人无血清输卵管上皮细胞模型与小鼠胚胎共培养的研究

目的了解２细胞期小鼠胚胎与人输卵管上皮细胞体外无血清共培养中的发育状况。方法配制无血清培养液,予小鼠超排卵并取２细胞期胚胎,将９０个２细胞期小鼠胚胎与人输卵管上皮细胞在无血清培养液中进行体外共培养,另以９０个同期胚胎在无辅助细胞的培养液中培养作对照,每日在显微镜下观察小鼠胚胎发育情况。结果共培养的胚胎有８２％发育到桑椹胚,７７％形成囊胚,６３％胚胎孵化。对照者仅４５％发育到桑椹胚,１３％到初级囊胚,无孵化胚胎。共培养胚胎发育速度快,碎片率明显低于对照者。结论人输卵管上皮细胞与小鼠胚胎共培养可以促进胚胎体外发育,并提高胚胎发育质量。     

人子宫内膜,早孕蜕膜单层细胞及其条件培养液对小鼠胚胎发育的作用

为研究人子宫内膜、早孕蜕单膜单层细胞及其培养液对早期胚胎发育的作用。本地将人增殖晚期－早分泌期子宫内膜及早孕蜕膜消化成单个细胞或细胞团块,分别在体外培养,并冷冻保存。建立单层细胞交制成条件培养液,由于人胚取材较困难,此采用小鼠早期胚胎共培养,结果显示,人子宫内膜及早孕蜕膜在体外发育良好,并可冷冻保存,复苏率〉４０％,小鼠胚胎的卵裂率、桑椹胚及囊胚形成率明显高于对照组。早孕且较子宫内膜组略高,但     

人子宫内膜、早孕蜕膜单层细胞及其条件培养液对小鼠早期胚胎发育的作用

为研究人子宫内膜、早孕蜕膜单层细胞及其条件培养液对早期胚胎发育的作用,本文应用酶消化法将人增殖晚期早分泌期子宫内膜及早孕蜕膜消化成单个细胞或细胞团块,分别在体外培养,并冷冻保存。建立单层细胞并制成条件培养液,由于人胚取材较困难,此文采用小鼠早期胚胎共培养,结果显示,人子宫内膜及早孕蜕膜在体外发育良好,并可冷冻保存,复苏率＞ ４０％ 。小鼠胚胎的卵裂率、桑椹胚及囊胚形成率明显高于对照组（Ｐ＜ ０．００１）,早孕蜕膜组较子宫内膜组略高,但无显著性差异。两种细胞的条件培养液均可刺激胚胎发育,但无显著性差异,而胚泡形成率在早孕蜕膜组明显高于对照组（Ｐ＜ ０．０５）。提示：人子宫内膜及早孕蜕膜细胞能释放某些刺激胚胎发育的物质有利于胚胎发育     

小鼠囊胚在子宫内膜体外共培养模型上着床的超微结构观察

目的:进一步证明已建立的体外着床模型的可行性。方法:将妊娠d4的小鼠囊胚培养在已建立的体外子宫内膜共培养模型上,电镜观察胚胎植入时囊胚与子宫内膜上皮细胞相互关系的超微结构。结果:小鼠囊胚能正常脱透明带、黏附和扩展。扫描电镜见黏附在子宫内膜上的囊胚呈椭圆形或扁平形;胚胎表面具微绒毛;在胚胎与子宫内膜细胞接触点可见胚胎滋养细胞黏附于胞饮突上。透射电镜观察显示,胚胎滋养细胞通过微绒毛黏附于上皮细胞上,有些滋养细胞通过微绒毛开始植入,局部滋养细胞与上皮细胞形成紧密连接。胚胎滋养细胞与上皮细胞间形成许多镶嵌连接。超微结构显示粗面内质网、线粒体、溶酶体和核糖体非常丰富。结论:小鼠囊胚在子宫内膜体外模型上生长发育良好,该胚胎与子宫内膜共培养系统是研究胚胎着床机理的理想体外模型。     

