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。     

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。     

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     

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。     

Stathmin与子宫蜕膜化关系的研究进展

Stathmin作为一种微管失稳蛋白影响了细胞周期进程,其在人类子宫内膜组织的腺上皮和基质细胞中有所表达。研究表明,stathmin在调节人子宫内膜基质细胞分化过程中的初始阶段起着必不可少的作用,和蜕膜化相关。Stathmin在子宫内膜蜕膜化、蜕膜的分化增殖与凋亡和蜕膜相关细胞的侵袭与黏附方面均起了重要作用。而蜕膜化作为正常着床和妊娠的一个重要特征,与子面滋养层共同构成母胎界面,从而影响正常早期妊娠,并可能与早期自然流产相关。     

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。     

人早孕期蜕膜和绒毛外滋养层细胞的免疫组织化学研究

侵入蜕膜组织的绒毛外滋养层细胞在HE染色标本上不易辨认。我们用免疫组织化学,ABC法,过氧化物-抗过氧化物酶标记蜕膜组织中的hCG与hPL阳性细胞。结果证明,子宫内膜中只要有一个绒毛外滋养层细胞存在就能被显示出来。这对提高妊娠诊断,防止残留滋养层细胞的增生与恶性病变很有意义。我们在蜕膜组织中还发现3类胎盘激素阳性细胞:hCG阳性细胞,hPL阳性细胞及hCG与hPL均为阳性的细胞。这一发现,证明了蜕膜有内分泌功能。     

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     

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     

sLe~X抗体对小鼠胚泡在E-选择素上黏附和扩展的影响

目的:探讨E-选择素及其配体在着床中的作用。方法:建立小鼠胚泡的体外培养模型,在倒置相差显微镜下观察不同条件下小鼠胚泡在细胞外基质E-选择素上黏附、扩展的改变。结果:小鼠胚泡能在一定浓度E-选择素上黏附、扩展,E-选择素对胚泡的脱透明带有促进作用。结论:确定了E-选择素体外实验的最佳作用浓度。小鼠胚泡在特定浓度的E-选择素上能黏附、扩展,但这种作用能被sLe~X 抗体所阻断,并呈浓度依赖性。     

Sera from women with histories of repeated pregnancy losses cause abnormalities in mouse peri-implantation blastocysts

Incubation of peri-implantation mouse blastocysts in the presence of untreated human sera resulted in destruction of the blastocysts. Heating the serum resulted in deactivation of the non-specific toxic factor. Whereas heat-treated serum from women with normal obstetrical histories, and men, supported normal trophoblast attachment and outgrowth, sera from women with reproductive dysfunction resulted in inhibition of attachment or disruption of the trophoblast cells. The inner cell masses were not adversely affected by the sera which were toxic to trophoblast. Fractionation of a serum sample by affinity chromatography resulted in removal of the toxic factor with the IgG fraction. Absorption of the toxic serum with human trophoblast membranes resulted in serum that supported trophoblast outgrowth indicating that the toxic factor was an antibody directed against trophoblast antigens.     

The role of leukemia inhibitory factor in tubal ectopic pregnancy

Introduction

Ectopic pregnancy is unique to humans and a leading cause of maternal morbidity and mortality. The etiology remains unknown however factors regulating embryo implantation likely contribute. Leukemia inhibitory factor (LIF) has roles in extravillous trophoblast adhesion and invasion and is present in ectopic implantation sites. We hypothesised that LIF facilitates blastocyst adhesion/invasion in the Fallopian tube, contributing to ectopic pregnancy.

Methods

We immunolocalised LIF receptor (R) in tubal ectopic pregnancy (N = 5). We used an oviduct cell line (OE-E6/E7) to model Fallopian tube epithelial cells and a trophoblast spheroid co-culture model (HTR-8/SVneo cell line formed spheroids) to model blastocyst attachment to the Fallopian tube. We examined LIF signaling pathways in OE-E6/E7 cells by Western blot. The effect of LIF and LIF inhibition (using a novel LIF inhibitor, PEGLA) on first-trimester placental outgrowth was determined.

Results

LIFR localised to villous and extravillous trophoblast and Fallopian tube epithelium in ectopic pregnancy. LIF activated STAT3 but not the ERK pathway in OE-E6/E7 cells. LIF stimulated HTR-8/SVneo spheroid adhesion to OE-E6/E7 cells which was significantly reduced after PEGLA treatment. LIF promoted placental explants outgrowth, while co-treatment with PEGLA blocked outgrowth.

Discussion

Our data suggests LIF facilitates the development of ectopic pregnancy by stimulating blastocyst adhesion and trophoblast outgrowth from placental explants. Ectopic pregnancy is usually diagnosed after 6 weeks of pregnancy, therefore PEGLA may be useful in targeting trophoblast growth/invasion.

Conclusion

LIF may contribute to the development of ectopic pregnancies and that pharmacologically targeting LIF-mediated trophoblast outgrowth may be useful as a treatment for ectopic pregnancy.     

