冷冻环冷冻促排周期未成熟卵母细胞效果的研究

目的:本文通过对冷冻环冷冻促排周期未成熟卵母细胞后成活率和体外成熟(IVM)能力的比较,及卵母细胞免疫荧光染色后纺锤体和染色体形态的分析,探索未成熟卵母细胞冷冻的方法。方法:获得的GV期卵母细胞随机分为3组:①对照组:GV期卵母细胞体外成熟为MⅡ期(n=68);②GV期冷冻组:卵母细胞在GV期冷冻后体外成熟(n=111);③MⅡ期冷冻组:卵母细胞IVM为MⅡ期予以冷冻(n=69)。获得的MⅡ期卵母细胞予以免疫荧光染色,激光扫描共聚焦显微镜(LSCM)观察纺锤体和染色体的形态。结果:GV期冷冻组卵母细胞存活率(89.11%)显著性高于MⅡ期冷冻组(77.05%),而GV期冷冻组卵母细胞的体外成熟率(77.78%)和对照组(86.74%)没有明显的差异(P>0.05)。GV期冷冻组成熟后卵母细胞正常形态的纺锤体(14.1%)和染色体(12.5%)都显著低于对照组(29.1%,32.7%,P<0.05),MⅡ期冷冻组正常形态的纺锤体(11.4%)显著性低于对照组(P<0.05),染色体(15.9%)也低于对照组,但是无显著性差异(P>0.05)。结论:玻璃化冷冻对GV期和MⅡ期卵母细胞纺锤体和染色体都会带来一定的损伤。IVM技术和玻璃化冷冻技术还需要进一步提高。     

冷冻环玻璃化法冷冻小鼠卵母细胞的效果评价

目的评价ED15(15%ethylene glycol,EG+15%dimethylsulphoxide,DMSO)冷冻环玻璃化法冻存小鼠成熟卵母细胞的效果,以及对其纺锤体形态、染色体分布和发育潜能的影响。方法分别以小鼠新鲜成熟卵母细胞为对照组,玻璃化冷冻复苏卵母细胞为冷冻组。玻璃化冷冻以冷冻环为载体,以乙二醇(EG)及二甲亚砜(DMSO)联合作冷冻保护剂。解冻后观察细胞存活情况并对解冻后培养0、1、2h的卵母细胞行纺锤体及染色体免疫荧光染色,激光共聚焦扫描显微镜观察;其余卵母细胞行体外受精培养,计算受精率、囊胚形成率。结果冷冻组成熟卵母细胞存活率为98.2%,复苏后卵母细胞培养0、1、2h的纺锤体形态正常率及染色体分布正常率与对照组比较无显著性差异(分别为87.0%、90.9%、90.3%vs95%,P>0.05;91.3%、95.4%、93.5%vs90%,P>0.05);冷冻组受精率及囊胚形成率与对照组比较均无显著性差异(81.3%vs85.2%,P>0.05;76.1%vs75.4%,P>0.05)。结论ED15冷冻环玻璃化法冻存小鼠成熟卵母细胞复苏存活率高,对其纺锤体形态、染色体分布及发育潜能无明显影响。     

两种不同玻璃化冷冻方案对小鼠卵母细胞纺锤体的影响

目的:探讨两种不同的玻璃化冷冻方案对小鼠第二次减数分裂中期卵母细胞纺锤体的影响。方法:实验分3组:方案A组、方案B组和对照组(新鲜卵母细胞)。均以冷冻环为载体,方案A组用乙二醇(EG)作为冷冻保护剂,方案B组用乙二醇联合二甲亚砜(DMSO)作为冷冻保护剂,用直接投液氮高速制冷冷冻小鼠卵母细胞;解冻后3h小鼠卵母细胞经固定并用间接免疫荧光法染色微管及染色体,观察纺锤体及染色体的形态。结果:两种玻璃化冷冻方案中,卵母细胞解冻后的存活率差异无显著性(80.3%vs87.5%,P>0.05);经玻璃化冷冻方案A冷冻的卵母细胞解冻后具有完整纺锤体结构的卵母细胞数显著低于对照组和方案B组(15.2%vs78.7%,15.2%vs77.5%,P<0.05),后两者差异无显著性(78.7%vs77.5%,P>0.05);方案A组解冻后具有正常染色体的卵母细胞数显著低于对照组和方案B组(17.4%vs76.6%,17.4%vs72.5%,P<0.05),后两者差异无显著性(76.6%vs72.5%,P>0.05);方案A组异常染色体数显著高于对照组和方案B组(82.6%vs19.1%,82.6%vs27.5%,P<0.05),后两者差异无显著性(19.1%vs27.5%,P>0.05)。结论:改变玻璃化冷冻方案有利于保持解冻后小鼠卵母细胞纺锤体结构完整性。     

