The presence of platelet-derived endothelial cell growth factor in human endometrium and its characteristic expression during the menstrual cycle and early gestational period

Decidualized tissues are characterized by the intensive outgrowthof the microvasculature. Several angiogenic factors are assumedto be involved during the drastic change in the vasculatureoccurring in the process of decidualization. We examined thepossible role of platelet-derived endothelial cell growth factor(PD-ECGF), a known angiogenic factor, during the process ofearly decidualization in humans. The expression of PD-ECGF inhuman endometrium was demonstrated by Western blot analysis,a marked increase being found in decidualized endometrium. Immunohistochemicalstaining showed that PD-ECGF immunoreactivity was present mainlyin decidualized endometrial stromal cells. We established aprimary cell culture of human endometrial stromal cells whichwere differentiated into decidualized cells in vitro by theaddition of progesterone. In this cell culture system, progesteroneaugmented the expression of PD-ECGF in a dose-dependent fashion.The addition of progesterone also resulted in an increased releaseof prolactin, a well-known marker for decidualization. Thesefindings suggest that PD-ECGF may play a physiological roleas a possible angiogenic factor in the process of decidualizationof human endometrium. angiogenesis/decidualization/human endometrium/PD-ECGF/prolactin     

Mechanisms regulating the expression of indoleamine 2,3-dioxygenase during decidualization of human endometrium

BACKGROUND: Expression of the tryptophan catabolizing enzyme, indoleamine 2,3-dioxygenase, in the mouse placenta has been shown to be critical in preventing immunological rejection of the fetal allograft. To clarify the physiological importance of indoleamine 2,3-dioxygenase in human pregnancy, we have studied how the expression of this enzyme changes during decidualization of human endometrium at both the cell and tissue level. METHODS AND RESULTS: The level of indoleamine 2,3-dioxygenase mRNA expression (determined by RT-PCR) was higher in decidual than in endometrial tissue. Uterine decidual tissue in ectopic pregnancy similarly showed increased mRNA expression. Immunohistochemistry demonstrated that indoleamine 2,3-dioxygenase protein immunoreactivity was found in glandular epithelium and in stromal cells. The intensity of this immunoreactivity was increased in decidualized tissue. In a cell culture model, the level of indoleamine 2,3-dioxygenase mRNA was suppressed specifically by progesterone-induced decidualization of isolated endometrial stromal cells. Indoleamine 2,3-dioxygenase protein abundance (determined by Western blot) was also decreased by progesterone-induced decidualization. However interferon-gamma, a potent stimulator of indoleamine 2,3-dioxygenase gene expression, increased the level of indoleamine 2,3-dioxygenase mRNA and protein in both non-decidualized and in decidualized cells. Indoleamine 2,3-dioxygenase activity (determined by measuring the concentration of tryptophan and its indoleamine 2,3-dioxygenase catabolite, kynurenine) was also decreased by progesterone-induced decidualization but enhanced following interferon-gamma treatment. Expression of other interferon-gamma inducible genes (STAT1 and tryptophanyl-tRNA synthetase) showed the same pattern as that of indoleamine 2,3-dioxygenase in tissue samples, but was not changed by decidualization in the cell culture model. CONCLUSIONS: These data suggest that despite suppression by progesterone, indoleamine 2,3-dioxygenase expression in endometrial stromal cells may increase during decidualization due to stimulation by interferon-gamma secreted by infiltrating leukocytes.     

Cyclic and characteristic expression of phosphorylated Akt in human endometrium and decidual cells in vivo and in vitro

