人类不同时期未成熟卵母细胞玻璃化冷冻后发育潜能的比较

目的:探索未成熟卵子的最佳冷冻时期。方法:收集卵胞质内单精子显微注射-胚胎移植(ICSI-ET)周期中未成熟的卵母细胞,按其成熟度分为生发泡期(GV组)卵子179枚和第一次减数分裂中期(MI组)卵子323枚,所有卵子均经玻璃化冷冻,解冻后行体外成熟(IVM)培养,ICSI受精,观察比较GV组和MI组解冻后存活、体外成熟、受精及胚胎发育情况。结果:GV组复苏存活率显著高于MI组(83.24%vs 75.54%,P=0.045),MI组体外成熟率、受精率、卵裂率、优质胚胎率均略均高于GV组,但无统计学差异(P0.05),MI组可利用胚胎率显著高于GV组(78.67%vs 60.53%,P=0.041)。结论:超促排卵周期中未成熟卵母细胞先玻璃化冷冻保存,再行体外培养是可行的。GV期卵母细胞复苏存活率高于MI期卵母细胞,但MI期卵母细胞冻融后发育潜能优于GV组卵母细胞。     

有无卵丘细胞对玻璃化冷冻后小鼠卵母细胞细胞骨架及体外发育能力的初步研究

目的:探讨卵丘细胞在玻璃化冷冻中对小鼠卵母细胞发育潜能及细胞骨架的影响。方法:采用玻璃化冷冻技术,保存带卵丘细胞或完全剥除卵丘细胞的小鼠MⅡ期和GV期卵丘复合体/裸卵(COC/DO),复苏且GV卵体外培养成熟后分别作体外受精或免疫荧光标记检查纺锤体和染色体的完整性。结果:MⅡ-COC和GV-COC的复苏率均显著高于MⅡ-DO和GV-DO(分别为86.49%vs60.92%和85.94%vs64.93%,P<0.01)。GV-COC组的受精率、囊胚率均高于GV-DO组,且MⅡ-COC组和GV-COC组的纺锤体和染色体均正常率均分别高于MⅡ-DO组和GV-DO组,但无显著性差异(P>0.05)。结论:卵丘细胞在玻璃化冷冻卵母细胞中能有效减少冷冻对细胞骨架的损伤,并改善卵子复苏及胚胎发育潜能。     

不同冷冻法对小鼠未成熟与成熟卵母细胞的成熟及胚胎发育的影响

本研究观察应用慢速冷冻．快速复温(常规冷冻法)与玻璃化冷冻法，对小鼠生殖泡期未成熟及第二次减数分裂中期(metaphageⅡ，MⅡ)成熟卵母细胞冷冻后的解冻、体外成熟、受精及早期胚胎发育的影响。     

人类未成熟卵母细胞玻璃化冷冻研究

目的:探讨玻璃化冷冻未成熟卵母细胞的有效性。方法:根据有无颗粒细胞将实施玻璃化冷冻的GV期卵母细胞分为含颗粒细胞(非裸卵)组和不含颗粒细胞(裸卵)组;将部分GV期卵母细胞体外培养至MⅡ期卵母细胞实施玻璃化冷冻,比较非冷冻IVM组与MⅡ卵玻璃化冷冻组间、裸卵组与非裸卵组间的存活率、成熟率、受精率、卵裂率及囊胚形成率。结果:非裸卵组的成熟率大于裸卵组(P<0.05),而存活率、受精率、2-细胞形成率、>2-细胞形成率之间均无统计学差异(P>0.05)。另外,非冷冻IVM组与GV玻化组间成熟率、受精率、卵裂率均存在显著性差异(P<0.05);非冷冻IVM组与MⅡ期卵玻化组间成熟率、受精率、卵裂率间均存在统计学差异(P<0.05);GV玻化组与MⅡ玻化组间存活率、成熟率、受精率、卵裂率间均无统计学差异(P>0.05)。结论:玻璃化冷冻未成熟卵母细胞需要保留颗粒细胞,同时初步构建了人GV期卵的玻璃化冷冻联合IVM技术的雏形。     

