人子宫内膜细胞共培养体系对早期鼠胚体外发育的影响

目的:研究人子宫内膜共培养体系对早期鼠胚体外发育的影响及移植后的妊娠情况。方法:将2-细胞小鼠胚胎与人子宫内膜细胞进行体外共培养,对照组为无营养细胞的单纯培养液,每日在显微镜下观察胚胎的发育情况。将培养到囊胚期的胚胎移植回小鼠的子宫腔,观察着床情况。结果:共培养体系中68.3％的2-细胞胚胎发育至桑椹胚期,50.8％发育至囊胚期,囊胚的孵化率为36.7％,胚胎的着床率为25.0％。而对照组只有24.8％的2-细胞胚胎发育至桑椹胚期,11.4％到达囊胚期,且其中大部分为早期囊胚即停止发育。另外对照组细胞碎片出现早且多,卵裂球不均匀,胚形态差,移植后胚胎的着床率仅为3.1％。结论:人子宫内膜细胞共培养体系可以促进小鼠胚胎的体外发育,改善胚胎的质量,提高着床率。     

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。     

小鼠囊胚在人羊膜上着床行为的超微观察

目的 :建立一种较理想的体外着床模型。方法 :将妊娠 d4的小鼠囊胚培养在人羊膜的基膜面。结果 :发现囊胚能在羊膜上正常脱带、贴附和扩展。光镜观察显示 ,滋养层细胞对羊膜有侵入 ,二者在结构上有交错。扫描电镜下 ,囊胚紧贴于羊膜表面 ,被无数长短一致、粗细均匀的微绒毛覆盖。透射电镜显示部分滋养层细胞及内细胞团细胞已侵入羊膜之中 ,滋养层细胞膜下和微绒毛内可见许多小泡样结构。结论 :小鼠囊胚在人羊膜上的发育生长可作为研究胚胎着床机理的体外模型     

小鼠容受性子宫内膜上皮细胞和基质细胞的共培养

建立一种较理想的体外子宫内膜立体共培养方法。方法:利用双室培养系统建立含有上皮细胞和基质细胞共培养的系统,光镜、电镜观察上皮细胞形态学变化;细胞免疫组化鉴定细胞来源。结果:扫描电镜显示,培养的上皮细胞生长汇合,可见到微绒毛细胞及局部有胞饮突的形成。透射电镜观察显示培养的上皮具有极性,表面有微绒毛,细胞间紧密连接及细胞间糖原沉积。结论:此方法可更好地模拟体内状态,是较理想的子宫内膜体外模型。     

子宫内膜血管生成与血流灌注指标在评价子宫内膜容受性中的价值

囊胚着床是一个动态过程,包括囊胚定位、黏附到子宫内膜上皮细胞,以及侵入到内膜基质。"种植窗"是指子宫内膜在空间上以及限定的时间内对囊胚着床处于容受状态,允许囊胚黏附、穿透,导致胚胎着床的状态,是保证受精卵着床、胎儿和胎盘正常发育的重要环节。"种植窗"一般为排卵后的6～8d,持续不到48h,即分泌中期和(或)黄体中期,并且以某些子宫内膜生长因子、细胞因子、黏附分     

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。     

TRAIL在小鼠胚胎着床过程中子宫内膜的表达

目的:检测TRAIL在小鼠胚胎着床过程中子宫内膜的表达,探讨它在蜕膜细胞凋亡中的作 用。方法:采用RT-PCR及免疫组化技术检测妊娠d 1-8小鼠子宫组织TRAILmRNA及蛋白的表 达情况。结果:妊娠d 1-8的小鼠子宫组织均有TRAIL mRNA的表达,且着床期间的表达较着床前 明显增加(P<0．05)。妊娠d 1-3,小鼠子宫内膜无TRAIL蛋白表达;妊娠d 4,TRAIL表达在小鼠胚 胎定位、黏附点的子宫内膜腔上皮细胞;妊娠d 5-6,TRAIL定位于胚胎着床点附近的蜕膜细胞中; 妊娠d 7-8,TRAIL表达在与子宫蜕膜邻近的胚胎滋养层细胞中。结论:在小鼠胚胎着床过程中, TRAIL诱导子宫内膜腔上皮细胞凋亡可能是胚胎跨越上皮屏障的重要机制之一,且TRAIL诱导的 蜕膜细胞和胚胎滋养层细胞的凋亡在滋养层细胞对子宫内膜的适度侵入过程中起重要作用。     

四跨膜蛋白超家族成员在胚胎植入中的作用

胚胎植入是哺乳动物生殖过程的关键环节.妊娠的正常建立和维持依赖囊胚与容受态子宫内膜间的相互作用,其中滋养细胞精确的侵入程度和子宫内膜的蜕膜化起重要作用.与肿瘤转移相似,滋养细胞侵袭子宫内膜也包括识别、黏附、侵袭、转移等过程,不同的是滋养细胞的侵袭行为具有＂自限性＂,是由一个复杂的网络系统精确调控的.四跨膜蛋白超家族成员以其在调控细胞黏附、侵袭、转移中独特的生物学作用,不仅在肿瘤转移中起重要作用,也在胚胎植入过程中扮演重要角色.     