BACKGROUND: Akt is activated by phosphorylation and plays an important role in cell survival and maintenance of structure. METHODS: We investigated whether phosphorylated Akt was characteristically expressed in human endometrium in vivo and whether insulin-like growth factor-I (IGF-I) can activate Akt using cultured decidualized human stromal cells in vitro, using immunohistochemistry and Western blotting analysis. RESULTS: The levels of phosphorylated Akt protein increased markedly in the decidual tissues from ectopic pregnancy. The expression of phosphorylated Akt protein in stromal cells increased with the decidualization. The decidual cells showed strong cytoplasmic staining for phosphorylated Akt. However, cultured decidualized human stromal cells diminished phosphorylated Akt expression compared to control cells. IGF-I administration to decidualized human stromal cells significantly recovered pAkt expression. The effect of IGF-I on decidualized human stromal cells was blocked by an inhibitor of phosphatidylinositol-3 kinase (PI3K) (LY294,002). These results suggest that IGF-I may activate Akt via PI3K in human endometrium and decidua. The expression of phosphorylated Akt in stromal cells was only detected in the functional layer, where tissue remodelling occurs during menstruation or implantation. CONCLUSIONS: Akt activation may be involved in cell survival and extracellular matrix remodelling in human endometrium and decidua.     

Insulin-like growth factors and insulin-like growth factor binding proteins in the endometrium. Effect of intrauterine levonorgestrel delivery

Insulin-like growth factor (IGF) system is one of the growth factor systems that are believed to modulate steroid hormone actions in the endometrium through autocrine/paracrine mechanisms. IGF-I and IGF-II stimulate proliferation and differentiation, and maintain differentiated cell functions in several cell types in vitro. Endometrial stromal cells produce IGF-I and IGF-II as well as the high affinity IGF-binding proteins (IGFBP), whereas epithelial cells and, in a lesser amount, stromal cells contain cell membrane receptors for IGF. Oestrogen stimulates IGF-I gene expression, and IGF-II gene expression is associated with endometrial differentiation. The mRNA of six high affinity IGFBPs, which can modulate IGF actions, are expressed in human endometrium. The most abundant IGFBP in human endometrium is IGFBP-1, which is secreted by predecidualized/decidualized endometrial stromal cells in late secretory phase and during pregnancy. The primary negative regulator of IGFBP-1 production is insulin. IGFBP-1 competes with type I IGF receptor for binding of IGF in the endometrium and in cultured human trophoblastic cells. IGF-I mRNA is suppressed and mRNA encoding IGF-II and IGFBP-1 are consistently up-regulated in decidualized endometrium in women treated with the intrauterine levonorgestrel system (LNG-IUS). Strong cytoplasmic staining for IGFBP-1 was detected in decidualized endometrium in women using LNG-IUS for contraception or for endometrial protection during post-menopausal oestrogen replacement therapy. Simultaneously, oestrogen receptors were present, while progesterone receptors were hardly detectable in the endometrium by immunohistochemistry. The latter findings suggest that suppression of IGF-I action by IGFBP-1 may be one of the molecular mechanisms accounting for progestagenic and anti-oestrogenic effects of LNG-IUS in the endometrium. Consequently, examination of local IGF-I, IGF-II and IGFBP-1 expression might provide additional information when evaluating the effect of different progestins on the endometrium at the molecular level.     

Endometrial stromal cell decidualization inhibits human chorionic gonadotrophin and human placental lactogen secretion by co-cultured trophoblasts

We have developed an in-vitro co-culture system to examine theinteraction between purified first trimester cytotrophoblastsand purified non-pregnant human endometrial stromal cells (ESC).ESC decidualization is an important step in endometrial maturationand may modulate embryo implantation. In order to investigatethe effects of ESC decidualization on trophoblast function,we examined human chorionic gonadotrophin (HCG), human placentallactogen (HPL), progesterone and oestrogen secretion by trophoblastsco-cultured in contact with ESC, either with or without decidualizationinduced by progesterone. Decidualized ESC inhibited basal HCGand HPL secretion for 3 days during the culture for HCG, andfor 5 days during the culture for HPL (P < 0.01 and P <0.03 respectively). After 5 days of co-culture, decidual transformationof ESC as indicated by prolactin production occurred in thecontrol cultures due to progesterone and oestradiol secretionby the co-cultured trophoblasts, but no significant differencesin HCG or HPL secretion were observed between the two groups.Although the type of trophoblast used in the present study isfar from implantation, our results clearly demonstrated thatHCG and HPL secretion by trophoblasts was inhibited by the presenceof co-cultured decidualized ESC, and suggested that ESC decidualizationmay regulate trophoblast function at the human fetal-maternalinterface.     

