人成熟卵泡液在人未成熟卵母细胞体外培养中的应用

卵母细胞体外成熟(IVM)培养主要用于治疗多囊卵巢综合征(PCOS)[1] 及诱导排卵中卵巢反应不良等症。虽然 IVM在临床已实践了 10 多年,但IVM卵母细胞的妊娠率远低于常规卵母细胞的妊娠率[2]。本研究旨在实验一种可用于临床的 IVM培养液,改善临床妊娠率。一、资料和方法1.对象: 2003年8~12月在我院生殖中心行未成熟卵IVM治疗 14 例、14 个周期的 PCOS 患者。诊断标准: 月经稀发或闭经、肥胖、不育;阴道超声提示双侧卵巢每一平面均有> 8 个最大直径< 10mm的小卵泡;月经第3天激素检查黄体生成素/卵泡刺激素(LH/FSH)≥2、泌乳素(P…     

未成熟卵母细胞体外成熟后体外授精-胚胎移植成功分娩1例

1991年始 ,国外有报道从人类卵巢中取到未成熟卵在体外成熟培养 (ｉｎｖｉｔｒｏｍａｔｕｒａｔｉｏｎ ,ＩＶＭ )后经体外授精 胚胎移植 (ｉｎｖｉｔｒｏｆｅｒｔｉｌｉｚａｔｉｏｎａｎｄｅｍｂｒｙｏｔｒａｎｓｆｅｒ,ＩＶＦ ＥＴ)可以获得妊娠[1～ 4] 。国内尚未见成功报道     

人卵母细胞体外成熟培养进展及临床意义

与其他许多哺乳动物一样，人卵母细胞在胎儿期间就开始了第一次减数分裂。出生时初级卵母细胞已经过第一次减数分裂前期，处于前期和中期之间的静止期，并长期停留在此阶段，直至青春期才开始分批分期完成第一次减数分裂。１９３５年，Ｐｉｎｃｕｓ和Ｅｎｚｍａｎ［１］观察到将兔卵母细胞从卵泡中移出后，卵母细胞可在体外自发地排出第一极体，此过程称为卵母细胞成熟。生殖工程的研究由此开辟了一个新的领域——卵母细胞的体外成熟（ｉｎｖｉｔｒｏｍａｔｕｒａｔｉｏｎ，ＩＶＭ）培养。一、生殖泡（ｇｅｒｍｉｎａｌｖｅｓｉｃａｌ，ＧＶ…     

时差监测培养系统在人未成熟卵母细胞体外成熟及其授精时机中的应用

目的观察时差监测培养系统在人未成熟卵母细胞体外成熟(IVM)中的应用,并探讨核成熟后最佳的授精时机。方法收集我生殖中心促排卵周期中捐赠用于科研的240枚GV期卵母细胞,应用时差监测培养系统联合IVM技术,观察卵母细胞核成熟的动力学参数,包括GV破裂时间、第一极体排出时间、MI持续和MⅡ静止的时间段。经过培养,对其中160枚卵母细胞根据第一极体排出后不同ICSI授精时间分为3组:3h组、3~6h组和6h组,比较各组的受精情况及胚胎发育情况。结果 240枚GV期卵母细胞体外成熟率为73.3%,平均GV破裂时间为实施IVM后的(7.0±0.3)h,MI持续平均时间为(14.9±0.2)h,第一极体排出平均时间为(22.7±0.5)h。不同授精时间3组的正常受精率、卵裂率和可利用胚胎率比较均无显著性差异(P0.05)。结论时差监测培养系统为未成熟卵母细胞IVM培养系统优化提供了新思路;第一极体排出后的3~6h实时ICSI可用胚胎率有增高的趋势,但其具体影响及最佳授精时间仍有待进一步研究探讨。     