冻融体外受精-胚胎移植周期人未成熟卵母细胞的体外成熟及纺锤体结局

目的:探讨冻融的不同状态人未成熟卵母细胞体外成熟后纺锤体状态与受精率的关系。方法:随机收集本中心108个体外受精-胚胎移植周期(IVF)中183枚不同状态废气的成熟卵母细胞,分为:卵丘-卵母细胞复合物组,48枚;裸卵组,135枚,其中第一次减数分裂中期(MI)65枚,生发泡期(GV)70枚。玻璃化冷冻保存,经解冻、体外培养成熟后,应用Polscope成像系统观察纺锤体,然后行卵胞浆内单精子显微注射受精,记录各指标情况。结果:①卵丘-卵母细胞复合物组与裸卵组的存活率、体外成熟率、纺锤体出现率、受精率比较,均无统计学差异(P>0.05);②GV组的存活率显著高于MI组(P<0.05),而前者的体外成熟率显著低于后者(P<0.05);③各组体外成熟后有纺锤体出现的卵母细胞受精率均显著高于无纺锤体组(P<0.05,P<0.01)。结论:冻融IVF周期不同状态的人未成熟卵母细胞都有一定发育潜能;有纺锤体出现的冻融人未成熟卵母细胞质量较高。     

激活素-抑制素-卵泡抑素系统对小鼠卵母细胞体外成熟及早期胚胎发育潜能的影响机制

目的:探讨激活素-抑制素-卵泡抑素(ACT-INH-FS)系统在小鼠生发泡(GV)期卵母细胞体外成熟及早期胚胎发育中的的影响机制。方法:取小鼠GV期卵母细胞,随机分成8组,A组:100ng/ml激活素A(ACTA);B组:100ng/mlACTA+100ng/ml卵泡抑素(FS);C组:100ng/ml抑制素A(INHA);D组:100ng/mlINHA+100ng/mlFS;E组:100ng/mlACTA+INHA;F组:100ng/mlACTA+100ng/mlINHA+100ng/mlFS;G组:100ng/mlFS;H组(对照组):人输卵管液(HTF)+10%血清蛋白代用品(SPS)。体外培养16-18h后,MⅡ期卵母细胞进行体外受精,观察ACTA-INHA-FS对GV期卵母细胞生发泡破裂(GVBD)、第一极体(PB1)排出及受精率、卵裂率、囊胚形成率的影响。使用MitoTrackerRed荧光探针对A组及E组MⅡ期卵母细胞内活性线粒体数目及分布进行检测。结果:与H组比,A、E、G组GVBD发生率、A、C、E组的MⅡ期卵母细胞发生率和4-细胞、8-细胞及囊胚形成率均显著增加,C组受精率下降,P均<0.05。A、E组卵母细胞内线粒体形态半周型及弥散型比例明显高于H组,整体分布存在显著性差异(P<0.01)。结论:①ACTA、INHA对GV期卵母细胞体外成熟及胚胎发育潜能具有显著的促进作用,并可以被FS所拮抗。②在ACTA/INHA缺乏或不足的条件下,FS可出现ACTA样作用,促进卵母细胞成熟。③ACTA-INHA对体外成熟卵母细胞核质成熟同步化具促进作用。     

玻璃化冷冻保存技术对体外成熟卵母细胞发育潜能和转录组的影响

目的探索玻璃化冷冻保存技术对体外成熟(in vitro maturation,IVM)卵母细胞发育潜能和转录组的影响。方法选择2014年9月—2016年3月期间在北京大学第三医院生殖医学中心接受腹腔镜手术联合经阴道穿刺取卵行卵母细胞IVM治疗的不孕症患者为研究对象进行前瞻性队列研究,将研究对象分为IVM组(A组,n=13)和IVM-冻卵组(B组,n=24)。分析卵母细胞的早期胚胎发育状况和单细胞水平的转录组学情况。结果 IVM后玻璃化冷冻-解冻对存活卵母细胞的早期胚胎发育没有明显影响,受精率、卵裂率、优质胚胎率和可移植胚胎率组间差异均无统计学意义。但IVM后行玻璃化冷冻-解冻对转录组存在一定影响,与A组相比,B组共有1 913个基因呈现差异表达,其中570个基因表达量升高,主要富集到59个生物学过程;1 343个基因表达量降低,主要富集到140个生物学过程,而且一些差异基因参与了卵母细胞质量、受精和胚胎发育潜能的调控。结论 IVM后行玻璃化冷冻-解冻对早期胚胎发育没有明显影响,但对转录组具有一定影响,且发生表达量变化的基因与卵母细胞质量、受精和胚胎发育潜能有关,提示玻璃化冷冻-解冻有可能影响子代安全性。     