小鼠4-细胞期胚胎单卵裂球体外发育潜能的实验研究

目的:探讨小鼠4-细胞期胚胎的单卵裂球(4-cell stage mice embryo derived-blastomeres, 4c-MEDB)的全能性。方法:将小鼠4-细胞期胚胎分割产生单个卵裂球,在体外无透明带培养条件下,不加外源性细胞因子,观察其发育潜能及特性,并将得到的囊胚与正常囊胚进行形态学、免疫学鉴定比较,同时将4c-MEDB来源囊胚(individual blastomere derived-blastocysts,IBDB)移植到假孕母鼠子宫,观察其发育情况。结果:①体外无透明带培养条件下,4c-MEDB较正常胚胎发育滞后,囊胚获得率低;②4c-MEDB具有发育成为完整囊胚的潜能;③IBDB移植入假孕小鼠子宫后具有一定发育能力,能发育到原条期,但未获得足月妊娠。结论:在未添加外源性细胞因子的体外无透明带培养条件下,小鼠4-细胞期胚胎单卵裂球具有发育为完整囊胚的能力,所获囊胚移植入假孕小鼠子宫后可以发育到原条期。     

重组人促性腺激素对早期鼠胚发育的影响

目的:探讨重组人促性腺激素对小鼠早期胚胎发育的影响。方法:将35只昆明种小鼠随机分为7组,实验组注射一定剂量的重组人促卵泡激素(rhFSH 20 IU)结合不同剂量的重组人促黄体激素(rhLH)对小鼠进行超促排卵,对照组注射孕马血清(PMSG10 IU)和hCG(10 IU),受精0.5 d处死各组雌鼠,收集2-细胞期的鼠胚进行体外培养,观察并记录鼠胚发育至各细胞期的数目。结果:10 IU和15 IU rhLH 组收集的胚胎数与对照组无差异,20 IU rhLH组则高于对照组,但20 IU rhLH组鼠胚发育至4-细胞、8-细胞、桑椹胚、囊胚、扩张期囊胚和孵出期的比率却较低,与对照组比有统计学差异,其余各组收集的胚胎数和胚胎发育至各细胞期数均低于对照组。结论:合适剂量的rhLH对小鼠早期胚胎的发育有明显的促进作用,但剂量过高则对胚胎发育有抑制作用。     

盐酸克仑特罗对小鼠胚胎体外发育的影响

目的:探讨盐酸克仑特罗对小鼠1-细胞胚胎和2-细胞胚胎体外发育的影响。方法:获取小鼠1-细胞和2-细胞胚胎,分别与盐酸克仑特罗10 ng/mL,3 ng/mL和1 ng/mL的3个剂量组共培养,观察胚胎各阶段的发育情况并计算胚胎的发育率。结果:1ng/mL和3ng/mL组的1-细胞小鼠胚胎,从4-细胞期到囊胚阶段的发育率与对照组相比差异显著(P<0.05),3 ng/mL组显现出比1ng/mL组更强的抑制作用(P<0.01),10 ng/mL组,2-细胞期就与对照组有差异(P<0.05,其中 4-细胞至囊胚阶段P<0.01),囊胚率只有2.4％。10 g/mL组及3 ng/mL组的2-细胞小鼠胚胎,分别从4-细胞期和8-细胞期与对照组相比差异显著(P<0.05),1 ng/mL组的各个阶段胚胎发育率与对照组相比未见统计学差异(P>0.05)。培养液中含有盐酸克仑特罗使胚胎的粗颗粒增多,部分印裂球碎裂、退化。结论:盐酸克仑特罗对小鼠胚胎的体外发育有毒性作用并呈一定的剂量效应。盐酸克仑特罗使1-细胞小鼠胚胎被抑制在2-细胞期,对处于晚2-细胞期胚胎的影响明显降低。     

人分泌早期子宫内膜共培养体系对早期鼠胚凋亡的影响

目的研究人分泌早期子宫内膜共培养体系对早期鼠胚凋亡的影响。方法将2一细胞期鼠胚与子宫内膜(A冻融、B传代)共培养以及单一培养液培养(C组)。72h后对桑椹期鼠胚用TUNNEL法进行凋亡检测。结果共培养组鼠胚的凋亡率明显低于单一培养组，且不受内膜细胞传代及冻融的影响；共培养组优质胚胎数均高于对照组，而共培养组之间无明显差异。结论共培养人分泌早期子宫内膜能减少早期鼠胚的凋亡，提高胚胎体外发育的质量，延长体外培养时间。     

