Presence of Meiotic Spindles Indicates Early Cleavage of Embryos

Objective To assess whether the detection of the meiotic spindle could anticipate the appear- ance 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 relation- ship 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.     

The role of brain-derived neurotrophic factor in mouse oocyte maturation <Emphasis Type="Italic">in vitro</Emphasis>

Brain-derived neurotrophic factor (BDNF) can promote developmental competence in mammalian oocytes during in vitro maturation (IVM),but the role of BDNF in oocyte maturation at cellular level is not still clear.In this study,mouse cumulus-enclosed oocytes subjected to IVM were fertilized and cultured to blastocyst stage.Meiotic spindle configuration and cortical granules distribution during oocyte maturation in vitro were assessed by using immunofluorescence and laser confocal microscopy.The results showed that BDNF contributed to the complete preimplantation development of mouse oocytes compared to the control oocytes (13.78% vs.5.92%;P<0.05).Further,BDNF did not accelerate nuclear maturation of IVM oocytes.For the BDNF-treated oocytes at meiosis Ⅰ,Meiotic spindle areas were significantly smaller and the number of cytoplasmic microtubule organizing centers was greater than that in the control,and the percentages of oocytes showed spindles positioned near the oolemma and a well-formed cortical granule-free domain were significantly higher than that of the control.These morphological characteristics of the BDNF-treated oocytes were much closer to the oocytes matured in vivo than those of the control oocytes.In conclusion,BDNF can promote the developmental competence of mouse IVM oocytes,by improving the meiotic spindle configuration and location and cortical granules distribution at meiosis Ⅰ.     

Effect of vitrification at the germinal vesicle stage on the global methylation status in mouse oocytes subsequently matured in vitro

Background It is still unclear whether the vitrification procedure itself is associated with the incidence of abnormal DNA methylation during oocytes vitrification.The purpose of this study was to evaluate the epigenetic profile of mouse oocytes,which went through vitrification either at a mature stage or at an immature stage following in vitro maturation （IVM） by analyzing the global DNA methylation.Methods Metaphase Ⅱ （M Ⅱ） stage and germinal vesicle （GV） stage oocytes were collected from adult female mice and were vitrified respectively.The M Ⅱ oocytes were assessed for cryo-survival and global DNA methylation.The GV oocytes were assessed for cryo-survival and only the surviving GV oocytes were cultured in vitro for subsequent assessment of global DNA methylation in mature oocytes.In vivo matured fresh M Ⅱ oocytes without undergoing vitrification were used as control.The level of global DNA methylation in the M Ⅱ oocytes was then examined by immunofluorescence using an anti-5-methylcytosine （anti-5-MeC） monoclonal antibody and fluorescein isothiocyanate （FITC）-conjugated goat anti-mouse IgG under a laser scanning confocal microscope.Results In terms of the effect of vitrification on global DNA methylation status in matured oocytes,in the M Ⅱ-v group,all the examined oocytes （90/90） were found with hypermethylation,including 63.3％ （57/90） of them displaying DNA methylation of a very high level,25.6％ （23/90） with a high level,and 11.1％ （10/90） with an intermediate level,whereas in the GV-v group,all the matured oocytes （129/129） were also examined with hypermethylation,including 67.4％ （87/129） of them displaying DNA methylation of a very high level,23.3％ （30/129） with a high level,and 9.3％ （12/129） with an intermediate level.Statistically,it was similar between both groups,which were similar to the control：68.6％ （83/121） of fresh M Ⅱ oocytes displayed DNA methylation of a very high level,21.5％ （26/121） with a high level,a     

The Effects of Murine Cytomegalovirus on the Maturation, Fertilization, Cleavage and Blastula Formation of Mouse Oocytes In Vitro

To study the effects of mouse cytomegalovirus （MCMV） on the in vitro maturation, fertilization, cleavage and blastula formation of mouse oocytes, the immature oocytes were infected in vitro by MCMVs of different dosages （100 TCID50, 10 TCID50 and 1 TCID50）. The oocytes were then observed for in vitro maturation, fertilization, cleavage and blastula formation and the ultrastructural changes after the culture with the viruses. Our results showed that no significant differences were found in IVM, IVF, cleavage and blastula formation among the groups treated with of virus of various dosages. And ultrastructural abnormality was observed in the oocytes treated by 100 TCID50 of viruses. It is concluded that MCMV did not have any conspicuous effects on IVM, IVF, cleavage and blastula formation of murine immature oocytes.     

