卵裂期胚胎中的凋亡征象及其与胚胎形态的关系

目的 评估体外授精的人卵裂期胚胎中是否存在凋亡,探讨卵裂期胚胎的凋亡征象与胚胎形态之间的关系.方法 结合末端脱氧核苷酸转移酶介导dUTP缺口末端标记法(TUNEL)原位检测DNA凋亡和测定Caspase 3的表达水平,检测人卵裂期胚胎中的凋亡.并分析卵裂期胚胎中胞质碎片占胚胎总体积的比例和发育速度与TUNEL阳性率和Caspase 3的表达强度之间的关系.结果 碎片比>20%组胚胎的凋亡率明显高于碎片比≤20%的胚胎(P<0.01),Caspase 3平均荧光强度在碎片比>50%组明显升高(P<0.05).发育正常、迟缓和停滞胚胎之间胚胎凋亡率差异无显著性意义.发育停滞组的Caspase 3平均荧光强度高于发育正常和迟缓组(P<0.05).结论 人体外授精的卵裂期胚胎中存在凋亡征象,凋亡主要与胞质碎片有关,胚胎发育停滞可能是胚胎凋亡的早期表现.     

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.     

早期原核分裂对于胚胎发育潜能预测的研究

目的通过比较移植早期原核与晚期原核分裂胚胎的临床妊娠率和种植率,探讨早期原核分裂与胚胎发育潜能之间的关系。方法回顾性分析05年11月至07年1月在生殖医学中心行体外受精胚胎移植的妇女240例,通过移植不同时期的原核分裂胚胎将其分为早期原核分裂组（143例）,晚期原核分裂组（47例）和混合组（50例）,比较三组的妊娠率和种植率。结果早期原核分裂组的受精率和早卵裂率分别为80．39％和54．16％,而晚期原核分裂组的受精率和早卵裂率分别为71．01％和8．99％,差异具有统计学意义（P〈0．001）。早裂组的妊娠率（47．55％）高于晚裂组的妊娠率（38．30％）,差异具有统计学意义（P〈0．001）。种植率前者为29．72％,高于后者（22．99％）,差异无统计学意义（P〉0．05）。结论早期原核分裂有更好的妊娠结局,能够预测胚胎发育潜能,评价早期原核分裂是筛选高质量胚胎的一种简单有效、非侵袭性方法。     

卵裂期无可移植胚胎患者行囊胚培养后早期囊胚的最佳移植策略

分析卵裂期无可移植胚胎的患者,在第5天新鲜移植Gardner I期,Ⅱ期囊胚的临床结局,探讨最佳的移植策略。     

麦管玻璃化法冻存人早期胚胎的临床应用

目的探讨麦管玻璃化法冷冻技术的临床应用效果。方法应用麦管玻璃化法冷冻技术保存17例患者的116枚4～8细胞期胚胎。结果复苏了7例患者的37枚胚胎,存活25枚,胚胎存活率为67.56%,其中完整胚胎24枚,移植23枚,种植2枚,种植率为8.69%(2/23),1例获得妊娠,妊娠率为12.5%(1/8),并足月剖宫产一健康女活婴。结论麦管玻璃化法是一种可应用于临床的冷冻保存人早期卵裂期胚胎的技术。     

卵裂期胚胎sHLA-G抗原表达及其与胚胎发育和种植的关系

【目的】通过对卵裂期胚胎培养液可溶性人类白细胞抗原G(sHLA-G)的定性和定量分析,了解卵裂期胚胎sHLA-G抗原表达及其与胚胎发育和种植的关系。【方法】应用免疫发光WesternBlot了解卵裂期胚胎sHLA-G和HLA-I类抗原的表达情况;应用双抗体夹心法ELISA了解卵裂期胚胎培养液中sHLA-I类抗原的表达量与胚胎发育和种植的关系。【结果】①胚胎培养液中以sHLA-G抗原为主,其它sHLA-I类抗原量极少或不存在,因此用培养液中sHLA-I类抗原的量来估计培养液中sHLA-G的含量是可行的。②卵裂期胚胎培养液中1级胚胎sHLA-I类抗原的绝对吸光度(A)值为0.077±0.006;2级胚胎为0.064±0.002;3级胚胎为0.043±0.002;3组比较差异有显著性(P=0.014)。胚胎种植率与胚胎sHLA-I类抗原绝对A值的相关系数为0.426,P=0.004。它们的表达量与胚胎发育和种植的有关。【结论】卵裂期胚胎培养液中sHLA-G的表达量与胚胎发育和种植的有关。     

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.     