Ghrelin影响小鼠胚泡着床的体外实验研究

目的:探讨Ghrelin在小鼠妊娠围植入期子宫表达的变化及其对胚胎着床的影响。方法:用免疫组化方法检测Ghrelin蛋白在小鼠妊娠围植入期子宫表达的变化;采用体外胚泡培养方法,观察重组Ghrelin蛋白对胚泡的孵化、黏附与扩展率的影响。结果:在小鼠妊娠的第1至2天,Ghrelin蛋白主要于腔上皮和腺上皮表达,随妊娠进展,其表达水平逐渐下降。体外胚泡培养48h时,与正常对照组相比[孵化率、黏附率和扩展率分别为(61.25±5.27)%、(71.45±6.56)%和(43.26±5.87)%],Ghrelin重组蛋白可明显抑制胚泡的孵化率[(20.36±4.78%),P<0.01]、黏附率[(53.23±4.98)%,P<0.01)]及扩展率[(18.34±7.12)%,P<0.05]。结论:Ghrelin可抑制胚泡孵化、黏附与扩展及抑制胚泡植入。     

Regulation of peri-attachment embryo development in the golden hamster: role of growth factors.

The molecular regulation of mammalian peri-implantation development is complex and difficult to study in vivo. We successfully cultured hamster blastocysts through hatching and peri-attachment stages, using a chemically defined medium, HECM-2h. Using this system, we showed that a species-specific, embryonic cysteine-like protease is involved in blastocyst hatching and that the process is modulated by growth factors. In particular, heparin binding-epidermal growth factor (HB-EGF) or leukemia inhibitory factor (LIF) enhance blastocyst hatching, and the former also improves attachment and trophoblast outgrowth. We observed interesting changing patterns of expression of mRNA and/or immunoreactive protein for EGF, HB-EGF, LIF and transforming growth factor-beta (TGF-beta) in the embryo and/or endometrial tissue, during peri-implantation development. Together, it appears that hamster blastocyst hatching, attachment and trophoblast outgrowth are regulated by autocrine and/or paracrine growth factors, produced by the embryo-endometrial tissues.     

In vitro models of human blastocyst implantation.

This paper reviews different in vitro models used for the study of blastocyst implantation in animals and the human. Furthermore, results from human blastocyst-endometrial interactions in vitro, investigated by scanning electron microscopy (SEM), light microscopy (LM) and transmission electron microscopy (TEM), are presented. SEM demonstrates the preference of human blastocysts to adhere to pinopode-presenting areas on endometrial cell cultures. LM and TEM show that the first morphological sign of cell contact, defined as junction formation, is present at the apical-to-lateral border of endometrial epithelial cells, whereas trophoblast attachment to apical endometrial epithelial plasma membranes was not observed. More advanced stages illustrate that the human blastocyst penetrates the epithelial lining by the intrusive penetration mechanism.     

Heparin binding-epidermal growth factor improves blastocyst hatching and trophoblast outgrowth in the golden hamster

The effect of heparin binding-epidermal growth factor (HB-EGF) on the in-vitro development of hamster 8-cell embryos was investigated. Supplementation of HB-EGF to culture medium accelerated zona escape of blastocysts (63 +/- 9% compared with 33 +/- 9% after 36 h; P < 0.05). Complete zona escape of blastocysts persisted even after 48 h (61 +/- 11% versus 30 +/- 4%) and 60 h (75 +/- 6% versus 42 +/- 8%). Addition of anti-HB-EGF antibody drastically reduced the percentage of zona escaped-blastocysts (30.0 +/- 5.0% versus 92.3 +/- 2.8%; P < 0.05). Interestingly, a significant increase in the area of trophoblast outgrowth occurred in the presence of HB-EGF (116 x 10(3) +/- 8 x 10(3) microm(2) versus 74 x 10(3) +/- 8 x 10(3) microm(2) at 48 h; P < 0.05). This, however, was not due to an increased number of trophectodermal cells in HB-EGF-treated blastocysts. Immunoreactive HB-EGF was localized in blastocysts and uterine sections, visible by intense immunostaining in the luminal epithelium, particularly on the apical surface. Moreover, the expression of HB-EGF in the uterus was maximum on day 4 of pregnancy, coinciding with the timing of zona escape and implantation. The receptor of HB-EGF, viz. EGF receptor was also detected in blastocysts and the luminal epithelium of day 4 pregnant uterus. These results show that HB-EGF improves blastocyst hatching and trophoblast outgrowth in hamsters.     

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.     

Treatment with gonadotropins impaired implantation and fetal development in mice

Purpose The effect of gonadotropins on implantation and fetal development in mice was investigated by superovulation with pregnant mare serum gonadotropin and human chorionic gonadotropin. In a previous study fetal growth was found to be highly retarded.Results Assessment of implantation in vivo revealed that late implantation did occur. Gestational length was highly extended, the mean number of live fetuses per pregnant mouse was lower and their mean weight significantly reduced. In vitro experiments revealed no significant difference in the rate of blastocyst adhesion and trophoblast outgrowth development. Immunohistochemical staining, however, showed that blastocysts from superovulated mice had smaller trophoblastic outgrowths than control embryos. Staging embryonic development at the time of flushing, however, revealed retarded embryo development in vivo in hormone-treated mice. After correlation with embryonic stage at the beginning of the culture, there was no difference in the size of trophoblastic outgrowths.Conclusion Treatment with gonadotropins impaired implantation and embryonic/fetal development. Changes in maternal milieu, rather than in embryo quality, may be responsible for the adverse effects observed.