用Polscope观察人类卵母细胞中MII期减数分裂的纺锤体能预测ICSI后胚胎发育能力

在卵母细胞减数分裂时,染色体排列和分离过程中,纺锤体起重要作用。通常认为,MII期纺锤体的位置与第一极体位置相关。对纺锤体位置不同的卵母细胞及ICSI时未见纺锤体的卵母细胞的胚胎发育能力进行研究。 用GnRHa和hMG长方案或短方案诱导排卵。当2个卵泡直径达18mm时,注射hCG10 000IU,36—38 h后取卵。

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。

不同冷冻方法对小鼠不同成熟时期卵母细胞纺锤体的影响

目的:探讨不同冷冻方法对小鼠成熟期(MⅡ期)及生发泡期(GV期)卵母细胞的纺锤体及胚胎发育的影响。方法:收集GV期和有纺锤体的MⅡ期小鼠卵母细胞,随机分为3组:慢速冷冻-快速复温组、超高速玻璃化冷冻组和对照组(未冷冻组)。Polscope观察解冻0、3、6h后存活的MⅡ期及体外培养成熟GV期卵母细胞的纺锤体,有明显纺锤体的行卵胞浆内单精子显微注射受精,评价胚胎发育。结果:(1)超高速玻璃化GV组的存活率、卵裂率均显著高于慢冻GV组(P<0.05);(2)两冷冻MⅡ组解冻后0、3及6h纺锤体出现率和优质胚胎率均显著低于对照MⅡ组(P<0.05);(3)超高速玻璃化GV组体外成熟后纺锤体的出现率、优质胚胎率均显著高于超高速玻璃化MⅡ组(P<0.05)。结论:慢速冷冻-快速复温法对小鼠不同成熟时期卵母细胞的纺锤体损伤较大;超高速玻璃化冷冻对小鼠生发泡期卵母细胞纺锤体的影响则较小,是一种简便、快捷、高效的冷冻方法。

人未受精MII期卵冻融过程中减数分裂纺锤体及透明带的变化

目的观察人类MII期卵母细胞冻融过程中纺锤体及透明带的变化。方法 50例非男性因素的不育妇女,将常规体外受精后未受精的MII期卵母细胞进行慢速冷冻/快速复苏,使用Polscope系统观察冻融过程中纺锤体及透明带的变化;将复苏存活的卵母细胞分别培养1、3、5h后观察纺锤体的变化。结果 95枚未受精卵中52枚冷冻前和冷冻过程中均能观察到纺锤体。卵母细胞进入复苏-1液后47.8%可观察到纺锤体,后逐渐消失。46枚复苏存活卵母细胞分三组体外培养,1h纺锤体检出率为40.8%;3h为100.0%;5h为88.1%,差异具统计学意义。另外,冻融前后卵母细胞透明带的双折射性也发生了改变。结论卵母细胞的冻融过程会引起纺锤体及透明带的损伤,卵母细胞复苏后纺锤体进行重建。复苏后培养3h左右纺锤体的重建率最高。

Presence of Meiotic Spindles Indicates Early Cleavage of Embryos

Objective To assess whether the detection of the meiotic spindle could anticipate the appearance of early cleavage. Methods Oocytes were obtained from stimulated ovaries of consenting patients undergoing oocytes retrieval for ICSI. Spindles were imaged with the Polscope. After ICSI, oocytes with or without spindles were cultured for examination of early cleavage and embryo development. A total of 328 oocytes from 50 cycles were examined with the Polscope and inseminated by ICSI. Results Spindles were imaged in 81.7% of oocytes. After ICSI, more oocytes with spindles （78.4%） fertilized normally than oocytes without spindles （53.3%） （P〈0. 001）. At 25-27 h post ICSI, more fertilized oocytes developed from oocytes with spindles （81.9%） were detected early cleavage than those from oocytes without spindles （28.1%） （P〈0.001）. Significantly more embryos with early cleavage （82.2%） developed to high quality embryos at d 3 compared with the embryos without early cleavage （48.3%） （P=0.001）. The value of rs related to the relationship between spindles and early cleavage was 0.420 （P〈0.001）. Conclusion The existing of the early cleavage may have a predictive value on the opportunity of high quality embryos and the existing of the spindle may have a predictive value in the appearance of early cleavage.

偏振光学显微成像技术在高分子结晶结构表征中的应用

光学成像技术是最为直观的一种表征手段,借助光学显微镜,根据物质、材料和光的相互作用,能够直接观察肉眼难以获得的微观结构信息.借助偏振光,又能看到在普通显微镜明场下无法观察到的晶体双折射等光学性质.本文主要介绍几种光学显微镜在高分子结晶表征方面的应用,从光通过偏振器件及样品的角度出发,介绍偏光显微镜、PolScope系统以及Müller矩阵显微镜的基本工作原理和在高分子结晶方面的应用研究,结合具体案例,阐述这些技术如何揭示高分子晶体的微观结构,并简述一些基础的操作要点及注意事项.依据晶体的双折射特点,偏光显微镜提供了晶体厚薄、取向等结构信息和晶体生长速率等动力学信息.但是偏光显微镜的透射光强是晶体双折射和主折射率方向的方位角两者耦合的结果,而拥有可变偏振方向的PolScope系统则可以进一步精确测得双折射的光程差及晶体主折射率方向的方位角.Müller矩阵显微镜则通过检测经过样品前后的圆偏振光变化来获得样品更为丰富的光学信息,如线性双折射、圆双折射、线性二色性和圆二色性等.

Vitrification of In vitro-matured Oocytes:Effects of Meiotic Spindle Morphology on Clinical Outcome

Objective:The meiotic spindle controls chromosome movement and mediates various functions essential for fertilization and early postfertilization events.This study aimed to examine whether vitrification causes meiotic damage in vitro-matured metaphase II(MII)human oocytes,and whether the meiotic spindle morphology influences the subsequent developmental outcomes.Methods:The spindle characteristics of MII human oocytes in vitro matured were studied before and after vitrification using PolScope imaging and immunofluorescence staining.The developmental competence of oocytes was also examined.Results:A total of 419 human MII oocytes were obtained from 593 intracytoplasmic sperm injection cycles at our hospital.Of these oocytes,54 were used for immunofluorescence staining,whereas the other oocytes were examined by PolScope imaging and classified into three groups according to the meiotic spindle morphology:(A)normal morphology,(B)weak refraction and short meiotic spindle,and(C)no detectable meiotic spindle.The three groups demonstrated statistically significant differences in terms of survival after vitrification.However,differences were not found in terms of oocyte chromosome structure and meiotic spindle morphology on immunofluorescence staining performed before and after vitrification.Oocyte survival,fertilization,and early embryonic development rates were significantly higher in Group A than in Groups B and C with or without vitrification.While vitrification had no effect on these metrics in Group A,Groups B and C demonstrated significantly lower fertilization and cleavage rates after vitrification/warming.Conclusions:Screening for normal meiotic spindle morphology and chromosome configuration before vitrification may increase the yield of healthy viable oocytes for various assisted reproductive technologies.