义.结论 载体的不同会对卵母细胞玻璃化冷冻效果形成一定程度的影响.     

玻璃化冷冻对不同成熟阶段小鼠卵母细胞的影响

目的：通过比较玻璃化冷冻对不同成熟阶段小鼠卵母细胞冷冻复苏、体外成熟、胚胎发育及细胞骨架的影响,寻求最佳的卵子冷冻方案。方法：玻璃化冷冻生殖泡期卵（GV）、成熟中期卵（MⅡ）及体外成熟卵（IVM-MⅡ）,解冻复苏后,分别作体外成熟、体外受精（IVF）、胚胎培养或固定作免疫荧光标记,统计复苏率、成熟率、受精率、囊胚率、细胞骨架正常率。结果：GV冷冻组、MⅡ冷冻组及IVM-MⅡ冷冻组三组复苏率差异无显著性（65%vs 60.92%vs 69.93%,P〉0.05）。GV冷冻组的成熟率显著低于对照组（79.6%vs 96.19%,P〈0.01）。GV冷冻组、MII冷冻组及IVM-MⅡ冷冻组三组受精率均明显低于对照组（P〈0.01）,且IVM-MⅡ冷冻组受精率显著低于MⅡ冷冻组（18.06%vs 31.34%,P〈0.01）。IVM-MⅡ冷冻组囊胚率低于MⅡ冷冻组和对照组,差异均有显著性（28.57%vs 52.38%,P〈0.05;28.57%vs 57.14%,P〈0.01）。GV冷冻组染色体和纺锤体均正常率均高于IVM-MⅡ冷冻组（53.85%vs 26.67%,P〈0.05）。IVM-MⅡ冷冻组细胞骨架三项指标均显著低于对照组,差异均有显著性（P〈0.05,P〈0.01）。结论：GV卵先玻璃化冷冻再体外成熟,其细胞骨架损伤较小,胚胎发育较好。     

小鼠卵母细胞程序化和玻璃化冷冻效果的比较

目的 本实验通过建立小鼠的实验模型,比较程序化和OPS玻璃化冷冻效果,探讨卵子冷冻的具体方法 ,为卵子冷冻技术应用于临床提供实验依据.方法 通过促排卵获取小鼠MII期卵母细胞,随机分组,观察不同冷冻方法 、高浓度的冷冻保护液,对卵子的成活及进一步发育潜能的影响;不同的IVF前培养时间对冻融卵子的受精及继续发育的影响.结果 (1)OPS玻璃化冷冻组与程序化冷冻组的存活率、受精率、卵裂率及6-8cell胚形成率均无显著差异.(2)玻璃化冷冻组与暴露组的卵母细胞存活率分别为35.3%,100%,有显著性差异(P＜0.05).暴露组与对照组比较的受精率、卵裂率,无显著性差异(P＞0.05).而暴露组与对照组的6-8cell的胚形成率分别为41%,63%,两组比较,有显著差异(P＜0.05).(3)无论OPS玻璃化冷冻法还是程序化冷冻法,冻融后的培养2 h组和培养1 h组的,受精率、卵裂率,两组比较,均有显著性差异(P＜0.05).结论 本实验证实,OPS玻璃化冷冻法至少可以达到程序化冷冻法同样的效果,而OPS玻璃化冷冻法有好于程序化冷冻法的趋势.加之,其简便、省时、经济等优点,因而,更有发展前景.     

Effect of Incubation Temperature and Warming Solutions on the Viability and Maturation of Vitrified-thawed Human Immature Oocytes

Objective To investigate the viability and maturation of frozen-thawed human immature oocytes exposed to different temperature of vitrification and warming solutions. Methods The immature oocytes in germinal vesicle (GV) and matephase I (MI ) stages were collected from our ICSI patients and exposed to different temperature of vitrification and warming solutions before frozen in the freezing/thawing procedures. The different temperature groups were as follows: Group A, equilibration solution at 37℃, vitrification solution at room temperature, warming solution at 37℃; Group B, both vitrification and warming solution at room temperature; Group C, both vitrification and warming solutions at 37℃; Group D, the frozen-thawed oocytes and the fresh oocytes were cultured for in vitro maturation. The survival rate and maturation rate were compared among groups. The oocytes were examined using immunofluorescent stainingand confocal microscopy to check the spindle configuration and chromosome arrangement. Results The survival rates and MII rates of GV stage oocytes in groups A, B, C were 100%(15/15), 81.3%(13/16), 68.8%(11/16) and 33.3%(2/6), 83.3%(10/12),72.7%(8/11), respectively. The survival rate of group C was significantly lower than that of the control (P<0.05). The normal spindle and chromosome configuration were only observed in group B, with the rates of 20% (2/20) and 10% (1/10), respectively. The survival rates of MI stage in groups A, B, C were 71.4%(10/14), 100% (12/12) and 83.3%(10/12), no significantly difference from that of the contro1(100%, 14/14). The MII rates of MI stage in groups A, B and C were 0%(0/14), 66.7%(8/12) and 80% (8/10), respectively. The MⅡ rate in group A was significantly lower than that in other groups (P<0.01). Only one oocyte in group C was found with normal spindle and chromosome configurations. Conclusion The appropriate operation temperature of vitrification and warming solutions can improve the outcomes of the vitrified-thawed human immature oocytes.     