玻璃化冷冻和程序化冷冻方法对人类卵母细胞骨架及其发育潜能的影响

目的探讨玻璃化冷冻和程序化冷冻对人卵母细胞纺锤体定位、细胞骨架及其发育潜能的影响。方法将第2日发育为M_II卵母细胞随机分为对照组、程序化冻融组、玻璃化冻融组(解冻0 h、1 h、3 h)。应用液晶偏振光显微镜(Polscope)成像系统观察卵母细胞纺锤体与第一极体(Pb)的夹角、表面积、卵透明带内层光阻值和外层光阻值。采用扫描电子显微镜和透射电子显微镜观察卵母细胞的表面和内部超微结构。统计2种冻融方法对卵母细胞发育潜能的影响。结果对照组、程序化冷冻解冻培养3 h组和玻璃化冷冻解冻后培养0 h、1 h、3 h组中的纺锤体可见率分别为92.4%、56.4%、11.2%、24.8%、61.1%。与程序化冻融组相比,玻璃化冷冻解冻培养3 h后卵母细胞中纺锤体与Pb的夹角更小(37.3°与68°,P=0.023)。对照组、程序化冻融组和玻璃化冻融后培养3 h组中卵母细胞的纺锤体面积、卵透明带内层光阻值和透明带外层光阻值差异无统计学意义(P0.05)。与程序化冻融组相比,玻璃化冻融后培养3 h组中卵母细胞表面突起丰富,微绒毛形态较为正常,倒伏在细胞表面,卵透明带边界较为清晰,与对照组较为接近。程序化冻融组的正常受精率(65.7%)明显低于对照组(79.2%,P=0.041),而卵裂率和囊胚形成率与对照组和玻璃化冻融组差异无统计学意义(P0.05)。玻璃化冻融后培养3 h组中正常受精率、卵裂率、囊胚形成率与对照组相比,差异无统计学意义(P0.05)。结论相比程序化冷冻,玻璃化冷冻对卵母细胞纺锤体和卵透明带的损伤及对卵母细胞的发育潜能的影响都较小,可以作为卵母细胞冷冻的一种有效方法。     

人成熟卵母细胞玻璃化冻存时长对体外受精-胚胎移植结局的影响

目的:探讨玻璃化冷冻人成熟卵母细胞的时长是否会影响解冻后卵细胞的复苏率、受精率和临床妊娠结局。方法:回顾性分析2013年5月至2018年6月就诊于北京大学第三医院生殖医学中心行成熟卵母细胞玻璃化冷冻,随后进行解冻受精形成胚胎培养移植的110例患者114个冷冻卵母细胞周期的临床资料。根据卵细胞冻存时间分为3组,A组(冻存时间≤3个月)42例,B组(3个月<冻存时间≤12个月)42例,C组(冻存时间>12个月)30例,比较3组的卵细胞解冻复苏率、受精率、可利用胚胎率、优质胚胎率、临床妊娠率和活产率等指标。结果:114个冷冻卵母细胞周期中最短冻存时间4天,最长32个月,共行胚胎移植周期114个,其中新鲜胚胎移植周期77个,解冻胚胎移植周期37个。共解冻成熟卵母细胞1578枚,复苏率为77.69%(1226/1578)、受精率为69.35%(774/1116)、卵裂率为93.41%(723/774)、可移植胚胎率为42.19%(305/723)、优质胚胎率为33.06%(239/723)、每冻卵活产率为2.47%(39/1578)。3组年龄、不孕年限、基础卵泡刺激素(FSH)水平、...     

细胞松弛素B预处理人卵行玻璃化冻融后纺锤体与染色体变化的研究

目的:探讨细胞松弛素B(cytochalasin B,CCB)预处理对改善人卵玻璃化冷冻的效果。方法:收集常规IVF-ET或ICSI-ET周期中成熟卵母细胞71枚,随机分成4组行玻璃化冷冻。A组(17枚)、B组(20枚)、C组(17枚)分别用CCB预处理20min、30min、40min后行玻璃化冷冻,D组(17枚)不用CCB处理直接玻璃化冷冻。冻存3周后融冻卵母细胞,复苏的卵母细胞行荧光染色固定,在共聚焦显微镜下观察各组卵母细胞纺锤体及染色体形态结构。结果:A、B、C、D各组间的复苏率(70.59%、64.71%、50.00%和64.71%)无统计学差异(P>0.05),4组间的纺锤体及染色体正常形态率(27.27%,30.00%,45.46%及33.33%)亦无统计学差异(P>0.05)。结论:冷冻使成熟卵母细胞纺锤体及染色体的正常形态结构受到一定程度的破坏,CCB并不能保护卵母细胞纺锤体及染色体在冻融过程中的损伤。     

