小鼠2-细胞胚胎卵裂球后代在囊胚中随机分布

目的 用异硫氰荧光素（FITC）-右旋糖苷和四甲基罗丹明（TMR）-右旋糖苷标记小鼠2-细胞胚胎的两个卵裂球,观察其发育,以探讨囊胚Em-Ab极性的形成。方法 向受精卵注射FITC-右旋糖苷确定标记物对胚胎的伤害性,向2-细胞胚胎两个卵裂球分别FITC-右旋糖苷分别和TMR-右旋糖苷,将标记后的胚胎体外培养发育至囊胚,观测两个卵裂球后代在囊胚中的分布情况。 结果 2-细胞胚胎两个卵裂球的后代在囊胚中分布并无规律。 结论 2-细胞胚胎卵裂球随机分布在囊胚的胚胎部分和胚外部分。     

玻璃化和程序化冻融人卵裂期胚胎结果分析

目的探讨冻融周期中玻璃化和程序化两种不同方法对人卵裂期胚胎的冷冻复苏效果。方法回顾性分析35例玻璃化冷冻复苏周期和123例程序化冷冻复苏周期资料,比较冷冻胚胎复苏后的胚胎存活率、完整性胚胎存活率,冻融前后的优质胚胎率,临床妊娠率、胚胎种植率。结果①玻璃化法与程序化法冷冻前优质胚胎率无显著性差异（70．83％和71．97％,P〉0．05）,冷冻后优质胚胎率差异显著（63．89％和47．75％,P〈0．05）,复苏后的胚胎存活率差异显著（95．83％和86．16％,P〈0．05）,完整性胚胎存活率差异极显著（90．28％和63．32％,P〈0．01）;②玻璃化法与程序化法临床妊娠率差异显著（48．57％和29．27％,P〈0．05）,种植率差异显著（31．88％和18．83％,P〈0．05）。结论在冻融人卵裂期胚胎的过程中,玻璃化优于程序化,可以更好的提高累积临床妊率。     

早期卵裂联合胚胎形态学评分选择胚胎移植妊娠结局探讨

目的探讨早期卵裂在体外受精-胚胎移植中对胚胎选择的临床价值。方法分析2010年12月至2011年9月在本中心接受体外受精((In Vitro fertilization,IVF)和卵胞浆内单精子显微注射-胚胎移植(intracytoplasmic sperm in-jection and embryo transfer,ICSI-ET)治疗的307个周期中根据移植的胚胎未观察到、观察到早期卵裂分为A、B两组,统计两组年龄、ICSI比例、内膜厚度、获卵数、移植胚胎数、移植优胚数、临床妊娠率、种植率。结果 B组临床妊娠率(54.1%vs40.8%)及种植率(34.5%vs25.7%)均高于A组,差异有统计学意义。结论早期卵裂联合胚胎形态评分运用简便、无创,可以在同等条件下改善体外受精-胚胎移植的临床结局。     

早期卵裂作为胚胎质量预测指标对体外授精结局的影响

目的探讨早期卵裂是否可作为评估胚胎质量、胚胎种植率、临床妊娠率的预测指标。方法对2006年1月～2006年12月的471对夫妇，480个周期随机分A组和B组（对照组）。A组：262个周期，即从授精25～27h后观察2PN受精卵是否出现早期卵裂，同时第二天、第三天观察胚胎质量并进行分级；B组：218个周期，第二天、第三天观察胚胎质量并进行分级，但授精25—27h后不观察2PN受精卵是否出现卵裂。同时根据移植胚胎中含有出现早期卵裂的个数分为四组A，（三个移植胚胎均出现早期卵裂），A2（移植胚胎含两个出现早期卵裂），A1（移植胚胎一个出现早期卵裂），A0（移植胚胎无出现早期卵裂）。比较四组胚胎种植率。结果A组种植率和临床妊娠率均高于未观察早期卵裂B组，但统计学分析无显著意义；A3、A2、A1、A0组间胚胎种植率分别为30．9％、26．1％、21．2％、11．2％，有显著性差异（P〈0．001）。结论（1）第2，3天胚胎形态学分级依然可作为评定胚胎质量的参数。（2）早期卵裂是可作为预测高种植率胚胎的指标之一。     