PVP对人卵母细胞玻璃化冻融效果的影响

目的 探讨在玻璃化冷冻液中添加大分子物质PVP(polyvinyl pyrrolidone)对人卵母细胞冻融效果的影响.方法 玻璃化液中添加6% PVP者为A组,不添加者B组,未冻融者为对照组(C组),冻融后存活及对照组卵母细胞固定后进行免疫荧光染色,然后用Nikon CISI激光扫描共聚焦显微镜观察纺锤体、染色体形态...     

体内、体外产生的小鼠桑葚胚玻璃化冻存后的发育能力

目的　探讨体内、体外产生的小鼠桑葚胚玻璃化冻存后的发育能力。方法体内、体外产生的小鼠桑葚胚分别被含有乙烯甘醇、Ficoll和蔗糖的玻璃化溶液处理后冻存。结果体内产生的小鼠桑葚胚单纯暴露于玻璃化溶液中 ,而未冻存时其发育到胚泡期的百分率为 97 3% (一步法 )和 98 4% (二步法 ) ,与对照组 (98 1% )均无显著差异 (P >0 0 5 ) ,体外产生的小鼠桑葚胚单纯被玻璃化溶液处理后发育到胚泡期的百分率分别为 81 8% (一步法 )、82 4% (二步法 )和 83 6 % (对照组 ) ,三者之间无显著差异 (P >0 0 5 )。但是 ,体外产生的小鼠桑葚胚经玻璃化冻存融解后 ,发育到胚泡期的百分率为 70 6 % (一步法 )、81 3%(二步法 )和 83 6 % (对照组 ) ,一步法玻璃化组明显低于二步法玻璃化组和对照组 (P <0 0 5 ) ,而在体内产生的小鼠桑葚胚玻璃化冻存融解后 ,发育到胚泡期的百分率为 93 1% (一步法 )和 95 7% (二步法 ) ,二者之间无显著差异 (P >0 0 5 )。所有玻璃化冻存的小鼠桑葚胚融解后 ,显微镜下观察均未见透明带破裂。结论体内、体外产生小鼠桑葚胚胎经玻璃化冻存融解后 ,可保持较高的存活率和发育能力 ,二步法玻璃化更适合冻存体外产生的小鼠桑葚胚。     

Experimental Study on Meiotic Spindles and Chromosomes of Mouse Mature (MⅡ) Stage Oocytes under Laser Scanning Confocal Microscopy

Taking the mouse as a model, the experimental method of observing the morphology of meiotic spindles and chromosomes in mature oocytes were investigated in order to evaluate the effects of various interventions on the quality of oocytes accurately and rapidly. Laser scanning confocal microscope (LSCM) was used to examine the meiotic spindles and chromosomes by the technologies of optical section and three-dimensional (3D) image reconstruction. The results showed that the configurations of meiotic spindles and chromosomes could be observed clearly by LSCM.The normal rate of meiotic spindles and chromosomes was 82% and 86% respectively. It was concluded that the LSCM was a valid instrument to observe the meiotic spindles and chromosomes of mature oocytes and could be used as a valid method to evaluate the quality of M Ⅱ oocytes.     

IVF受精失败卵子纺锤体、染色体的立体结构分析

目的：探索受精失败卵子纺锤体、染色体的立体结构变化及其相关因素。方法：选自本所受精卵子的材料分两组实验组：卵子来源于IVF取卵后24～40h受精失败的MⅡ期卵子。对照组：为未授精卵子。两组卵子通过免疫荧光染片后,用Nikon激光共聚焦显微镜观察染色体、纺锤体的立体结构,根据年龄段、促排卵药的差别进行分组,用t和χ^2检验进行统计学分析。结果：年龄小于30岁组的纺锤体、染色体正常率分别为：13．3％、13．3％,与对照组相比差异均无显著性,（P〉0．05）。30～35岁组的纺锤体、染色体正常率分别为：16％,12％,纺锤体正常率与对照组差异相比,无显著性,（P〉0．05）,染色体正常率与对照组相比,差异有显著性,（P〈0．05）。年龄大于35岁组的纺锤体、染色体正常率分别为：0％、0％,与对照组相比均差异有显著性,（P〈0．05）。合计年龄小于35岁患者受精失败卵子的纺锤体、染色体正常率分别为：15％、12．5％,与对照纽比,无显著性差异。果纳芬组的纺锤体及染色体的正常率均为12．5％,与对照相比,无显著性差异,（P〉0．05）。丽申宝组纺锤体及染色体的正常率分别为11．1％,5．6％,纺锤体正常率与对照组相比,差异无显著性,（P〉0．05）,染色体正常率与对照组相比,差异有显著性,（P〈0．05）。实验组的总的纺锤体和染色体正常率为12％、10％,与对照组相比均差异有显著性,（P〈0．05）。结论：高龄妇女受精失败卵子的纺锤体和染色体的正常率下降;用丽申宝进行超促排卵的受精失败卵子染色体正常率下降;染色体非整体倍增加的一个相关因素,可能对纺锤体正常梭形结构的破坏。     