The IGF system in in-vitro human decidualization

Decidualization of endometrial stromal cells (ESCs) is criticalfor a successful pregnancy but the molecular mechanisms of theprocess are poorly understood. In this study, we investigatedwhether the insulin-like growth factor (IGF) network is involvedin this cellular process. Expression kinetics of members ofthe IGF system was examined at both mRNA and protein levelsduring in-vitro decidualization of cultured human ESCs. We founda significant up-regulation of IGF-II as well as of IGF-I receptorand the A and B insulin receptor (InsR) isoforms. In addition,levels of the key adaptor proteins insulin receptor substrate1 (IRS-1) and IRS-2 increased, suggesting a potential involvementof the IGF signalling pathway in the decidualization process.Expression of two IGF binding proteins, IGFBP-1 and IGFBP-4,which can inhibit IGF action, also increased. In order to determinewhether IGF signalling was activated during decidualization,the phosphorylation status of the receptors and the adaptorproteins was estimated. Only IRS-2 was slightly phosphorylatedin decidualized cells and was further activated by the additionof exogenous IGF-II. These results suggest that the IGF signallingpathway could play a crucial role in the functions of decidualizedendometrial cells.     

Expression of interleukin-11 during the human menstrual cycle: coincidence with stromal cell decidualization and relationship to leukaemia inhibitory factor and prolactin

Interleukin-11 (IL-11) is crucial in the decidualization response of the uterine stroma to the implanting blastocyst in the mouse. This study examined the localization and expression of IL-11 in human endometrium throughout the menstrual cycle and of prolactin and leukaemia inhibitory factor (LIF) in secretory phase endometrium. The mRNA expression of IL-11 receptor alpha and the signalling component, gp130, in endometrial tissue were also determined. Immunoreactive IL-11 was highest in the secretory phase and present in decidualized stromal cells, glandular epithelial cells, endothelial and smooth muscle cells, and the mRNA expression was verified by in-situ hybridization. Decidual cells showed the most intense staining. IL-11 receptor alpha and gp130 mRNA were detected throughout the cycle with minimal variation. Expression of IL-11 mRNA and protein preceded that of prolactin. While immunoreactive prolactin was found in stromal, decidual and glandular epithelial cells, prolactin mRNA was confined to decidual cells. In contrast, endometrial LIF expression preceded IL-11 but was largely confined to the glandular epithelium. The sequence of appearance of LIF, IL-11 and prolactin suggests a synchronized role for each in the differentiation of the endometrium. The cyclical changes and cell type specific expression of IL-11 suggests a potential role in the decidualization of stromal cells.     

Corticotrophin-releasing hormone (CRH) interacts with inflammatory prostaglandins and interleukins and affects the decidualization of human endometrial stroma

The hypothalamic neuropeptide, corticotrophin-releasing hormone (CRH), which is also produced by human endometrium, has been shown to induce its decidualization in vitro. This process, induced mainly by progesterone, has characteristics of an aseptic inflammatory reaction, and is modulated by locally produced pro-inflammatory factors. In humans, prostaglandin E(2) (PGE(2)) enhances while interleukin (IL)-1 inhibits the decidualizing effect of progesterone. The aim of the present work was to test the hypothesis that CRH might affect the decidualization of human endometrium interacting with these factors. Therefore, we studied its effects on the production of pro-inflammatory interleukins IL-1, IL-6 and of PGE(2) from human endometrial stromal cells in primary culture. The results strongly suggest that CRH decidualizes stromal cells, as judged by the appearance of cytokeratins and the production of prolactin, two established markers of decidualization. In parallel to its effect on decidualization, CRH also decreased the production of PGE(2), while it increased the production of IL-1 and IL-6. Exposure of endometrial stromal cells to IL-6 also caused decidualization. The data presented here suggest that endometrial CRH regulates the production of local modulators of decidualization, i.e. PGE(2), IL-1 and IL-6. We postulate that, through the regulation of these factors, CRH acts as a local fine-tuner of decidualization initiated by progesterone.     