Immunofluorescent Study of Human MⅡ Oocytes Failed to Fertilize after IVF and ICSI

Objective To discuss the reason why human M Ⅱ oocytes failed to fertilize after IVF and ICSI. Methods The unfertilized human MⅡ oocytes were collected 24-48 h after IVF and ICSI and stained for immunoflurescence and PI counterstain. The types of fertilization failure were identified under the fluorescence microscopy. Results About 55.8% oocytes in IVF were found no sperm in them, which were more than that in ICSI （9.7%） （P〈0.01）. About 14.9% oocytes in IVF and 58.1% in ICSI displayed oocyte activation failure. The difference was significant （P〈0.01）. Defects in pronuclear formation and or migration was found in a similar proportion of oocytes both after IVF （25.3%） and ICSI （32.3%）（P〉0.05）. There were 3.9% oocytes with other abnormalities were observed in IVF but none in ICSI. Conclusion The main reason of fertilization failure after IVF was no sperm penetration. However fertilization failure after ICSI was mainly associated with incomplete oocyte activation.     

Effect of Mouse Oocyte Vitrification on Mitochondrial Membrane Potential and Distribution简

The effects of mouse oocyte vitrification on mitochondrial membrane potential and distribution were explored in this study. The collected mouse oocytes were randomly divided into vitrification and control groups. Ethylene glycol（EG） and dimethylsulphoxide（DMSO） were used as cryoprotectants in the vitrification group. The mitochondrial function and distribution in the oocytes were examined by using the fluorescent probes, JC-1 and Mito Tracker green. The results showed that the ratio of red to green fluorescence in mouse oocytes was significantly decreased after thawing in the vitrification group as compared with the control group（1.28 vs. 1.70, P0.05）. The percentage of polarized distribution of the mitochondria in oocytes was conspicuously reduced in the vitrification group when compared with the control group（31% vs. 63%, P0.05）. It was suggested that vitrification significantly affects the mitochondrial function and distribution in oocytes and reduces the potential of oocyte fertilization and embryo development.     

N-hexane Alters the Maturation of Oocytes and Induces Apoptosis in Mice

Objective This study was aimed to determine the effects of n-hexane on the maturation of mouse oocytes.Methods Cell culture was used to observe the maturation of mouse oocytes and CLSM was employed to determine their apoptosis.Results Germinal vesicle breakdown(GVBD) and extrusion of the first polar body in mouse oocytes were significantly inhibited by n-hexane.After fertilization,the number of eggs in the mouse was significantly reduced by n-hexane.Mitochondrial membrane potentials(ΔΨm) were altered in mouse oocytes that were leading to apoptosis of the oocytes.Conclusion N-hexane might have affected the maturation of oocytes,causing alteration of ΔΨm and leading to apoptosis which maybe one of the most important mechanisms.     

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.     

Follicular development, oocyte maturity and fertilization in vitro.

Forty-four infertile women in 44 cycles were stimulated by CC/hMG/hCG (39 cycles) and by CC/hCG (5 cycles) for the purpose of in vitro fertilization (IVF, 37 cases with 35 transfers) and gamete intrafallopian transfer (GIFT, 7 cases). Intraoperative ovum pick-up was performed 32-36 hours after hCG injection. Blood E2 and P levels were determined. Blood E2 was found to be proportional to the number of mature and intermediate oocytes obtained, and correlated with the number of follicles (greater than 10 mm) and the total follicular fluid volume. Altogether 213 follicles aspirated were analyzed. There was a highly significant linear correlation (r = 0.72, P less than 0.01) between the follicular diameter (X cm) on the operation day and the aspirated follicular fluid volume (FFV, Y). The equation of the line was Y = 3.8 X-4.4. The higher concentrations of follicular E2, P, FSH, LH and lower T concentration were crucial to the maturing process of oocytes. It was found that mature oocytes could be retrieved from 26.5% of the small follicles, so it is worthwhile to aspirate the small ones. The fertilization rate was much higher with the mature oocytes than the immature ones. But the morphologically mature oocytes might function differently, i.e. the mature oocytes retrieved from the small follicles or from follicles with unbalanced hormonal microenvironment usually give a low fertilization rate.

     

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.     

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.     

OPS玻璃化冷冻小鼠MII期卵效果观察

目的　以小鼠为研究模型 ,探讨MII期卵母细胞冷冻后纺锤体和染色体改变的有效观察方法。研究OPS玻璃化冻存成熟卵的可行性。　方法　运用激光扫描共聚焦显微镜 (LSCM)的光学切片和三维图像重建技术 ,系统研究了OPS玻璃化冷冻对小鼠MII期卵纺锤体和染色体的影响。　结果　冷冻组卵复苏后存活率达 5 8% ,纺锤体和染色体形态正常率分别为 46%和 5 2 % ,与对照组 ( 76%和 70 % )相比 ,差异具显著性 (P <0 0 5 )。暴露组卵纺锤体和染色体结构正常率较对照组略有下降 ,但无统计学差异。　结论　LSCM能够清晰有效地观察冷冻对卵母细胞纺锤体和染色体影响。应用OPS玻璃化方法能够有效冻存卵母细胞     