Experimental Study on Meiotic Spindles and Chromosomes of Mouse Mature （MⅡ） Stage Oocytes under Laser Scanning Confocal Microscopy

The in vitro culture,maturation and cryopr-eservation of oocytes are all the i mportant parts inthe assisted reproductive technology.Mature oo-cyte(i.e.MⅡoocytes)is arrested at the meta-phase stage of meiosisⅡ,in which chromosomesare attached to the spindle microtubles and line uponthe equatorial plateinthe normal spindles.Dis-organized structure of spindles can not onlylead tosuch morphologic changes as unwound chromo-somes but also the chromosome nondisjuction andresultant aneuploid.When t…     

Spindle and Chromosome Analysis of Non-Fertilized Oocytes from Conventional in vitro FertiliTation for Non-Male Factor Infertiliy： Significance of Rescue ICSI？

Objective To determine whether rescue intracytoplasmic sperm injection (ICSI) is associated with improved outcomes for non-male factor infertility. Methods The changes of micro-structure, including meiotic spindle and chromosome distribution, sperm penetration, as well as oocyte activation were compared in IVF (in vitro fertilization) fertilization failure (IVF FF) patients, ICSI patients with non-fertilized oocytes (ICSI NF) and oocytes in vitro maturation (IVM). Results A total of 164 unfertilized oocytes (93 oocytes in IVF and 71 oocytes in ICSI) and 56 IVM oocytes were available for this study. The abnormality of spindle and chromosomes was significantly higher in IVF FF group than that in ICSI NF group or IVM group (abnormal spindle rates were 80.6%, 64.8% and 57.1%, respectively; abnormal chromosome rates were 91.4%, 80.3% and 75.0%, respectively). No sperm penetration after IVF and sperm expulsion after ICSI were 78.5% and 40.8%, respectively. Activation failure occurred in 16.1% of the IVF FF cases and 49.3% of ICSI NF oocytes. Conclusion Rescue ICSI of fertilization-failed oocytes fails to lead outcome improvement due to the internal defects of oocytes.     

Study on the developing potentiality of mouse morula produced in vitro or in vivo after vitrification

Great horovement haS been made in the cryopreservation Of inalnlnalian elnbmp since wnttingham['] reported his Pioneering wold about successful cryOPreservationof mouse embryo. It results in the availability of cryOPreserVation of embryos of all stages[']. HOwever, cryopreserved embryos can not avoid the damages by ice crystal,which is a major cause of cen death in the freezing andthawing ProceduresL',']. SO it is necesseq to seareh for anew and more efficient method for embryO cryOPrese…     

不同浓度冷冻保护剂乙二醇在人扩张囊胚玻璃化冷冻中的应用

目的研究不同浓度冷冻保护剂乙二醇(ethlene glycol,EG)在人扩张囊胚玻璃化冷冻中的应用。方法不同浓度EG 15%,30%,40%,联合15%二甲基亚砜(dimethylsulphoxide,DMSO),0.5 mol/L蔗糖(sucrose),采用两步法,利用改制半麦管为冷冻载体,玻璃化冷冻人扩张囊胚。比较解冻后囊胚存活率,扩张率及孵出率;并比较微吸管吹吸、辅助孵化针刺破胞膜辅助脱水及未辅助脱水对冷冻扩张囊胚的影响。结果由1原核(pronucleus, PN)发育而来的扩张囊胚玻璃化冷冻解冻后囊胚存活率15%EG组明显高于30%及40%EG组(P<0.05)。由2PN 发育成的扩张囊胚玻璃化冷冻解冻后囊胚孵出率15%EG组明显高于30%及40%EG组(P<0.05)。在冷冻过程中给予人工辅助脱水组解冻后囊胚孵出率明显高于未辅助脱水组(P<0.05)。微吸管吹吸组与辅助孵化针刺破胞膜辅助脱水组囊胚孵出率差异无显著性(P>0.05)。结论15%EG为人扩张囊胚玻璃化冷冻中有效和合适浓度。玻璃化冷冻中给予人工辅助脱水,能提高解冻后囊胚的发育潜能。