不同冷冻方法对小鼠卵母细胞发育潜能及细胞骨架的影响

目的比较不同冷冻方法对不同成熟阶段小鼠卵母细胞复苏、胚胎发育及细胞骨架的影响,寻求最佳的卵母细胞冷冻方法。方法分别以SOPS玻璃化及慢速法冷冻小鼠中期(MII)和生发泡期(GV)卵母细胞。解冻复苏后,分别作体外培养成熟(IVM)、体外受精(IVF)或固定作免疫荧光标记,统计复苏率、成熟率、受精率、囊胚率及纺锤体等细胞骨架指标。结果玻璃化冷冻组MII卵和GV卵母细胞的复苏率及囊胚率均显著高于慢速冷冻组(P<0.05)。慢速冷冻组中MII卵复苏率显著低于GV卵(40.4%vs 57.1%,P<0.01)。但同一种冷冻方法保存的MII卵和GV卵的受精率及囊胚率相比,均无显著性差异(P>0.05)。两组的GV卵成熟率无显著差异。玻璃化冷冻组GV卵的纺锤体、染色体及纺锤体和染色体正常率均高于慢速冷冻组GV卵但无显著差异。结论玻璃化冷冻能有效改善冻融卵母细胞的复苏及胚胎发育;与MII卵比较,冷冻GV卵对细胞骨架损伤较小;两种冷冻方法均不影响GV卵冻融后成熟。     

比较四种冷冻载体在水牛卵母细胞快速玻璃化冷冻保存中的效果

目的探讨不同冷冻载体对水牛卵母细胞快速玻璃化冷冻保存效果的影响。方法以体外成熟培养18 h的水牛卵母细胞为实验材料,分别以0.25 ml冻精塑料细管(A)、自制拣卵针尖端部分制成的极细玻璃微管(D)和两种制作核移植工具针的玻璃细管(B、C)为冷冻载体卵母细胞,解冻后统计成熟率及早期胚胎发育率等冷冻保存效果指标。结果A组卵母细胞冷冻保存效果最差(成熟率:A vs B、C、D为43.9%vs 51.7%、51.9%、55.9%,2~4细胞率:A vs B、C、D为34.1%vs 40.0%、40.7%、41.2%),D组卵母细胞冷冻效果最好,但解冻时卵母细胞的回收率显著低于其他组(D vs A、B、C为69.2%vs 87.0%、94.1%、91.9%)。B、C两组在解冻后的成熟率及早期发育率上显著高于A组,低于D组,但差异不明显,且其解冻后卵母细胞回收率显著高于D组(P<0.05)。结论综合回收率和解冻后早期胚胎发育率,B、C两种玻璃细管是一种水牛卵母细胞快速玻璃化冷冻保存中可行的冷冻载体。     

卵母细胞玻璃化冷冻对胚胎发育及妊娠结局的影响

目的探讨玻璃化冷冻对卵母细胞体外受精、胚胎发育以及妊娠结局的影响。方法将193枚玻璃化冷冻卵母细胞解冻复苏,采用卵胞浆内单精子注射法受精,观察胚胎发育形态,经72h体外培养后移植。结果成熟卵母细胞解冻复苏率为75．6％,正常受精率为72．6％,卵裂率为88．7％,优质胚胎率为48．9％。共移植19例,9例妊娠,已分娩2例,临床妊娠率为47．3％。新鲜周期与冻卵解冻周期相比,卵裂率、可移植胚胎率和优质胚胎率均无显著性差异（P〉0．05）。结论玻璃化冷冻方法对成熟卵母细胞的胚胎发育及妊娠结局无显著影响。     