冷冻保存人类未成熟卵母细胞对其发育潜能的影响

目的 评价冷冻保存人类未成熟卵母细胞对其发育潜能的影响。方法 收集常规胞质内单精子显微注射-胚胎移植（ICSI-ET）中不成熟的卵母细胞168个,根据成熟程度分为生发小泡期（GV）卵母细胞103个和第1次减数分裂中期（MI）卵母细胞65个,再将各期卵母细胞分别分为冷冻组和对照组,冷冻组细胞经慢速冷冻-快速融解（慢冻-速融）后在体外培养成熟,对照组直接进行体外培养成熟。最后进行免疫荧光染色并观察。结果 GV冷冻组和GV对照组卵母细胞之间体外成熟率（分别为69．1％、74．3％）比较,差异无统计学意义（P〉0．05）;GV冷冻组和GV对照组卵母细胞之间纺锤体形态正常率（分别为28．9％、53．9％）和染色体形态正常率（分别为23．7％、50．0％）比较,差异有统计学意义（P〈0．05）;MI冷冻组和MI对照组卵母细胞之间体外成熟率（分别为68．6％、88．0％）比较,差异无统计学意义（P〉0．05）。MI冷冻组和MI对照组卵母细胞之间纺锤体形态正常率（分别为20．8％、54．6％）和染色体形态正常率（分别为25．0％、63．6％）比较,差异有统计学意义（P〈0．05）;GV冷冻组和MI冷冻组之间各项结果比较,差异均无统计学意义（P〉0．05）。结论 应用常规慢冻．速融方案并不能很好地保存未成熟卵母细胞的体外成熟后的纺锤体形成能力,未成熟的卵母细胞的发育潜能从而受损。     

Maintenance of the meiotic spindle during vitrification in human and mouse oocytes

Vitrification appears to be a viable method for the cryopreservation of human metaphase II (MII) oocytes, but concerns regarding the concentration of cryoprotectants used during vitrification have been raised. In an attempt to circumvent this potential problem, the majority of protocols are carried out at room temperature. Exposing oocytes to temperatures below 37 degrees C, however, leads to rapid microtubule depolymerization. Polarized light microscopy was used to measure meiotic spindle retardance following exposure to cryoprotectants and vitrification in human and mouse oocytes. To quantify the extent of depolymerization, spindle retardance was determined before and after each treatment. Exposure to vitrification and warming solutions at room temperature (21-22 degrees C) caused the spindle of mouse MII oocytes to depolymerize. In contrast, no measurable changes in the meiotic spindle were detected by maintaining the temperature at 37 degrees C during the exposure regimen. By carrying out the entire vitrification and warming procedure at 37 degrees C, the spindle was also unaffected. Comparable results were obtained with vitrification of human MII oocytes at 37 degrees C. Analysis of sibling human oocytes demonstrated that slow freezing, in contrast to vitrification, was unable to preserve the meiotic spindle. Using a vitrification protocol employing 37 degrees C impacts negligibly on the meiotic spindle. Thus, fertilization can proceed without having to await spindle reformation.     

Interactions of the meiotic spindle with mitotic chromosomes in GV mouse oocytes

During mitosis, a spindle checkpoint detects chromosome misalignment and halts the cell cycle progression. In meiosis of female germ cells, however, it is debatable whether such a checkpoint is present. This research employed a unique model in the mouse, mitotic chromosomes transferred to meiotic cytoplasts to investigate whether a meiotic oocyte's microtubule apparatus can effectively separate mitotic metaphase chromosomes, and whether a spindle checkpoint exists during its division. The intact germinal vesicle (GV) oocytes, enucleated GV cytoplasts, and enucleated GV cytoplasts at 15 h in-vitro maturation were transferred with a metaphase fibroblast cell. When mitotic chromosomes were transferred into enucleated or intact mouse GV oocytes, the first bipolar meiotic spindles were established and the reconstructed oocytes were able to extrude polar bodies. However, none of the reconstructed oocytes showed complete and accurate alignment of chromosomes, except the enucleated GV cytoplasts reconstructed after maturation. The spindle formation and polar body extrusion suggest that the first meiotic spindle was functional, and the chromosome misalignment did not prevent the onset of anaphase. The data indicate that a spindle checkpoint, providing surveillance of misaligned chromosomes, was overridden or compromised by the incompatibility between somatic chromosomes and meiotic spindles during the first meiotic division.     