着床研究模型:离体胚泡与子宫内膜“共培养”

本工作用小鼠和豚鼠的离体胚泡与子宫内膜共培养方法,为研究着床与抗着床机理提供了一个体外研究模型。子宫内膜取自妊娠小鼠第5天上午8时前和妊娠豚鼠第4天下午,分别与小鼠或豚鼠胚泡共培养。经培养2～3天后可见胚泡从透明带逸出并附着于子宫内膜,部分植入内膜。     

子宫内膜容受性评价进展

<正>妊娠的发生要求子宫内膜允许囊胚定位、黏附和侵入,这需要通过多种细胞因子和黏附分子的共同协调来实现,胚胎植入的关键是子宫内膜容受性(endometrial receptivity,ER)的建立。影响胚胎种植成功的关键因素有胚胎质量、子宫内膜因素等,提高胚胎质量和改善ER是治疗的两大关键问题。ER是指子宫内膜对胚胎的接受能力,与胚胎种植窗开放关系密切。正常生理状态下,胚胎于排卵后6～8天着床于子宫内膜,着床子宫内膜仅在较短暂的一段时间内     

补肾益气和血方中药对胚泡着床障碍小鼠子宫内膜表面胞饮突表达的影响

目的　观察补肾益气和血方中药对胚泡着床障碍小鼠子宫内膜表面胞饮突表达的影响。方法　选择 8～ 12周龄昆明小鼠 ,于妊娠第 4天 (Pd4 )晨 ,皮下注射米非司酮 ,制成胚泡着床障碍模型。然后将妊娠小鼠随机分为正常组、模型组和治疗组 ,治疗组从Pd1开始灌服中药 (补肾益气和血方 ) ,正常组和模型组则给予等量生理盐水灌服 ,模型组和治疗组于Pd4晨皮下注射米非司酮 ,正常组则注射等量生理盐水。采用扫描电镜观察各组小鼠在Pd4晚 2 1时 30分～ 2 2时 (第 1时间点 ,T1)和Pd5上午 9时 30分～ 10时 (第 2时间点 ,T2 ) ,共两个时间点子宫内膜表面胞饮突的表达。结果　模型组妊娠率 ( 2 5 % )较正常组 ( 90 % )显著降低 ,差异有显著性 (P <0 0 5 ) ,模型组平均着床胚泡数为 ( 7 7± 1 3)个 ,正常组为 ( 14 7± 1 4 )个 ,两组比较 ,差异有极显著性 (P <0 0 0 1) ;治疗组的妊娠率 ( 5 5 % )及平均着床胚泡数 [( 12 3± 0 8)个 ]均较模型组显著增加 (P <0 0 5 ,P <0 0 1)。T1时正常组子宫内膜表面表达大量发育中的胞饮突 ,至T2时则表达大量发育完全的胞饮突 ;T1时模型组子宫内膜发育不同步 ,胞饮突仅在局部少量表达 ,至T2时则表达完全消失 ;T1时治疗组子宫内膜表面表达大量发育中的胞饮突 ,与正常组相比略延     

输卵管积水妇女种植窗期子宫内膜胞饮小泡及部分种植因子的变化

目的:探讨输卵管积水对种植窗期子宫内膜胞饮小泡及种植因子integrinβ3、MUC1及LIF表达的影响。方法:选择接受IVF治疗的20例输卵管积水妇女(积水组)及21例因男性因素所致不孕的妇女(对照组),在种植窗期间通过扫描电镜观察子宫内膜表面胞饮小泡的形态、密度,免疫组织化学分析种植因子integrinβ3、MUC1及LIF的表达。结果:对照组成熟期胞饮小泡所占比例以及胞饮小泡的密度与积水组相比无统计学差异(P>0.05)。integrinβ3、LIF及MUC1在正常对照组的腺上皮细胞和腔上皮细胞中的表达强度均明显高于积水组,integrinβ3在间质细胞中表达亦明显高于积水组。结论:integrinβ3、MUC1及LIF受积水的影响较胞饮小泡更为敏感,先于子宫内膜表面超微结构受到改变,可能是造成胚胎种植率下降的主要原因之一。     