神经生长因子在人胚神经管早期发育中的定位表达

目的 研究捷安肽素的抗真菌作用机理.方法 采用形态学方法和同位素标记法.显微形态观察经捷安肽素处理后的供试真菌的形态学变化.进一步采用14C同位素标记的特异底物"尿苷二磷酸-(14C)-葡萄糖"示踪,研究捷安肽素对真菌(1,3)-β-D-葡聚糖合成酶活性反应的影响.结果 研究神经生长因子(NGF)在早期人胚神经管发育过程中的定位表达.方法 采用免疫细胞化学ABC法染色,研究35天人胚的发育情况.结果 在人胚神经管的室管带中,神经元的细胞质和细胞核NGF免疫反应阳性;在中间带,一部分神经元的细胞核NGF免疫反应阳性,另外一部分神经元的细胞核NGF免疫反应阴性,而其突起NGF免疫反应阳性;在边缘带NGF的表达与中间带相似.在神经管的头侧NGF阳性反应较强,神经管的尾侧NGF阳性反应较弱.结论 NGF在人胚神经管免疫反应阳性,表明NGF可能是诱导神经管分化发育的重要信号分子,提示NGF可能在人胚神经管的发育中具有十分重要的作用.     

胚胎玻璃化冷冻技术在辅助生殖治疗中的应用

目的探讨人类卵裂期胚胎玻璃化冷冻在临床治疗中的应用效果。方法经患者知情同意后,将实施体外受精一胚胎移植术患者第3天移植后剩余的符合冷冻标准的胚胎进行玻璃化冷冻保存。如果新鲜周期妊娠失败,根据患者情况解冻胚胎,观察胚胎复苏后的临床妊娠效果。结果玻璃化冷冻84个周期,解冻胚胎207枚,解冻后存活胚胎198枚,存活率为95．7％;完整存活胚胎187枚,完整存活胚胎率为90．3％;种植胚胎41枚,种植率为19．8％;临床妊娠28倒,临床妊娠率为33．3％。早期流产3例,足月分娩5例,20例继续妊娠。结论玻璃化冷冻法适于人类早期胚胎的冷冻保存,获得了理想的复苏率、种植率和妊娠率。     

Effect of Insulin on Development oflCR Mouse Embryos in Vitro

Objective To study the effects and the appropriate concentration of insulin on the development of mouse embryos in vitro, and the effects of insulin on the development of different stages of embryos.
Methods Mouse embryos were cultured in vitro in the mKSOM media supplemented with insulin at different concentrations and with insulin during different stages of embryos. The blastoeyst rates and the cell numbers were counted.
Results Additions of insulin significantly increased the rates of blastocyst and the total cell numbers. The concentrations of 0.005 μg/ml and 0.05 μg/ml insulin caused a significant increase in the total cell numbers compared with the control and experimental groups. Addition of insulin from 2-cell to 4-cell stage or from the 4-cell to morula stage, significantly increased the blastocyst rates compared with control and experi- mental groups.
Conclusion Insulin can promote the development of mouse embryos in vitro. The appropriate concentration of insulin added in mKSOM was 0.005 μg/ml and 0.05μg/ml. Exposure of embryos to insulin, beginning at the 2-cell and extending to the 4-cell stage or beginning at the 4-cell and extending to the morula stage, is important for the development of lCR mouse embryos in vitro.     

Effect of Insulin on Development of ICR Mouse Embryos in Vitro

Objective To study the effects and the appropriate concentration of insulin on the development of mouse embryos in vitro, and the effects of insulin on the development of different stages of embryos.Methods Mouse embryos were cultured in vitro in the mKSOM media supplemented with insulin at different concentrations and with insulin during different stages of embryos. The blastocyst rates and the cell numbers were counted.Results Additions of insulin significantly increased the rates of blastocyst and the total cell numbers. The concentrations of 0.005 μg/ml and 0.05 μg/ml insulin caused a significant increase in the total cell numbers compared with the control and experimental groups. Addition of insulin from 2-cell to 4-cell stage or from the 4-cell to morula stage, significantly increased the blastocyst rates compared with control and experi-mental groups.Conclusion Insulin can promote the development of mouse embryos in vitro. The appropriate concentration of insulin added in mKSOM was 0.005 μg/ml and 0.05 μg/ml.Exposure of embryos to insulin, beginning at the 2-cell and extending to the 4-cell stage or beginning at the 4-cell and extending to the morula stage, is important for the development of ICR mouse embryos in vitro.     