以细胞筛为载体的玻璃化冷冻和慢速程序化冷冻保存人卵巢组织的效果比较

目的比较以细胞筛为载体的玻璃化冷冻与慢速程序化冷冻用于人卵巢组织冷冻的效果。方法人卵巢组织取皮质切块后,随机分为3组:新鲜组织对照组(F组)、慢速程序化冷冻组(S组)及以细胞筛为载体玻璃化冷冻组(V组)。卵巢组织冷冻复苏后,固定切片后行苏木素-伊红染色,观察卵泡形态;使用TdT介导的dUTP缺口末端标记技术,观察卵泡凋亡情况;部分卵巢组织体外培养,隔日采集培养液后检测雌二醇(E2)浓度。比较三组卵泡正常形态率、卵泡凋亡率及E2浓度。结果与F组原始卵泡正常形态率(91.1%)相比,V组原始卵泡正常形态率(88.1%)无显著差异(P0.05),而S组原始卵泡正常形态率(79.6%)显著下降(P0.001);V组初级卵泡正常形态率(74.2%)与S组(73.6%)相似,但两组均低于F组(89.0%)(P0.05);凋亡检测中,3组凋亡率无显著差异(P0.05);体外培养2周,各组E2浓度持续上升,F组E2浓度显著高于S组、V组(P0.001),而S组与V组E2浓度无显著差异(P0.05)。结论以细胞筛为载体的玻璃化冷冻能较好地保存人卵巢组织,复苏后组织体外培养后,还可持续分泌E2。     

玻璃化冷冻不同来源卵母细胞的临床研究

目的 探讨玻璃化冷冻对卵巢刺激（ovarian stimulation,OS）和控制性超促排卵（controlled ovarian hyperstimulation,COH）来源的卵母细胞影响.方法 对OS和COH来源的卵母细胞进行玻璃化冷冻,解冻后观察存活率,继续发育潜能及移植后的妊娠率并与COH来源的新鲜卵母细胞进行比较.结果 OS和COH来源的卵母细胞玻璃化冻融后的存活率和继续发育潜能无统计学差异（P＞0.05）,与新鲜卵母细胞差异无统计学意义（P＞0.05）.COH冷冻组和OS冷冻组的妊娠率和种植率分别是25.0%（3/12）、20.8%（5/24）和33.3%（1/3）、12.5%（1/8）.结论 玻璃化冷冻技术可以应用于OS和COH来源的成熟卵母细胞的冷冻保存,对其继续发育潜能和妊娠结局无显著影响.     

梯度平衡法改善猪卵巢组织玻璃化保存效果

目的研究梯度平衡法对卵巢组织玻璃化冷冻后组织形态学及凋亡的影响。方法取新鲜猪卵巢切成小块,未进行任何处理的为对照组、采用梯度平衡法冷冻的为梯度平衡组、采用直接平衡法冷冻的为直接平衡组。HE染色观察3组组织形态,原位末端转移酶标记(TUNEL)技术检测细胞凋亡情况。结果与对照组相比,梯度平衡组和直接平衡组卵泡的正常率均下降(均P<0.05),但后两组比较无显著差异(P>0.05)。与对照组相比,梯度平衡组与直接平衡组的细胞凋亡率均显著增加(P<0.05),但梯度平衡组相对于直接平衡组,卵母细胞、颗粒细胞、基质细胞凋亡率(分别为14.8%vs.17.6%、13.8%vs.15.2%和15.7%vs.18.5%)均显著降低(P<0.05)。结论梯度平衡法可以减少玻璃化冻融卵巢组织过程中的细胞凋亡。     

冻融兔卵母细胞培养不同时间再激活效应的研究

目的探讨冻融兔卵母细胞行卵胞浆内单精子注射(ICSI)后,不同培养时间、不同激活方法,卵母细胞受精及胚胎发育能力。方法共收集兔卵母细胞470枚,玻璃化冷冻,解冻行ICSI后培养半小时,分为3组激活:组1(n=46):钙离子激活;组2(n=40):氯化锶(SrCl_2)激活;组3(n=33):无水酒精激活;再同样方法解冻一部分卵母细胞,ICSI后培养1 h,分为2组激活:组4(n=30):钙离子激活;组5(n=26):氯化锶激活;对照组(n=39):ICSI后直接培养,不激活。各组得到的囊胚玻璃化冷冻,受体兔HCG准备后,胚胎解冻后移植。结果卵母细胞解冻行ICSI后,组5的卵母细胞受精率、分裂率和囊胚形成率最高,分别为53.8%,26.9%和7.7%,所获囊胚的母兔可能怀孕。结论冷冻卵母细胞解冻行ICSI后,适当延长培养时间,使用合适的激活方法可能会提高卵母细胞的受精以及早期胚胎发育能力。     