体外着床模型中人孵化后早胚细胞角蛋白和肌动蛋白的表达

目的:建立理想的体外胚胎着床模型,并检测模型中人孵化后早胚细胞角蛋白、肌动蛋白和hCG。方法:孵化后早胚与人子宫内膜蜕膜化的基质细胞共培养,观察胚泡在基质细胞层上的定位、黏附、铺展和侵入过程;用免疫荧光染色技术,测定共培养系统中的细胞角蛋白和肌动蛋白;用免疫荧光分析技术,测定培养液中的hCG水平。结果:胚泡和基质细胞共培养5h起,胚泡开黏附在基质细胞层上,最终侵入蜕膜化的基质细胞间。共培养48h后,细胞角蛋白仅仅在滋养层细胞中表达;肌动蛋白在人蜕膜化的基质细胞和滋养层细胞中均有表达。囊胚与子宫内膜基质细胞共培养的培养液中的hCG水平明显高于囊胚单独培养的(P<0.01)。结论:成功建立了一个能反映人胚泡黏附、铺展及侵入到人子宫基质细胞的体外着床模型,细胞角蛋白、肌动蛋白和hCG在着床早胚细胞中起相应变化。     

表皮生长因子(EGF)和胰岛素样生长因子(IGF-Ⅱ)对小鼠1-细胞胚胎体外发育的影响

目的:研究表皮生长因子(epidermalgrowthfactor,EGF)在小鼠早期胚胎体外发育中的作用,以及EGF联合胰岛素样生长因子-Ⅱ(insulin-likegrowthfactor-II,IGF-Ⅱ)对小鼠早期胚胎体外发育是否有协同促进作用。方法:①在mKSOM培养液中添加0ng/ml(对照组)、0.1ng/ml、1ng/ml、10ng/ml、100ng/ml的EGF、10ng/mlIGF-Ⅱ、1ng/mlEGF+1ng/mlIGF-Ⅱ、1ng/mlEGF+10ng/mlIGF-Ⅱ、10ng/mlEGF+1ng/mlIGF-Ⅱ、10ng/mlEGF+10ng/mlIGF-Ⅱ培养小鼠1-细胞胚胎,每组胚胎在培养箱中连续培养120h,每24h观察胚胎发育情况,分别计算2-细胞率、4-细胞率、桑椹胚率、囊胚率和孵化率,并进行囊胚细胞计数。结果:添加1ng/ml、10ng/mlEGF组的囊胚率及孵化率显著增加;EGF+IGF-Ⅱ组较单一添加组、对照组囊胚率及孵化率显著增加(P<0.05),且EGF+IGF-Ⅱ组囊胚细胞计数显著高于其他组(P<0.05),其中10ng/mlEGF+10ng/mlIGF-Ⅱ组的孵化率及囊胚细胞数最高(P<0.05)。结论:EGF浓度在1￣10ng/ml范围内可以提高体外培养鼠胚的囊胚率及孵化率;EGF及IGF-Ⅱ对体外培养鼠胚的发育有协同促进作用;本实验范围内10ng/mlEGF+10ng/mlIGF-Ⅱ为最优组合。     

Development of mammalian embryos exposed to mixed-size nanoparticles

Inhaled or ingested ultrafine nanoparticles and their effects on early pregnancy remain polemic. The objectives of the study were: (a) to determine the embryotoxic effects of nanoparticles at the 2-cell stage and (b) to localize the internalized nanoparticles in the blastocyst. Thawed mouse 2-cell embryos (no. = 128) were exposed to either mixed-size polystyrene-based nanoparticles (11 million/ml) or control G1.3 medium and assessed after 72 hours. Additionally, blastocysts (no. = 146) were exposed to nanoparticles and analyzed. The results showed that the nanoparticles did not inhibit 2-cell embryo development to the blastocyst stage (89.4 vs 96.8%; treated vs control). There were no differences in hatching (34.8 vs 43.5%), implantation (13.6 vs 24.2%) and degeneration (10.6 vs 3.2%). Delayed exposure to nanoparticles showed similar percent hatching (40.7 vs 47.3%) and implantation (17.6 vs 20.0%). Although nanoparticles were internalized, embryo development was not inhibited suggesting a lack of embryotoxicity. During hatching, the larger nanoparticles adhered to the extruding blastocyst, preferentially on trophoblasts, but interference was insignificant. Exposure to polystyrene-based nanoparticles at the concentration tested are not associated with embryonic loss.     