褪黑素对老化卵母细胞体外发育能力的改善效果评价

目的探讨体外培养阶段(IVC)添加褪黑素(MT)对卵母细胞老化引起发育受损的改善效果。方法采用不同浓度的过氧化氢(H_2O_2)处理小鼠MII期卵母细胞诱导老化,浓度分别为0(对照组)、10、50、100、150μmol/L,体外受精(IVF)后统计各组二细胞率、囊胚形成率,检测老化卵母细胞的线粒体活性及丰度(Mitotracker Red、JC-1)、活性氧(ROS)水平、线粒体拷贝数等指标;在100μmol/L H_2O_2处理条件下,老化卵母细胞在IVF后的培养阶段分别添加不同浓度褪黑素(10-5、10-7、10-9 mol/L),统计二细胞率、囊胚率,并且分别检测各组获得囊胚的细胞数及凋亡率。结果不同浓度H_2O_2诱导卵母细胞老化后,囊胚发育率随H_2O_2浓度的升高而降低,50μmol/L和100μmol/L H_2O_2组囊胚形成率分别为(26.27±0.06)%和(28.46±3.45)%,相比对照组的(34.90±1.77)%显著降低,差异有统计学意义(P0.05);在H_2O_2诱导老化卵母细胞中,100μmol/L、150μmol/L H_2O_2浓度时,ROS水平相比对照组显著升高,差异具有统计学意义(P0.05);而活性线粒体的丰度及拷贝数呈现下降趋势,膜电位呈上升趋势,但相比对照组无显著性差异(P0.05);100μmol/L H_2O_2处理诱导卵母细胞老化后,在培养液中添加10-9 mol/L褪黑素组与老化对照组相比,囊胚率[(29.42±2.39)%vs.(20.87±4.12)%]、囊胚细胞数(39.36±9.78vs.37.91±4.25)均显著升高,凋亡率显著下降(2.57%vs.3.18%),差异均有统计学意义(P0.05);并且这些指标均达到与未经老化处理组的相似发育水平。结论老化卵母细胞体外受精后发育率和发育质量偏低的现象,可以通过在受精后体外培养阶段添加褪黑素得到改善。     

表皮生长因子和促性腺激素对人类卵母细胞体外成熟的影响

目的 :研究表皮生长因子 (EGF)和不同浓度促性腺激素 (Gn)对未成熟卵母细胞体外成熟的影响。　方法 :将EGF添加至卵母细胞培养液中 ,观察EGF Gn联合应用与单独使用Gn对卵母细胞体外成熟的影响 ;分别改变绒毛膜促性腺激素 (hCG)和卵泡刺激素 (FSH)的浓度 ,观察不同浓度的hCG、FSH对卵母细胞体外成熟的影响。结果 :添加EGF可显著提高卵母细胞的体外成熟率 (P <0 .0 5 ) ;提高hCG和FSH的浓度对卵母细胞的培养、受精及卵裂无影响。　结论 :EGF可促进卵母细胞体外成熟 ,明显提高卵母细胞体外成熟率 ;提高Gn浓度对卵母细胞的培养无进一步的促进作用。     

卵母细胞体外成熟治疗多囊卵巢综合征不孕妇女

多囊卵巢综合征是最常见的无排卵性不孕的原因.如果在常规诱导排卵的方法失败后,可采用辅助生育技术.而卵母细胞体外成熟是一种有效、经济、简便而且没有卵巢过度刺激综合征的好方法.     

体外成熟的卵母细胞胞质成熟的评价指标

卵母细胞的体外成熟培养(in vitro maturation,IVM)是辅助生殖技术的重要过程之一.优质的IVM的卵母细胞必须是细胞核和细胞质同步成熟,以保证成功受精、胚胎正常发育乃至健康子代的出生.目前,细胞核成熟的评价标准已得到共识,而细胞质的成熟却表现复杂,尚缺乏有效的观察评价指标.近年来研究发现,卵母细胞成熟时其超微结构、一些信号分子和细胞因子均发生某些变化,这为评价细胞质成熟提供了依据.     