胚胎早裂联合胚胎发育速度及形态评分在胚胎选择中的应用

目的探讨胚胎早裂联合胚胎发育速度及形态评分对于体外授精／卵母细胞单精子显微注射（IVF／ICSI）周期中胚胎选择的意义。方法IVF／ICSI610周期,随机分为A、B两组。A组（269周期）单以授精后72h（D3）胚胎发育速度及形态评分选择胚胎;B组（341周期）在此基础上联合胚胎早裂情况进行胚胎选择。比较两组的临床妊娠率和单胚着床率;比较B组移植胚胎中有早裂胚胎和无早裂胚胎者的妊娠结局。结果B组临床妊娠率和单胚着床率均高于A组（P〈0．05）;B组移植胚胎中有早裂胚胎者的临床妊娠率和单胚着床率均高于无早裂胚胎者（P〈0．01）。结论与单纯的胚胎发育速度及形态评分比较,胚胎早裂联合胚胎发育速度及形态评分进行胚胎评价可显著改善IVF／ICSI的临床结局。     

胚胎早裂联合胚胎发育速度及形态评分在胚胎选择中的应用

目的 探讨胚胎早裂联合胚胎发育速度及形态评分对于体外授精/卵母细胞单精子显微注射(IVF/ICSI)周期中胚胎选择的意义.方法 IVF/ICSI 610周期,随机分为A、B两组.A组(269周期)单以授精后72 h(D3)胚胎发育速度及形态评分选择胚胎;B组(341周期)在此基础上联合胚胎早裂情况进行胚胎选择.比较两组的临床妊娠率和单胚着床率;比较B组移植胚胎中有早裂胚胎和无早裂胚胎者的妊娠结局.结果 B组临床妊娠率和单胚着床率均高于A组(P<0.05);B组移植胚胎中有早裂胚胎者的临床妊娠率和单胚着床率均高于无早裂胚胎者(P<0.01).结论 与单纯的胚胎发育速度及形态评分比较,胚胎早裂联合胚胎发育速度及形态评分进行胚胎评价可显著改善IVF/ICSI的临床结局.     

两种不同培养体系对卵母细胞体外受精及胚胎体外发育的影响

目的　挑选出一种较为理想的体外受精(IVF)培养体系。方法　90例(91个周期)接受IVF/单精子胞浆内注射(ICSI)技术治疗的患者,卵母细胞的IVF及胚胎的体外培养分别采用GⅢ体系(A组, 51周期, )和Quinn体系(B组,40周期)。结果　A组和B组分别获得683枚和652枚卵母细胞,IVF后,受精率分别为63 .25% ( 432 /683 )及66 41%(433 /652),无显著性差异(P>0. 05)。体外培养24h后,A组414枚受精卵发生卵裂,卵裂率为95. 83% ( 414 /432 );B组397枚受精卵发生卵裂,卵裂率为91 69% (397 /433) ,无显著性差异(P>0 .05)。继续培养,行第3天胚胎移植,A、B两组分别获得优质胚胎220及140枚(53. 14%和35. 26% )。最终,A组共有26例获得临床妊娠,B组15例获得临床妊娠,妊娠率为50. 98%和37. 5%,两者之间有显著性差异(P<0 .05)。另外,B组中受精率、卵裂率、优质胚胎率及临床妊娠率方面,IVF周期与ICSI周期之间无显著性差异(P>0. 05),而A组中优质胚胎率及临床妊娠率方面,有显著性差异(48. 97%和63 .11%, 45 .71%和56 25%,P<0 05),受精率和卵裂率方面两周期之间无显著性差异(P>0. 05 )。结论　Quinn系列似乎有助于卵母细胞的体外受精,而GⅢ体系更有利于胚胎的早期发育及后期的着床方面。对于GⅢ系列采取ICSI授精方式似乎更能提高试管婴儿的     

Oct4基因在猪孤雌激活和体外受精早期胚胎中的表达特征

摘 要：[目的]研究植入前胚胎发育重要基因Oct4在猪孤雌和体外受精胚胎中的表达特征。[方法]收集成熟卵母细胞、孤雌和体外受精2细胞、4细胞、8细胞和囊胚，做荧光定量PCR，以成熟卵母细胞做对照分析相对表达量。[结果]孤雌组和体外受精组在8细胞期Oct4表达量均最高（p<0.05），在孤雌和体外受精组囊胚相对于其他时期Oct4表达量最低（p<0.05）。在同一时期孤雌和体外受精Oct4表达并没有差异。[结论]多能性基因Oct4在卵裂发育时期表达量动态变化，孤雌胚胎在一定程度上可作为体外胚胎基因表达的模型，且不同的胚胎培养条件可能导致基因表达的不同。     

Selective Short-term Fertilization Combined with Early Rescue ICSh An Optimal Strategy for Patients at High Risk for Fertilization Failure