人体外与体内成熟卵母细胞发育潜能及妊娠结局的观察

目的观察体外成熟(IVM)与体内成熟人卵母细胞的发育潜能和妊娠结局。方法69例多囊卵巢或多囊卵巢综合征不育患者,分别行IVM治疗49个周期(IVM组)和体外受精-胚胎移植(IVF-ET)治疗29个周期(IVF组)。结果IVM组卵母细胞成熟率为65.4%。IVM组和IVF组受精率和卵裂率分别为62.1%和77.2%,60.2%和91.2%,两组受精率无显著性差异(P>0·05),IVM组较IVF组卵裂率降低非常显著(P<0.01)。IVM组和IVF组临床妊娠率和种植率分别为26.5%和12.6%,41.4%和22.9%,IVM组临床妊娠率和种植率较低,但两组无显著性差异(P>0.05)。IVM组和IVF组卵母细胞受精后D2≥4细胞和D3≥6细胞胚胎数占受精卵数(2PN)的比例分别为24.5%和19.4%,67.0%和66.4%,IVM组较IVF组胚胎发育速度慢,两者有非常显著性差异(P<0.01)。IVM组D3优质胚胎形成率(23.7%)较IVF组(58.2%)为低,亦有非常显著性差异(P<0.01)。结论人卵母细胞IVM将成为一种选择性的辅助生殖技术。人卵IVM较体内成熟卵母细胞受精后2PN至卵裂期发育阻滞发生率高,胚胎发育速度慢和优质胚胎形成率低。     

The application of in vitro maturation of oocytes in the infertile patients with polycystic ovarian syndrome

<正> Objective:To evaluate the effects of in vitro maturation(IVM)of oocytes in the infertile pa-tients with polyeystic ovarian syndrome(PCOS).Methods:The infertile patients with PCOS who underwent IVM or IVF/ICSI from January2004 to August 2005 were studied retrospectively.68 unstimulated cycles(48 cases)underwentIVM as IVM group,42 cycles(39 cases)underwent IVF/ICSI as control group.Main outcomesincluding the number of oocytes retrival,the rates of fertilization,embryo cleavage,implanta-tion,pregnancy,miscarriage,ovarian hyperstimulation syndrome(OHSS)and multiple pregnan-cy were assessed.Results:No FSH was administered in IVM group and the mean number of FSH used was(25±6.2)ampoules in control group.When compared with control group,women in IVM grouphad significant increase in fertilization rate(70.7% versus 63.9%)and decrease in cleavage rate(87.9% versus 99.4%)and ovarian hyperstimulation syndrome(0 versus 7.1%).No significantdifferences between IVM group and control group were found in the number of oocytes obtained,implantation rate,clinical pregnancy rate,miscarriage rate and multiple pregnancy rate.Conclusion:Our results suggested that for infertile PCOS women who required assisted con-ception treatment,IVM is a more economical method with less OHSS complication than that ofconventional IVF treatment.     

卵母细胞体外成熟技术在多囊卵巢综合征不育妇女中的应用

目的评价未成熟卵母细胞体外成熟(IVM)技术对多囊卵巢综合征(PCOS)不育患者的治疗效果。方法对2004年1月至2005年8月PCOS不育患者在本中心行IVM或常规体外受精-胚胎移植(IVF-ET)、卵胞浆内单精子注射(ICSI)的周期作回顾性分析。分为无药物刺激的IVM观察组共48例、68个周期,与IVF/ICSI组39例、42个周期,比较两组的获卵数、受精率、卵裂率、种植率、临床妊娠率、流产率、卵巢过度刺激综合征(OHSS)及多胎发生率。结果IVM组与IVF/ICSI组比较,IVM组未采用卵泡刺激素(FSH),IVF/ICSI组FSH用量为(25±6.2)支;受精率分别为70.7%及63.9%(P<0.01);卵裂率分别为87.9%及99.4%(P<0.01);OHSS分别为0及7.1%(P<0.05),差异均具统计学意义。两组的获卵数、种植率、妊娠率、流产率、多胎率差异均无统计学意义(P>0.05)。结论IVM技术对难治性PCOS不育患者是一种较为节约,且不发生OHSS的有效辅助生育技术。     