小鼠子宫内膜基质细胞分离培养、鉴定及体外蜕膜化的诱导

背景：国内已建立许多有关人的子宫内膜基质细胞分离培养及蜕膜化的诱导方法,但人的子宫标本存在取材困难等问题。 目的：建立小鼠子宫内膜基质细胞的分离培养及体外蜕膜化的诱导方法,为进一步研究子宫内膜蜕膜化的机制提供一种简单、有效的模型。 方法：通过胰酶与胶原酶Ⅱ消化及差时贴壁等方法,分离和纯化小鼠妊娠第4天的子宫内膜基质细胞;用细胞免疫化学方法鉴定基质细胞;通过孕酮和雌二醇联合处理诱导子宫内膜基质细胞的蜕膜化,然后用苏木精染核、实时定量PCR和免疫印迹方法检测蜕膜化标志分子的变化。 结果与结论：①分离培养的子宫内膜基质细胞的存活率为(90.1±2.3)%。②细胞免疫化学方法染色显示,分离纯化的子宫基质细胞Vimentin染色呈阳性,而Cytokeratin染色呈阴性,细胞纯度可达(92.3±2.4)%。③孕酮和雌二醇处理48,72 h后,苏木精染色显示细胞呈现多核化,蜕膜化标志分子dPRP、周期蛋白D3、孕酮受体mRNA随着培养时间延长均明显增加(P < 0.05)。结果可见采用酶消化和差时贴壁方法,可获得高纯度的子宫内膜基质细胞;子宫内膜基质细胞在体外通过孕酮和雌二醇联合诱导可产生蜕膜化。     

Mechanisms of normal and abnormal endometrial bleeding

Expression of tissue factor (TF), the primary initiator of coagulation, is enhanced in decidualized human endometrial stromal cells (HESC) during the progesterone-dominated luteal phase. Progesterone also augments a second HESC hemostatic factor, plasminogen activator inhibitor-1 (PAI-1). In contrast, progestins inhibit HESC matrix metalloproteinase (MMP)-1, 3 and 9 expression to stabilize endometrial stromal and vascular extracellular matrix. Through these mechanisms decidualized endometrium is rendered both hemostatic and resistant to excess trophoblast invasion in the mid-luteal phase and throughout gestation to prevent hemorrhage and accreta. In non-fertile cycles, progesterone withdrawal results in decreased HESC TF and PAI-expression and increased MMP activity and inflammatory cytokine production promoting the controlled hemorrhage of menstruation and related tissue sloughing. In contrast to these well ordered biochemical processes, unpredictable endometrial bleeding associated with anovulation reflects absence of progestational effects on TF, PAI-1 and MMP activity as well as unrestrained angiogenesis rendering the endometrium non-hemostatic, proteolytic and highly vascular. Abnormal bleeding associated with long-term progestin-only contraceptives results not from impaired hemostasis but from unrestrained angiogenesis leading to large fragile endometrial vessels. This abnormal angiogenesis reflects progestational inhibition of endometrial blood flow promoting local hypoxia and generation of reactive oxygen species that increase production of angiogenic factors such as vascular endothelial growth factor (VEGF) in HESCs and Angiopoietin-2 (Ang-2) in endometrial endothelial cells while decreasing HESC expression of angiostatic, Ang-1. The resulting vessel fragility promotes bleeding. Aberrant angiogenesis also underlies abnormal bleeding associated with myomas and endometrial polyps however there are gaps in our understanding of this pathology.     