核抑制剂(Roscovitine)对促排周期人未成熟卵母细胞体外成熟安全性的初步研究

目的 观察核抑制剂(Roscovitine)对人未成熟卵母细胞体外成熟(in vitro maturation,IVM)安全性的影响.方法 获取促排周期的GV期未成熟卵随机分为两组,实验组(经二步法):未成熟卵母细胞先用含有200 vmol/LRoseovitine的培养液培养6 h,然后转入IVM成熟培养液培养24～36 h;对照组:直接用IVM成熟培养液培养24~36h.观察两组卵母细胞成熟情况并应用激光扫描共聚焦显微镜观察培养成熟后MⅡ期卵母细胞纺锤体和染色体的形态.结果 实验组共获未成熟卵母细胞61个,经体外成熟培养后50个卵成熟,其中39个卵可观察到纺锤体与染色体,纺锤体的形态异常者为15个,染色体异常者11个;对照组共获未成熟卵母细胞60个,经体外成熟培养后43个卵成熟,其中32个卵可观察到染色体与纺锤体,纺锤体形态异常者14个,染色体异常者11个.结论 Roscovitine能可逆性抑制体外培养的人卵母细胞的核成熟,但未对纺锤体及染色体形态产生不良影响.     

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。     

四种显微技术观测大鼠颌下腺细胞与丝素-壳聚糖体外共培养的形态学特点

目的 采用倒置显微镜、扫描电镜(scanning electron microscopy,SEM)、荧光显微镜和激光共聚焦显微镜(( laser scanning confocal microscopy,LSCM))技术对大鼠颌下腺细胞(rat submandibular gland cells,RSMGs)与丝素-壳聚糖( silk fibroin-chitosan,SFCs)的体外复合培养进行形态学观察.为观测、评估种子细胞在三维支架的内部生长情况提供技术支持.方法 取0～8d龄SD大鼠的颌下腺,对大鼠颌下腺细胞进行原代培养、分离纯化并传代;用抗细胞角蛋白单克隆抗体( CK8)及淀粉酶抗体的免疫细胞化学染色鉴定细胞来源.选取传至第二代的对数生长期的RSMGs作为种子细胞,选取SFCs共混膜(5×5×2)mm作为支架材料构建组织工程化涎腺样结构.将种子细胞与支架材料复合培养并分别于倒置显微镜、SEM、荧光显微镜和LSCM下观察二者复合生长情况.结果 倒置显微镜可以直接观察活细胞与支架复合生长情况,方法简单易行.SEM可以较精确的展示细胞支架复合生长的表面超微结构.经过荧光染料的着色,荧光显微镜和LSCM都可以观察到支架上锚定的种子细胞.荧光显微镜可见细胞核的荧光信号均匀的分布在支架孔隙内.LSCM通过层扫描及三维重建技术对较厚的标本获取图像;并可以通过旋转图像,从不同角度观察细胞支架复合物的三维剖面或整体结构,得到更为准确的定位信息.结论 四种显微技术均可应用于RSMGs与SFCs体外共培养的形态学观测.LSCM的三维重建技术结合荧光染料标记可以较好地获得RSMGs与SFCs复合生长的情况,有着较广泛的应用价值.     

昆明小鼠成熟卵母细胞体外受精及受精卵体外培养的研究

目的通过比较获能受精液成分及成熟卵母细胞的处理方法，观察对昆明小鼠体外受精(IVF)效果的影响和用于早期胚胎培养的三种发育培养液培养效果，探讨并建立起适合于昆明小鼠卵子体外受精与受精卵体外发育的实验体系。方法用T6或FM两种获能受精液对采自昆明雄鼠附睾尾的精子与成熟卵母细胞进行体外受精(IVF)，受精前对成熟卵母细胞选用透明质酸酶去除卵丘细胞和不去卵丘细胞(对照组)两种方法进行处理，受精卵分别用M16、改良的CZB液(mCZB)和改良的M16液(mM16)三种发育培养液对体外受精胚进行培养。结果选用FM获能受精液的受精率极显著高于T6液(92．3％对64．7％)，且体外受精前先用透明质酸酶处理的成熟卵母细胞比对照组成熟卵母细胞受精率要低(59．2％对64．7％)，但差异不显著；用mM16培养液进行培养时桑胚率及囊胚率(55．3％和35．6％)显著高于mCZB(13．3％和8．1％)和M16培养液(17．3％和14．7％)，后两者差异不显著。结论昆明小鼠卵子体外受精时宜选用FM受精获能液，不宜用透明质酸酶去除卵丘细胞，且宜选用mM16培养液对昆明小鼠体外受精卵进行早期培养，其中的亚硫磺酸钠可有效的克服2-细胞胚后的体外发育阻滞现象。