Clinical Assisted Reproduction: An Analysis of Spontaneous Hatching in a Human Endometrial Epithelial Coculture System: Is Assisted Hatching Justified?

Purpose: To evaluate spontaneous embryo hatching in an endometrial epithelial coculture system, and compare it with cases where coculture was performed because of maternal age, previous repeated implantation failures, or both. To clarify in which cases assisted hatching would be appropriate.Methods: Individual human embryos were cocultured on an endometrial epithelial cell monolayer until Day 6.Results: Blastocyst hatching rate at Day 6, depending on maternal age, was 9.1% (age <37 years) and 3.4% (age 37 years). However, blastocyst hatching rates depending on number of previous IVF failures were similar.Conclusions: Maternal age and previous implantation failures are factors affecting the ability of human embryos to reach the blastocyst stage in coculture. However, assisted hatching is not justified in these populations because of the absence of hatching rate differences between blastocysts obtained from these two groups and the control group.     

人卵泡液对小鼠胚胎生长发育影响的研究

目的：探讨人卵泡液对小鼠胚胎生长发育的影响。方法：在培养液中分别加入人卵泡液（卵泡液组）和人血清（血清组）,观察和检测2细胞期鼠胚在体外发育至8细胞期、囊胚期以及鼠胚孵出的比率;用放射免疫方法测定人卵泡液和血清中表皮生长因子（EGF）含量。结果：卵泡液组有72.9%的2细胞期鼠胚能发育到8细胞期,血清组仅有48.0%到达8细胞期;继续培养显示,卵泡液组细胞分裂较快,有50.9%的鼠胚可发育至囊胚期,并有26.3%孵出,明显高于血清组（24.5%,6.9%),两组比较,差异有显著性（P＜0.05)。卵泡液中EGF的含量为0.50μg/L,血清中为0.26μg/L,两组比较,差异有显著性（P＜0.05）。结论：卵泡液对早期胚胎发育有促进作用,在早期胚胎培养中,添加人卵泡液比添加人血清对提高早期胚胎培养质量更为有效。     

Effect of laser-assisted hatching and necrotic blastomere removal on the development of vitrified–warmed four-cell mouse embryos

PURPOSE: To investigate the effect of laser-assisted hatching and necrotic blastomere removal on the development of vitrified-warmed mouse embryos. METHODS: The vitrified-warmed four-cell stage mouse embryos were divided into five groups; vitrified intact with no laser-assisted hatching, vitrified intact with laser-assisted hatching, vitrified damaged with neither laser assisted hatching nor necrotic blastomere removal, vitrified damaged with laser-assisted hatching, and vitrified damaged with necrotic blastomere removal. Thereafter blastocyst formation, blastomere and apoptotic cell number within all groups were statistically compared. RESULTS: The rate of blastocyst formation showed a significant improvement in the group vitrified intact with laser-assisted hatching. However, neither laser-assisted hatching nor necrotic blastomere removal can improve a delayed vitrified-warmed damaged embryos in term of blastocyst formation and total cell number. Nevertheless, apoptotic cell number was significantly reduced after application of both techniques. CONCLUSIONS: Laser-assisted hatching can improve the development of vitrified-warmed intact four-cell stage mouse embryos, whereas necrotic blastomere removal has no significant effect on the development of vitrified-warmed four-cell stage damaged embryos.     

Effect of glutamine on preimplantation mouse embryo development in vitro

OBJECTIVES: The purpose of this research was to study the independent effect of the amino acid glutamine on preimplantation mouse embryo development in vitro. STUDY DESIGN: Two-cell stage mouse embryos were cultured in human tubal fluid medium in the presence and absence of 1 mmol/L of glutamine. Outcomes for morphology and cleavage rates were compared with Fisher's and Mann-Whitney's tests, respectively. RESULTS: Glutamine increased the proportion of embryos that developed to the blastocyst stage (86.4%) compared with those cultured without glutamine (59.1%) (P =.052). The percentages of embryos developing to the morula or hatching blastocyst stages were comparable in the 2 groups. Blastocyst total cell numbers were significantly higher in the glutamine group (34+/-1.7 vs 18.5+/-3.5, respectively; values are mean+/-SEM, P =.044). CONCLUSION: The amino acid glutamine independently improves preimplantation mouse embryo development in vitro. Further studies are needed to examine the applicability of these results to humans.     