Effect of Cytochalasin B on spindle and chromosome configuration of human in vitro matured oocytes after vitrification

Objective: To investigate the effect of Cytochalasin B(CCB) on the spindle and chromosome configuration of human in vitro matured oocytes after vitrification.Methods: Immature oocytes which were collected in intracytoplasmic sperm injection (ICSI) cycles (including stage GV and M I )after ovarian stimulation, followed in vitro matured(IVM) for 24-48 h. 170 oocytes were matured according to the extrusion of first polarbody, were randomized into five groups for vitrification. Group A:37 oocytes were treated with CCB for 20 mins before vitrification; Group B:38 oocytes with CCB for 30 mins; Group C:33 oocytes with CCB for 40 mins. Group D:36 oocytes without CCB before vitrification. Group E:26 ooctyes whitout vitrification and CCB as the controlled group. 71 in vivo matured oocytes were collected after ovarian stimulation in in vitro fertilization-embryo transfer (IVF-ET) cycles and ICSI cycles, and randomized into four groups for vitrification. Group A1:17 oocytes treated with CCB for 20 mins before vitrification; Group B1:20 oocytes with CCB for 30 mins. Group C1:17 oocytes with CCB for 40 mins. Group D1:17 oocytes without CCB before vitrification. All the oocytes were thawed after three weeks and then immunoftuorescence staining were perforned for tubu-lin and chromatin and at last visualized by confocal laser scanning microscopy.Results: There are no significant differences of survival rates among group A, B, C, D (80.39%, 64.86%, 55.36% and 78.79%, P>0.05) and group A1, B1, C1, D1 (64.7%, 50.0%, 64.7% and 70.6%, P>0.05). There are no significant differences in the frequencies of normal spindle and chromosome in group A, B, C, D (21.05%, 26.3%, 27.78% and 23.81%, P>0.05), but significantly lower than group E (61.54%, P<0.05). There were no significant differences in the frequencies of normal spindle and chromosome in group A1, B1, C1, D1 (27.3%, 30.0%,45.5% and 33.3%, P>0.05). No statistical differences were found in survival rate and frequencies of normal spindle and chromosome between in vitro matured groups treated with CCB and in vivo matured groups after vitrification (P>0.05).Conclusion: Spindle and chromosome configuration of human in vitro matured oocytes was damaged in vitrification, which can not improved by CCB pre-treatment before vitrification. Oocytes in vitro matured or in vivo matured have the same tolerance to the cryoinjury.     

体外受精失败MII期人卵母细胞的免疫荧光研究

目的探讨体外受精中MII期人卵母细胞受精失败的原因。方法收集体外受精后24～48h仍未受精的MII期卵母细胞,进行免疫荧光染色和碘化丙啶(PI)复染,在荧光显微镜下对其失败原因进行分类。结果卵母细胞内未见精子的在常规体外受精(IVF)周期有55.8%,显著多于卵胞浆内单精子注射(ICSI)周期中的9.7%(P<0.01);卵母细胞活化失败两者分别为14.9%和58.1%,有显著性差异(P<0.01);原核形成和(或)迁移缺陷的在两者分别为25.3%和32.3%(P>0.05);其他异常两者分别为3.9%和0.0%。结论IVF中MII期卵母细胞的受精失败主要是缺乏精子的穿透,ICSI周期中的主要原因是卵母细胞活化不完全。     

注射人绒毛膜促性腺激素与否对体外受精周期中改行未成熟卵体外培养结局的影响

目的探讨不育患者在常规体外受精一胚胎移植（IVF-ET）周期中改行未成熟卵体外培养（IVM）,取卵前注射人绒毛膜促性腺激素（hCG）与否对其实验室及临床结局的影响。方法回顾分析2008年1月至2010年11月在温州医学院附属第一医院生殖医学中心行常规IVF治疗的138个周期,在促排卵5～7d后,双侧卵泡数目≥20个,或用药8～13d后,卵泡发育缓慢且两侧卵泡数目≥15个,最大卵泡直径平均≤12mm,根据患者意愿,停药改行IVM治疗,其中63个周期在取卵前12h注射hCG（A组）,75个周期未注射hCG（B组）。比较两组的实验室及临床结局。结果A组的卵成熟率（64．56％）显著高于B组（56．3％）,但是平均获卵数（8．42±0．52）个低于B组（11．32±0．82）个,差异均有统计学意义（P〈0．05）。两组的受精率、卵裂率、移植率、临床妊娠率、流产率,多胎妊娠率比较均无统计学差异（P〉0．05）。结论常规IVF周期改行IVM后可以获得良好的妊娠结局,取卵前注射hCG虽然可以提高卵子的成熟率,但并不能提高妊娠结局。     