体外受精-胚胎移植周期未成熟卵体外成熟培养的临床价值

目的探讨体外受精-胚胎移植(IVF-ET)周期未成熟卵体外成熟培养(IVM)的临床价值。方法对106例不孕患者IVF/ICSI-ET治疗周期中357枚未成熟卵(MⅠ期,217枚;GV期,140枚)进行24h和48h体外培养,比较不同发育阶段未成熟卵的体外成熟率、受精率、卵裂率、优质胚胎率及囊胚形成率。结果 MⅠ期卵24h和48h总成熟率均显著高于GV期卵(分别为59.91%vs.24.29%和72.35%vs.42.86%)(P0.05);体外成熟MⅡ卵平均每取卵周期可利用胚胎数只有(0.50±0.84)枚,且受精率、卵裂率和优胚率、可利用胚胎数均显著低于同一促排卵周期体内成熟MⅡ卵(P0.05)。结论 IVF-ET周期中所获未成熟卵经IVM能发育成熟,并具备一定的受精和胚胎发育潜能,但相关指标明显低于体内成熟卵,临床应用价值有限。     

颗粒细胞复合体在卵母细胞体外成熟培养中作用的研究

目的探讨颗粒细胞复合体应用于未成熟卵母细胞体外成熟培养(IVM)中的作用。方法收集2015年1月至2017年6月在我院生殖医学科接受ICSI-ET治疗的156例不育患者促排卵周期中废弃的未成熟卵母细胞为研究对象。共收集MⅠ期和GV期未成熟卵母细胞305枚,以随机方式入组到对照组(C组,培养时不添加颗粒细胞复合体)和实验组(T组,培养时添加颗粒细胞复合体)进行培养,并按照MⅠ期和GV期未成熟卵母细胞的不同分为4个亚组:MⅠ-C组、MⅠ-T组和GV-C组、GV-T组。观察各组的体外成熟及受精后胚胎发育情况等。结果 MⅠ-T组体外培养48h后的总成熟率显著高于MⅠ-C组(88.42%vs.73.62%,P0.01),体外培养24h的成熟率(80.00%vs.65.93%)、正常受精率(69.74%vs.53.33%)、受精率(75.00%vs.55.00%)及优质胚胎率(35.56%vs.12.00%)均显著高于MⅠ-C组(P0.05),且MⅠ-T组的卵母细胞利用率亦显著高于MⅠ-C组(21.05%vs.5.00%,P0.01);MⅠ-T组获得4个囊胚,多于MⅠ-C组(1个囊胚)。GV-T组体外培养48h的总成熟率显著高于GV-C组(65.00%vs.35.59%,P0.01);GV-T组培养24h的各项指标与GV-C组比较均无显著性差异(P0.05);GV-T和GV-C两组均未获得囊胚。结论含有颗粒细胞复合体的共培养体系可以提高促排卵周期中不同来源未成熟卵母细胞的体外成熟率,改善MⅠ期成熟卵母细胞的发育潜能,但是否能改善GV期卵母细胞的发育潜能尚需进一步研究。     

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。     

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.     

卵母细胞体外成熟在IVF—ET周期中降低OHSS风险中的应用

目的探讨卵母细胞体外成熟培养（IVM）在体外受精-胚胎移植（IVF—ET）周期中降低卵巢过度刺激综合征（OHSS）风险的应用价值。方法51例IVF长方案超排卵过程中出现OHSS早期征象时,充分知情后按患者意愿分成两组,实验组为即刻停药并于当天注射人绒毛膜促性腺激素（HCG）36h后取卵,改行卵母细胞IVM治疗的21个周期;对照组为继续按常规治疗的30个周期。比较两组促性腺激素（Gn）用药天数、用药量、受精率、周期取消率、临床妊娠率及OHSS发生率。结果两组年龄、不育年限、基础内分泌、受精率、优质胚胎率、临床妊娠率均无统计学差异（P〉0．05）,但实验组的卵泡刺激素（FSH）总用量、FSH用药天数、获卵数、OHSS（中、重度）发生率均低于对照组（P％0．05）,并且实验组周期ET取消率也显著低于对照组（P〈0．05）。结论在常规IVF周期超排卵中,当出现OHSS早期征象时改行卵母细胞IVM可显著减少促排卵药物的使用量,降低OHSS的发生风险及周期移植取消率,同时获得较好的临床妊娠率。     

The effect of helium-neon laser irradiation on in vitro maturation and fertilization of immature bovine oocytes