Pregnancy and delivery after the transfer of one blastocyst with enzymatically removed zona pellucida

OBJECTIVES: The embryonic development up to the blastocyst stage and hatching from zona pellucida are prerequisites for implantation and successful pregnancy. It is suggested that one of the possibilities limiting the implantation rate is impaired hatching. To overcome this problem an artificial alteration of the zona pellucida have been carried out in many laboratories. DESIGN: The report of the first pregnancy in Poland obtained after the transfer of enzymatically zona removed blastocyst. MATERIALS AND RESULTS: The patient was previously treated three times in in vitro fertilisation program without success. In the fourth program the embryos were cultured in co-culture of Vero cells to the blastocyst stadium. On the day 5 after insemination the zona of cavitating and expanding blastocyst was removed by pronase. The zona free blastocyst was transferred to the uterus. As a result of implanted blastocyst the ongoing pregnancy developed normally and the patient delivered healthy baby. CONCLUSION: Enzymatic zona pellucida hatching probably increases the rate of implantation. It is simple, safe and economic techniques.     

Peptides extracted from vero cell cultures overcome the blastocyst block of mouse embryos in a serum-free medium

Purpose The aim of this work was to evaluate the effect of a Vero cell coculture system on the development of mouse embryos. Methods Mouse embryos were randomly divided and cultured in human tubal fluid (HTF) medium with/without Vero cell monolayers, conditioned medium (CM) obtained from Vero cell cultures, and HTF medium supplemented with peptides extracted from CM. The concentrated CM was examined by SDS/PAGE. Results The development of mouse embryos was blocked at the blastocyst stage in pure HTF medium (1.4% hatching at day 5). This “blastocyst block≓ was overcome by coculture with Vero cell monolayers (48.1% hatching at day 5; 1.4 vs 48.1%; P<0.001). CM and the addition of 5% fetal bovine serum (24.1 and 34.9% hatching, respectively, at day 5) were also able to enhance the process of hatching. In the other experiment, the addition of peptides extracted from Vero cell cultures also overcame the blastocyst block (12.5%) compared with pure HTF medium (2.1%) (P<0.05). Electrophoretic separation revealed several classes of polypeptides consistently secreted into CM obtained from Vero cell cultures. Most peptides occurred in the M_r range between 6.5 kd and 35.9 kd. Conclusion A developmental block (blastocyst block) of mouse embryos in a serum- and protein-free medium (HTF) was discovered in this study. This block was effectively overcome by HTF plus serum and coculture with Vero cell monolayers and also by the peptides extracted from Vero cell-conditioned medium. We speculate that certain factors secreted or converted by Vero cells may be critical in hatching of mouse embryos. Further study of these factors may be helpful in delineating its mechanism.     

肿瘤转移抑制因子CD82/KAI1对植入前小鼠胚胎体外发育的影响

目的:研究CD82/KAI1mRNA及蛋白质在小鼠胚胎中的表达规律及其对小鼠胚胎体外发育的影响。方法:①应用RT-PCR及免疫荧光技术观察CD82/KAI1mRNA及蛋白质在小鼠不同发育时期胚胎中的表达规律。②在不同CD82/KAI1抗体浓度的培养液中,培养小鼠8-细胞胚胎,观察囊胚发育率、孵化率和胚胎细胞数的变化。结果:①CD82/KAI1mRNA在小鼠不同时期胚胎中均有表达,于8-细胞期及桑葚胚表达较丰富,CD82/KAI1蛋白表达于各期胚胎细胞的胞膜和胞浆,在桑葚胚期CD82蛋白还强表达于胚胎细胞的胞核。②一定浓度的CD82/KAI1抗体可明显抑制胚胎的发育速度(1∶800,P<0.05),降低囊胚的形成率(1∶400,P<0.05)和孵出率(1∶800,P<0.05),减少囊胚细胞数(1∶800,P<0.05)。结论:CD82/KAI1在小鼠胚胎的发育过程中可能发挥重要作用。