体外受精周期中卵泡发育阻滞患者应用未成熟卵体外成熟培养的临床观察

目的观察常规体外受精(IVF)中卵泡发育阻滞患者采用未成熟卵体外成熟(IVM)培养治疗结果。方法常规IVF治疗患者11例,在促排卵过程发生卵泡发育阻滞时,取卵行IVM培养成熟后,卵母细胞行卵胞浆内单精子注射(ICSI)以获得受精卵并最终实现胚胎移植。统计分析未成熟卵的成熟率、卵母细胞的受精率、胚胎的发育情况及临床结果。结果共获取卵母细胞128枚,其中成熟卵21枚,行ICSI后15枚正常受精,获10枚正常卵裂胚胎,全部移植3例,均未妊娠;107枚未成熟卵,经IVM、ICSI和体外培养后,成熟率、受精率、正常卵裂率及优质胚胎率分别为72.9%、75.6%、78%及39.1%,胚胎移植后3例临床妊娠(新鲜周期2例,冻融周期1例)。结论常规IVF控制性超促排卵(COH)过程中,发育阻滞的卵泡在体外适宜的培养条件下仍有成熟、受精、继续发育的能力,并最终实现临床妊娠。IVM技术是改善IVF中发生卵泡发育阻滞患者结局的措施之一。     

Human umbilical cord blood serum in culture medium on oocyte maturation In vitro

In vitro maturation (IVM) of immature oocytes for infertile patients is an attractive treatment. It can avoid side effects of ovarian stimulation with gonadotropins. However, at the present the successful results of IVM treatment are lower than conventional in vitro fertilization (IVF) treatment. The key issue may be the IVM medium for immature oocyte maturation. In the present study, we compared 20% (v/v) human follicular fluid (hFF) and 20% (v/v) human umbilical cord serum (hUS) as a supplement to IVM medium. A total of 47 patients with polycystic ovary syndrome (PCOS) underwent 47 IVM treatment cycles. Immature oocytes (349) collected from 32 patients were cultured in IVM medium supplemented with hFF, and immature oocytes (160) collected from 15 patients were culture in IVM medium supplemented with hUS. The results indicate that the final maturation rate of oocytes cultured in IVM medium supplemented with hUS (93.8%) is significantly higher than those cultured in IVM medium supplemented with hFF (77.1%). The percentage of high-quality embryos produced from IVM medium supplemented with hUS (50.0%) is significantly higher than IVM medium supplemented with hFF (23.8%). In addition, the results also indicate that the final maturation rate of oocytes is higher in IVM medium supplemented with hUS and the time course of oocyte maturation is hastened. Following transfer 6 out of 15 patients (40.0%) become pregnant when IVM medium was supplemented with hUS, and 7 out of 31 patients (22.6%) were pregnant when IVM medium was supplemented with hFF. These results suggest that IVM medium containing hUS appears to be a more effective means to stimulate in vitro oocyte maturation and is capable of achieving a promising clinical outcome.     

Oocyte membrane maturation and oocyte-sperm relationship: transmission electron microscopy study

The success of in vitro maturation (IVM) depends greatly on the acquisition of immature oocytes. Immature oocytes in prophase I (PI) and metaphase I (MI), aspirated after controlled ovarian hyperstimulation, were incapable of fertilization, leading to a lower fertilization rate. Therefore, they must be evaluated on a fine structure level for their in vitro maturation (IVM) processes and their relationship with sperm. Oocyte membrane maturation and oocyte-sperm relationship were studied using transmission electron microscopy. A total of 55 human oocytes obtained from 20 patients at various times and 83 oocytes obtained from the dissected ovarians of female Wistar rats were used for transmission electron microscopy (TEM) evaluation. Despite being in either prophase I and metaphase I or in metaphase II, the oocytes were not fertilized after 48 h of incubation. At the various stages of maturation between PI and MII, the number and the size of microvilluses on the oocyte membrane increased as MII approached and decreased after full maturation. Oocyte activation was related to oocyte membrane maturation and has an effect on the oocyte sperm penetration.