A study was made of the effects of low power laser irradiation on in vitro oocyte maturation rates and subsequent fertilization of immature bovine oocytes. Immature bovine oocytes from cows sacrificed at a slaughterhouse were irradiated with Helium-Neon laser (HeNe laser) irradiation at an energy density of 0.4 and 2.0 J cm^–2. An oocyte group was left untreated, serving as control group. All oocytes were matured and fertilized in TCM-199 medium supplemented with 20% fetal calf serum (FCS). Maturation and fertilization rates obtained in the irradiated oocytes group were lower (p<0.001 andp<0.05, respectively) than those of the control group. Furthermore, the laser-treated oocytes showed important degenerative changes on both cytoplasm and chromosomes in comparison with untreated control oocytes which showed a homogenous cytoplasm and disperse chromosomes. It is concluded that the application of HeNe laser irradiation at 0.4 and 2.0 J cm^–2 energy densities has a detrimental effect on in vitro maturation and fertilization process of immature bovine oocytes.     

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。     

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.     

褪黑素对人卵裂期胚胎发育的影响及机制研究

目的探讨褪黑素对人第3天(D3)卵裂期胚胎发育成囊胚的影响及其作用机制。方法收集2012年10月至2014年10月在本中心行IVF-ET助孕治疗患者捐赠的D3玻璃化冷冻胚胎,复苏后存活率100%且未发生卵裂球溶解的胚胎纳入研究(n=143),随机分为褪黑素组(培养液中加入1×10-7 mol/L褪黑素,n=71)和对照组(不添加褪黑素,n=72)。两组胚胎均放于37℃、5%CO2培养箱中培养72h,统计囊胚及优质囊胚形成率;采用鲁米诺化学发光法检测培养液中总活性氧(ROS)水平;实时荧光定量PCR法分析抗氧化酶类基因CAT、GPx、SOD1、SOD2及凋亡相关基因Bax、Bcl-2的表达。结果褪黑素组的囊胚形成率(42.25%)显著高于对照组(26.38%)(P0.05),优质囊胚形成率(30.00%)略高于对照组(26.32%),但无显著性差异(P0.05)。培养72h后两组胚胎培养液中的ROS水平比较(513.33vs.556.67RLUs)无显著性差异(P0.05)。褪黑素处理后,胚胎内过氧化氢酶基因CAT的相对表达量(1.68±0.43)较对照组(1.00±0.03)显著上调(P0.05),其他基因包括Bax、Bcl-2、SOD1、SOD2、GPx的表达则没有显著性差异(P0.05)。结论褪黑素能够促进人卵裂期胚胎继续发育形成囊胚,其机制可能与上调胚胎中过氧化氢酶基因CAT的表达有关。     

Follow-up of pregnancies after treatment of in vitro maturation of immature oocytes retrieved from PCOS patients with unstimulated cycles

Objective: To follow up and analyze pregnancy outcomes obtained from treatment of in vitro maturation (IVM) of immature oocytes retrieved from women with polycystic ovary syndrome (PCOS) in unstimulated cycles.Methods; Data were collected from the Reproductive Medical Center of First Affiliated Hospital of Wenzhou Medical College. 140 IVM cycles of 118 women with PCOS were treated. All children born were followed up. The multiple pregnancy rate, birth weight, premature rate, pregnancy complications, mode of delivery, growth retardation and congenital abnormalities were observed.Results: The clinical pregnancy rate was 40.17% (57/140), including 2 cases of ectopic pregnancies (3.5%),35 cases of singletons (61.4%), 17 cases of twins (29.82%) and 3 cases of triplets(5.26%). Among singletons,7 cases were aborted at gestation of 7-14 weeks (12.28%). 31 babies were delivered from 17 cases of twins, among them, one case was aborted at 22 week gestation, another case was received selective reduction to become singleton.3 cases of triplets were all reduced into twins and 6 babies were delivered alive. All together, 65 babies were born from 47 women in this study. The rate of multiple pregnancies, premature and infants with low weight were 36. 36%(20/55), 21.28%(10/47), and 26. 15%(17/65), respectively. The general pregnancy complications rate was 26. 32%(15/57).Conclusion: Our results showed no increasing pregnancy complications and congenital malformations in 65 babies born from women with PCOS in unstimulated IVM cycles.     

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。