Interleukin 11 advances progesterone-induced decidualization of human endometrial stromal cells

Differentiation of endometrial stromal cells into decidual cells is crucial for embryo implantation and placentation. Interleukin (IL)-11 signalling is essential for adequate decidualization in the mouse uterus. We examined the role of IL-11 during progesterone-induced decidualization of human endometrial stromal cells over a 10-12 day period, using prolactin (PRL) production as a decidual marker. These cells produced biologically active IL-11 and expressed IL-11, IL-11Ralpha and PRL mRNA during decidualization. Neutralization of endogenous IL-11 with an anti-human (hu)IL-11 antibody (AB) reduced production of PRL from day 8 and insulin-like growth factor binding protein (IGFBP)-1, another marker of decidualization, from day 10 of culture. Following AB washout, PRL and IGFBP-1 secretion increased. Addition of recombinant (r)huIL-11 (10 or 100 ng/ml) to endometrial stromal cells increased secretion of PRL from day 4 and IGFBP-1 from day 6 compared with progesterone alone. Morphological signs of differentiation accompanied biochemical differentiation in the progesterone-treated cells and were further induced by exogenous rhuIL-11. Our observations demonstrate that human endometrial stromal cells produce biologically active IL-11, which promotes progesterone-induced decidualization. These results suggest that IL-11 has both paracrine and autocrine actions on human endometrial stromal cells and plays an important role in preparing the human endometrium for implantation.     

Functional characterization of prostaglandin transporter and terminal prostaglandin synthases during decidualization of human endometrial stromal cells

BACKGROUND: Decidualization of endometrial stromal cells is essential for successful implantation and pregnancy. Prostaglandins (PG) have been shown to be required for the initiation and maintenance of decidualization in animal models. The transport of PG across the plasma membrane is mediated by carriers such as prostaglandin transporter (PGT). Our recent data have shown the expression of human PGT (hPGT) in the endometrium during the menstrual cycle. The objective of the present study was to characterize hPGT in decidualized stromal cells. METHODS AND RESULTS: Human endometrial stromal cells were treated with a combination of cAMP and medroxyprogesterone acetate to induce decidualization. Decidualization was confirmed by morphological differentiation and increased secretion of prolactin. A large increase in hPGT mRNA level, as measured by real-time PCR analysis, was observed in decidual cells compared with control. Similarly, a 2-fold up-regulation of hPGT and 3-12-fold increase in PG biosynthetic enzymes were obtained at the protein level. Decidual cells exhibited a higher isotopic PGE2 uptake and greater intracellular PG levels than control. CONCLUSIONS: The higher uptake of PG by decidual cells is highly likely to be mediated via hPGT. PGT is a newly identified regulator of PG action at the cellular level and likely contributes to the regulation of PG action in female reproductive processes.     

Prokineticin 1 induces Dickkopf 1 expression and regulates cell proliferation and decidualization in the human endometrium

Prokineticin 1 (PROK1) signalling via prokineticin receptor 1 (PROKR1) regulates the expression of several genes with important roles in endometrial receptivity and implantation. This study investigated PROK1 regulation of Dickkopf 1 (DKK1) expression, a negative regulator of canonical Wnt signalling, and its function in the non-pregnant endometrium and first trimester decidua. DKK1 mRNA expression is elevated during the mid-secretory phase of the menstrual cycle and expression increases further in first trimester decidua. DKK1 protein expression is localized to glandular epithelial and stromal cells during the proliferative, early- and mid-secretory phases, whereas expression is confined to the stroma in the late-secretory phase and first trimester decidua. PROK1 induces the expression of DKK1 in endometrial epithelial cells stably expressing PROKR1 and in first trimester decidua explants, via a Gq-calcium-calcineurin-nuclear factor of activated T-cells-mediated pathway. Endometrial epithelial cell proliferation is negatively regulated by PROK1-PROKR1 signalling. We demonstrate that this effect on cell proliferation occurs via DKK1 expression, as siRNA targeted against DKK1 reduces the PROK1-induced decrease in proliferation. Furthermore, decidualization of primary human endometrial stromal cells with progesterone and cyclic adenosine monophosphate is inhibited by miRNA knock down of PROK1 or DKK1. These data demonstrate important roles for PROK1 and DKK1 during endometrial receptivity and early pregnancy, which include regulation of endometrial cell proliferation and decidualization.     