促排卵移植周期中不同卵裂球数目优势胚胎移植结局的比较

目的探讨体外受精-胚胎移植周期中移植不同卵裂球数目的优势胚胎对妊娠结局的影响。方法对我中心2008年1月-2009年10月270例来曲唑促排卵方案第三天移植的胚胎进行回顾性分析,根据不同卵裂球数目分为4组：至少一个优势胚胎8细胞组（A组）,至少一个优势胚胎7细胞组（B组）,至少一个优势胚胎6细胞组（C组）,小于6细胞组（包括4细胞和5细胞）（D组）计算各组移植后临床妊娠率、种植率,流产率。结果优势胚胎为8细胞的妊娠率和种植率最高（37.5%,19.9%）,其次是7细胞组（25%,15.9%）和6细胞组（23.8%,12.5%）,4细胞和5细胞最低（13.3%,12.1%）,各组具有统计学差异（P〈0.05）。各组流产率无显著统计学差异（P〉0.05）。结论第三天移植优势卵裂球8细胞胚胎获得更高的临床效果,细胞数目少的胚胎仍具有一定的发育潜能,不能轻易丢弃。     

植入前遗传学诊断中卵裂球固定技术改进

目的改进植入前遗传学诊断卵裂球固定技术,减少卵裂球细胞丢失,获得更好的固定效率、更高的荧光原位杂交成功率,简化操作程序.方法体外授精治疗周期废弃胚胎的96个卵裂球细胞,分别用3种不同的固定技术进行固定,固定后使用X、Y着丝粒探针进行荧光原位杂交,比较其固定效率和杂交效率.结果改进方法固定率和杂交率均为100%.而两种传统技术的固定率分别为90%,83.3%;杂交率为80%,73.3%.结论这种新的固定卵裂球技术从根本上消除了卵裂球标本的丢失,使PGD结果更为可靠;同时简化了操作程序,值得推广.     

人类胚胎早期发育潜能的研究

目的　本研究采用卵裂球分离技术获取不同发育阶段人类早期胚胎单个卵裂球 ,对分散的人类早期胚胎单个卵裂球进行体外培养。对其进一步发育和分化进行研究 ,发现其影响因素。结果　 2 0个 4-细胞、2个 3-细胞人类早期胚胎分散后获得单个卵裂球 83个 ,经体外培养后有 35个发育成为扩张囊胚。结论　 4-细胞人类胚胎卵裂球具有发育成为完整囊胚的能力     

小鼠第一次卵裂周期中线粒体分布的变化

用线粒体专一性活体荧光染色剂罗丹明１２３显示小鼠受精卵在第一次卵裂周期中Ｍ的分布变化，雌原核和雄原核在汇合之前，Ｍ在细胞质中呈弥散状随机分布，两原核汇合后，Ｍ在核周围略显聚集，第一次卵裂后期，Ｍ沿纺锤体微管和２个子核周围集中，但在赤道区域内明显稀少，预示出细胞分裂面的定位，这说明细胞质分裂是收综环收缩和细胞结构调整共同作用的结果。２－细胞阶段，Ｍ在细胞核周围明显聚集。２－细胞胚受秋水仙素作用后，Ｍ     

胚胎密度对卵裂期优质胚胎率的影响

目的 通过调整协变量来探索培养的胚胎密度与第3天卵裂期胚胎发育结果之间的相关性.方法 回顾性分析206对夫妇1196个胚胎体外受精-胚胎移植(IVF-ET)治疗的资料.培养条件为低氧,培养液每微滴体积为30 μl.以第3天卵裂期胚胎的优质胚胎率为终点指标对胚胎质量进行评价.选择女方年龄,男方年龄,窦卵泡数,抗苗勒管激素...     