宫腔粘连模型大鼠子宫内膜胞饮突发育和整合素β3表达

目的:建立宫腔粘连大鼠模型,观察子宫内膜胞饮突的发育和整合素β3的表达,探讨宫腔粘连对子宫内膜容受性的影响。方法:选取健康SD大鼠30只,将每只大鼠左侧子宫作为模型组(宫腔注入95%无水乙醇,持续5min),右侧子宫作为对照组(注入等量生理盐水)。术后两个动情周期取子宫,光镜观察子宫内膜形态学变化及纤维化程度,扫描电镜观察大鼠子宫内膜胞饮突的发育情况,免疫组化法检测子宫内膜整合素β3表达。结果:模型组大鼠子宫内膜明显变薄,上皮细胞排列紊乱,间质内胶原纤维增多,子宫内膜纤维化评分为(4.69±0.22)分,明显高于对照组。扫描电镜观察,对照组大鼠子宫内膜可见大量发育完全的胞饮突,而模型组子宫内膜胞饮突分布稀少,表面皱缩,发育不同步。模型组的整合素β3表达明显低于对照组,差异有统计学意义(P0.05)。结论:注射无水乙醇可建立稳定的大鼠宫腔粘连动物模型,抑制子宫内膜胞饮突和整合素β3表达,降低子宫内膜的容受性。     

Endometrial receptivity: changes in cell-surface morphology

Ovulation and fertilization trigger embryonic development and endometrial differentiation by corpus luteum progesterone production. These two synchronous processes couple about 1 week later, when the blastocyst begins to implant in the now receptive endometrium (implantation window). Receptivity is a state of endometrial differentiation marked by a change in epithelial morphology: the hairy-like cell microvilli fuse to a single flower-like membrane projection called the "pinopode." Scanning electron microscopy of sequential endometrial biopsies shows that pinopodes form briefly (1-2 days), and their numbers correlate with implantation. On average, the formation of pinopodes is earlier in stimulated (days 19-20) and later in artificial (days 21-22) compared with natural cycles (days 20-21). There is, however, a wide (up to 5 days) variation between women in the cycle days on which pinopodes form. These results suggest the existence of a narrow and discrete implantation window in humans. Detection of pinopodes is a potential clinical marker to assess endometrial receptivity.     

Ultrastructural markers of tissue endometrial receptivity in patients with recurrent implantation failure

Abstract

The scanning electron microscopy of the endometrial surface epithelium during the ‘implantation window’ was performed in 119 patients with uterine factor of infertility or recurrent miscarriage due to endometrial hypoplasia. Ultramorphological picture of the surface endometrial epithelium was characterized by aplasia and hypoplasia of pinopodes (67.39%), dense cell – cell contacts (69.53%), heteromorphy of secretory cells (15.22%) in combination with atypia of microenvironment cells (50%) in patients with infertility. The asynchronous development of pinopodes (46.67%) and the absence of intercellular contacts separation during the ‘implantation window’ (84.44%) was observed in patients with recurrent miscarriage. The revealed disturbance determines the mechanisms of the blastocyst adhesion violation and trophoblast invasion in the different stages of implantation in patients with uterine factor of infertility and recurrent miscarriage.     

Early human pregnancy in vitro utilizing an artificially perfused uterus

The penetration of luminal epithelium in the uterine cavity represents the crucial event that triggers the failure of embryo implant, thus limiting the possibility of fertility control. The purpose of our study was to implant a human blastocyst, cultured in vitro, into a human uterus extracorporeally perfused with an oxygenated medium. For this purpose, human blastocysts, collected from patients who underwent IVF program because of irreparable tubal infertility, were injected under the luminal epithelium of human perfused uteri. Light and electron microscopy showed that human blastocyst can successfully undergo the stage of implantation and trophoblastic invasion in 52 hours of extracorporeal perfusion.     

Rabbit endosalpinx inhibits implantation in vitro.

OBJECTIVE: To evaluate the ability of rabbit endometrial or endosalpingeal cells to support implantation in vitro and to assess the effects of endosalpinx and endometrium-conditioned media (CM) on blastocyst-endometrial cell interaction. DESIGN: In one experiment, rabbit blastocysts were co-cultured in vitro with endometrial or endosalpingeal cells growing on Matrigel-coated plastic culture plates or Millicell-HA inserts. In a second experiment, rabbit blastocysts were co-cultured with endometrial cells in the presence of fresh medium or of endosalpinx- or endometrial-CM. After 48 or 72 hours attachment to the cell monolayer was evaluated. RESULTS: Blastocysts in co-culture attached to endometrial but not to endosalpingeal monolayers. The addition of CM from cultured endosalpinx significantly decreased embryo attachment to endometrial cells in culture. CONCLUSIONS: These findings in vitro agree with the observation that rabbit endosalpinx in vivo does not support embryo implantation and support the hypothesis that rabbit endosalpinx secretes a factor that prevent tubal implantation.