Objective To investigate clinical outcomes in patients who were at more precise criteria risks for fertilization failure and were treated with selective, short-term fertilization （oocytes and sperm co-incubated for 4 h） and early rescue intracytoplasmic sperm injection （ICSI）. Methods A retrospective analysis was performed on 2023 women undergoing assisted reproductive technology （ART）. They were assigned to 4 groups： short-term in vitro fertilization （short-term IVF,, group A, n=217）, regular IVF （oocytes and sperm coincubated overnight, group B, n=1475）, short-term IVF and early rescue ICSI （shortterm ICSI, group C, n=94）, and regular ICSI （group D, n=237）. Results In group A, 69.8% （217/311） achieved normal fertilization rates, and the complete fertilization failure rate （fertilization rate was 0%） was 12.9% （40/311）. But all of the fertilization failure oocytes got rescue ICSI. In group B, the complete fertilization failure rate was 1.1% （19/1 692）. The fertilization rate, 2 PN （pronucleus） rate, and i PN rate were significantly lower in group A than those in group B （70.9% vs 80.8%, 57.8% vs 66.3%, and 3.5% vs 6.2%, respectively）. No significant differences were observed in clinical pregnancy rates and birth defect rates between groups A and B. The fertilization rates in groups C and D did not significantly differ （77.9% vs 76.2%）, which was also true for birth defect rates. The clinical pregnancy rate of group C was higher than that of group D （51.2% vs 42.3%）, but this difference was not significant （P〉0. 05）.Conclusion These results suggested that selective, short-term fertilization can result in effective outcomes for patients who were at high risk for fertilization failure.     

In-vitro Developmental Potential of Single Blastomeres Derived from Day 3 Discarded Human Embryos

Objective To investigate whether isolated blastomeres of discarded human embryos could develop into blastocysts cultured in vitro without zona pellucida. Methods Total discarded 60 embryos, which were not suitable for transplanting or freezing, were collected from 21 patients. Of 60 embryos, 10, 8, 24, 12, and 4 embryos were at 2-, 3-, 4-, 5- and 6-cell stage, respectively. These embryos were split by 0.5% protease combined with mechanical method. The resulting single blastomere was cultured individually, evaluated daily and counted blastocyst development. Pluripotency of inner cell mass （ICM） of the blastocyst was analyzed by the expression of alkaline phosphatase（AKP）. Results A total of 229 single blastomeres were isolated from 60 embryos. Defining the day when the embryos were split as the first day （d 1）. The majority of the blastomere- derived embryos followed the normal pattern of development with compaction on d 3 and cavitationon d 4 and developed into small blastocysts on d 5. Rates of division, compaction, cavity and expansion of single blastomeres from 4-cell embryos were higher than those in other groups （P 〈O.05）, and AKP was expressed in ICM. Conclusion Some of the blastomeres from discarded human embryo are flexible and able to develop into blastocysts, the potential was related to their donor embryos closely. They seem to follow development pattern of their donor embryos. The blastomere- derived blastocysts have smaller size and fewer cells compared with regular in vitro cultured human embryos. However, AKP expression showed ICM cells with pluripotency. We believe that the value of single blastomere of discarded embryos will be further confirmed in the future.     

Co-Culture of Early Embryo with Human Decidual Stromal Cells in vitro by Improvement of Early Embryo Development

The quality of the in vitro culture conditionsfor human pre--implantation embryos is one of themost critical aspects of successful in vitro fertilization and embryo transfer (IVF--ET) and other biology techniques, such as embryo clone, geneknock--out, pre--implantation genetic diagnosis etc.The culture of pre--im:7lantation mammalian embryos can take place in a range of embryo culturemedia, ranging from simple chemically defined media to more complex media. In vitro pre--implantation embryo …     

卵丘细胞线粒体移植对年老小鼠早期胚胎发育的影响

目的探讨线粒体对早期胚胎发育的影响。方法采用昆明小鼠为动物模型,以其卵丘颗粒细胞、卵母细胞、受精卵为试验材料,提取年老小鼠卵丘细胞线粒体,将其注入年老小鼠卵母细胞和体内冲出的受精卵中。结果与结论通过卵丘细胞线粒体移植,给卵子补充了一定量的线粒体,可以部分弥补卵母细胞因变异引起的功能不足。为第2次减数分裂、受精卵和胚胎发育提供足够的能量,明显改善了胚胎的质量。     

Effect of an Improved Mechanical Method for Assisted Hatching on the in vitro Development of Mouse Embryos

Implantation of embryo occurs after its escape from the zona pellucida (ZP), the pro- cess of which is called hatching. In vivo hatching is viewed as a prerequisite for successfuldialogue between the embryo and the endometrium. It has been speculated that…