早期人胚胎视网膜色素上皮细胞的体外培养及生物学特性

目的探索早期人胚视网膜色素上皮(RPE)细胞体外培养以获得高纯度的RPE细胞的方法,并进一步研究其生物学特性。方法取意外流产4h以内人胚(6～10周)眼球,通过混合酶消化法获取RPE细胞。用F12培养液体外培养并作细胞形态学、S-100和Keratin免疫组织化学鉴定;通过Trizol一步法提取总RNA,逆转录聚合酶反应(RT-PCR)分析体外培养RPE细胞酪氨酸酶基因的表达。结果用F12培养液可获得纯度高、活性好的人胚RPE细胞;体外培养的RPE细胞不表达酪氨酸酶基因。结论早期人胚RPE细胞具有在体RPE细胞的部分特征;体外培养的RPE细胞未检测到酪氨酸酶RNA表达,故随着细胞分裂增殖,色素颗粒逐渐减少,提示体内外RPE细胞在功能上存在某些差异。     

DNA fingerprinting of sister blastomeres from human IVF embryos

BACKGROUND: Previously published single cell DNA fingerprinting systems have been plagued by high rates of allele drop-out (ADO) and preferential amplification (PA) preventing clinical application in preimplantation genetic diagnosis. METHODS: Tetranucleotide microsatellite markers with high heterozygosity, known allelic size ranges and minimal PCR stutter artefacts were selected for chromosomes X, 13, 18 and 21 and optimized in a multiplex fluorescent (FL)-PCR format. FL-PCR products were analysed using the ABI Prism 377 DNA sequenator and Genescan software. Validation of the DNA fingerprinting system was performed on single diploid (n = 50) and aneuploid (n = 25) buccal cells and embryonic blastomeres (n = 21). RESULTS: The optimized pentaplex PCR DNA fingerprinting system displayed a high proportion of successful amplifications (>91%) and low ADO and PA (<6%) when assessed on 50 human buccal cells. DNA fingerprints of single cells from a subject with Down's syndrome detected the expected tri-allelic pattern for the chromosome 21 marker, confirming trisomy 21. In a blind study on 21 single blastomeres, all embryos were identifiable by their unique DNA fingerprints and shared parental alleles. CONCLUSIONS: A highly specific multiplex FL-PCR based on the amplification of five highly polymorphic microsatellite markers was developed for single cells. This finding paves the way for the development of a more complex PCR DNA fingerprinting system to assess aneuploidy and single gene mutations in IVF embryos from couples at genetic risk.     

Short Report. The study of early human embryos using interactive 3-dimensional computer reconstructions

Tracings of serial histological sections from 4 human embryos at different Carnegie stages were used to create 3-dimensional (3D) computer models of the developing heart. The models were constructed using commercially available software developed for graphic design and the production of computer generated virtual reality environments. They are available as interactive objects which can be downloaded via the World Wide Web. This simple method of 3D reconstruction offers significant advantages for understanding important events in morphological sciences.     

Ultrastructure of the developing stomach in human embryos

Summary Ultrastructural development of the stomach was studied by light, scanning electron and transmission electron microscopy, using 19 human embryos at Carnegie stages from 14 to 23 (6.8–28.0 mm in crown-rump length, 5 to 8 weeks of gestation). The precise time of appearance of differentiated characteristic structures was examined electron microscopically. The first gastric pit, with radially arranged epithelial cells beneath which the basement membrane bulged into the mesenchyme, was observed on the lesser curvature at stage 22. Although the mesenchymal condensation which would develop into the inner circular muscle layer appeared at stage 18 onward, cytoplasmic myofibrils were not observed until stage 22. Nerve fibers were first observed at stage 16, and at later stages they gathered into bundles to form a nerve plexus external to the developing inner circular muscle layer. On the basis of accurate timing of the appearance and the mode of development of these structures, possible relations between developing gastric layers were discussed. Histocytochemically, glycogen or other carbohydrates were demonstrated in the cytoplasm of the gastric epithelium throughout the stages examined. These carbohydrates were localized mainly in vacuole-like spaces in the basal part of the epithelial cells. This subcellular localization, and the amount of carbohydrate, did not change significantly during the observed embryonic period. In the serosa, carbohydrates were not detected at stages 14 and 15, but observed consistently within the vacuoles in the cytoplasm from stage 17 onward. No other layer of the embryonic stomach had detectable carbohydrates. These observations suggest that carbohydrates in the gastric epithelium at an early developmental stage are not directly related to the developing mucin secretory activity of the epithelium, but may serve as an energy source for cell growth and differentiation of the epithelium and/or for mesenchyme-epithelial interactions.     