Oncostatin M inhibits decidualization of normal human endometrial stromal cells

It is well known that oncostatin M binds and activates leukemia inhibitory factor-specific receptors while leukemia inhibitory factor cannot bind oncostatin M-specific receptors. In this study, the differential effects of oncostatin M and leukemia inhibitory factor on cell survival and decidualization of normal human endometrial stromal cells were investigated by using 8-Br-cAMP-induced decidualization assay. Oncostatin M did not affect the viable cell numbers or the prolactin release of unstimulated stromal cells. However, oncostatin M dose-dependently suppressed prolactin releases from 8-Br-cAMP-stimulating stromal cells but enhanced their viable cell numbers. Although oncostatin M significantly enhanced viable cell numbers of 8-Br-cAMP-stimulated stromal cells, it did not affect prolactin secretion from 8-Br-cAMP-induced decidualized cells. Leukemia inhibitory factor significantly enhanced viable cell numbers of 8-Br-cAMP-stimulating cells in a dose-dependent manner as did oncostatin M, while leukemia inhibitory factor did not show any significant suppression of PRL secretion from 8-Br-cAMP-stimulating cells. These results indicate that oncostatin M inhibits the 8-Br-cAMP-induced decidualization process via oncostatin M-specific receptors and that oncostatin M and leukemia inhibitory factor enhance cell survival of 8-Br-cAMP-stimulated prolactin-non-secreting stromal cells mainly via leukemia inhibitory factor receptors. Thus, oncostatin M may autoregulate human endometrial stromal cell survival and decidualization in a paracrine manner.     

Insulin-like growth factor binding protein-1 induces decidualization of human endometrial stromal cells via alpha5beta1 integrin

Progesterone is known to induce decidualization of human endometrial stromal cells in vitro. Decidualized stromal cells produce insulin-like growth factor binding protein-1 (IGFBP-1) as well as prolactin (PRL). In this study, we tested the possibility that IGFBP-1 directly stimulates endometrial stromal cell decidualization. Endometrial stromal cells were obtained from normal menstruating patients with uterine myoma at hysterectomy. Stromal cells were cultured for up to 4 weeks with estradiol (E(2)) and/or medroxy progesterone acetate (MPA) in the presence or the absence of IGFBP-1 and, LR(3)-IGF-I (an IGF-I analogue) that binds to the IGF-I receptor but has reduced affinity for IGFBPs. Decidualization of endometrial stromal cells was evaluated by morphological changes and PRL release into culture media. The binding of IGFBP-1 to endometrial cells was analysed using a biosensor. MPA and E(2) induced decidualization of stromal cells, while LR(3)-IGF-I inhibited decidualization by MPA and E(2) as well as PRL and IGFBP-1 secretion into medium. IGFBP-1 induced decidualization of stromal cells in the absence of MPA and E(2) in the medium. IGFBP-1-induced decidualization was not inhibited by the addition of LR(3)IGF-1 but was inhibited by the addition of an RGD peptide, however, the RGD peptide had no effect on decidualization when added alone. The binding analysis showed that IGFBP-1 bound to the surface of endometrial stromal cells and an anti-alpha5beta1 integrin antibody inhibited its binding. These results suggest that IGFBP-1 produced by endometrium can mediate progesterone-induced decidualization possibly by interacting with alpha5beta1 integrin on the surface of endometrial stromal cells.     

Angiogenesis, vascular endothelial growth factor and the endometrium

Angiogenesis is an essential component of endometrial renewal. The formation of new vessels depends on interactions between various hormones and growth factors, and this review focuses on the expression of angiogenic growth factors in the human endometrium. Peptide and non-peptide angiogenic factors interact during endometrial renewal, including epidermal growth factor (EGF), transforming growth factors (e.g. TGF-beta), platelet-derived endothelial growth factor/thymidine phosphorylase (PD-ECGF/TP), tumour necrosis growth factors and vascular endothelial growth factor (VEGF). Their role in the proliferation and migration of endothelial cells from pre-existing vessels is described, concentrating on VGEF and its receptors (VEG-R1 and -R2), and the fibroblast growth factor (FGF) family. The actions of the products of the VEGF gene are outlined, and the hormonal and non-hormonal control of their localization in the human endometrium and biological actions on vasculature and coagulation are described. Finally, the role of VEGF in menorrhagia is assessed.