Resumption of meiosis-I tissue to enucleated preovulatory oocytes: a preliminary report

Purpose : Ovarian tissue banking may be the best strategy to preserve female fertility. But optimal method to obtain viable mature oocytes remains challenging. In order to bypass the long in vitro oocyte growth period, we developed this study to test whether reconstruction of thawed primordial oocytes with enucleated preovulatory germinal vesicle (GV) oocytes could induce dictyate nuclei to undergo chromosomal condensation and meiotic maturation. Methods : Isolated primordial oocytes from thawed mouse ovarian tissue were reconstructed with enucleated GV oocytes. After electrofusion and in vitro maturation, the reconstituted oocytes were assessed for first polar body extrusion, cytoskeleton configuration, and chromosome abnormalities. Results : Primordial oocytes from thawed ovarian tissue showed a high survival rate. Following transfer and electrofusion, they could be fused with enucleated GV oocytes (35.6%, 36/101) and extruded a first polar body (52.8%, 19/36). These mature oocytes showed a normal spindle configuration and chromosome number. Conclusions : We successfully established a mouse cell model to prove that omitting the whole growth and maturation period by transfer of primordial oocytes to developmentally older enucleated oocytes would bypass the long growth period required to the preovulatory stage. Polar body extrusion could also ensue after in vitro growth. This study provided an alternative approach for future investigations in oocyte maturation.     

Congélation lente et vitrification des ovocytes humains matures et immatures

Performance and security questions in human oocyte cryopreservation have been taking researchers for about two decades. Oocytes are usually frozen at metaphase II. Immature oocytes cryopreservation is still a research alternative. Two techniques are currently available for oocyte cryopreservation: slow freezing and vitrification. Experimental data suggest that vitrification has less impact on oocyte physiology than classical slow freezing. After slow freezing of mature oocytes, survival and fertilization rates reach 70 to 80% whereas cleavage rates are around 90%, leading to five implantations and 1.2 births per 100 thawed oocytes. After vitrification of mature oocytes, survival and cleavage rates reach 90% leading to 11 implantations and 1.8 births per 100 thawed oocytes. The obstetrical and neonatal prognosis of these pregnancies is reassuring. No increased risk of congenital anomalies has been observed. However, further evaluation is needed to guarantee the safety of cryopreservation procedures. Immature oocyte cryopreservation is not currently perfected but some indications appear of great interest.     

Effect of different cryopreservation protocols on the metaphase II spindle in human oocytes

The aim of this study was to evaluate the impact of different cryopreservation protocols on the repolymerization of metaphase (M)II spindles in human oocytes. Fresh aspirated donor oocytes were cryopreserved 3-4 h after retrieval using four different protocols: slow freezing using 1.5 mol/l 1,2-propanediol (PROH) + 0.2 mol/l sucrose (n = 36); 1.5 mol/l PROH + 0.3 mol/l sucrose (n = 34); 1.5 mol/l PROH + 0.3 mol/l sucrose with Na(+) depleted-choline replaced media (n = 27), and vitrification by the Cryotip method (n = 23). The control group comprised 34 fresh oocytes. Three hours after thawing, surviving and control oocytes were fixed for meiotic spindle/chromatin assessment. Survival rates were 63.8, 73.5, 74.1 and 86.9% respectively for the four protocols described above. Survival for vitrified oocytes was higher than that observed for slow freezing with 0.2 and 0.3 mol/l sucrose (P < 0.05). The proportion of oocytes showing normal spindle configuration was similar for the four protocols (81, 73.9, 88.9 and 81.3% respectively) and 88.5% for controls, showing that the MII spindle returns to its normal configuration after 3 h of post-thawing incubation under standard conditions, irrespective of the cryopreservation technique used.