输卵管积水对小鼠胚胎体外发育能力的影响

目的探讨输卵管积水对小鼠胚胎体外发育能力的影响。方法收集人输卵管积水，小鼠促超排卵，收集2细胞胚胎和囊胚，随机分配到含有不同浓度输卵管积水的培养液中，观察胚胎的发育，计算成囊率，囊胚孵出率。结果输卵管积水组的胚胎成囊率和囊胚孵出率低于无积水组，并呈剂量依赖性。结论输卵管积水能显著影响胚胎的早期发育潜能。     

Fertilization and early embryology: The presence of multinucleated blastomeres in human embryos is correlated with chromosomal abnormalities

The purpose of the present study was to determine whether thepresence of one or more multinucleated blastomeres during earlyembryonic development is associated with chromosomal abnormalitiesIn sibling blastomeres of that embryo. Embryos with multinucleatedcells (n = 47) detected on day 2 or 3 of development were comparedto dividing embryos without multinucleation. Arrested embryoswere excluded from this study. Chromosome abnormalities weredetected using fluorescent in-situ hybridization (FISH) withX, Y, 18 and 13/21 chromosome- specific probes. Of 47 embryosincluded in this study, 76.6% were chromosomafly abnormal, comparedto 50.9% in the control group (P < 0.001). Excluding aneuploidy,which is originated in the gametes and not the embryo, the differenceswere even higher, with 74.5% of multinucleated embryos beingchromosomafly abnormal compared to 32.3% of non-multinucleatedembryos (P < 0.001). Day of multinucleation appearance, numberof nuclei per cell, number of multinucleated cells per embryoand developmental quality of the embryos as well as the typeof fertilization (intracytoplasmic sperm injection versus standardinsemination) were not found to affect the rate of chromosomalabnormalities In embryos with multinucleated cells. These resultssuggest that embryos with multinucleated cells may not be suitablefor replacement and should be excluded unless no other embryosare available.     

The frequency and developmental capability of human embryos containing multinucleated blastomeres

The frequency of multinucleated blastomeres (MNB) in 2- and 4-cell stage human embryos was recorded immediately before embryo transfer using a high-power inverted microscope. About 44% of patients (150/338) possessed embryos exhibiting MNB. The appearance of this nuclear abnormality was not correlated with maternal age. Overall, 15% of the otherwise good quality embryos (274/1885) that developed after monospermic fertilization contained several multinuclei (from two to seven) in at least one cell. Quite often MNB were found within all cells of the embryo (50% in 2-cell embryos). Blastomere multinucleation was significantly higher in 2-cell than 4-cell embryos (P <0.0001). This suggests that a considerable number of human embryos become abnormal during the first embryonic division. The embryos containing MNB were usually excluded for uterine transfers, with the exception of 19 cases when only such embryos could be replaced (6%; 19/338 patients). The results demonstrated that embryos with MNB may implant (4/19 cases; 21%) and they can lead to both spontaneous abortions and the successful birth of healthy infants (two cases). The fact that in the successful cases, 2-cell stage embryos with a mononucleated and a binucleated blastomere were transferred also suggests that due to the cell totipotency, development of a healthy baby is possible from one normal blastomere. Since multinucleation in early embryos may reflect gross chromosomal abnormalities or development of mosaic embryos, it is advisable not to replace embryos with MNB. Occasional transfers, however, can be considered because defective embryos may sometimes develop normally.