Human Endometrial Endothelial Cell Research Articles

Xiaoyao powder improves endometrial receptivity via VEGFR-2-mediated angiogenesis through the activation of the JNK and P38 signaling pathways

Ethnopharmacological relevanceXiaoyao powder (XYP) is a traditional Chinese medicine formula which has wide scope of indications related to liver stagnation, reconcile qi and blood in TCM syndrome. Infertility can induce similar symptoms and signs to the clinical features of liver stagnation syndrome, the treatment of infertility by soothing the liver is obvious. XYP can increase the clinical pregnancy rate, follicle development, oocyte quality and improve endometrial receptivity. However, its underlying pharmacological mechanism of improving endometrial receptivity is unclear. Aim of the studyThe aim of the study was to investigate the effect of XYP on pregnancy rates and endometrial angiogenesis, to determine the potent mechanism in association with the pro-angiogenic behavior which closely related to improving endometrial receptivity. Materials and methodsWe established an animal model exhibiting decreasing endometrial receptivity by controlled ovarian hyperstimulation and a human endometrial microvascular endothelial cell (HEMEC) model. Endometrial morphology was observed by hematoxylin-eosin staining and Scanning electron microscopy. Western blot and qRT-PCR analysis were used to detect expression of PCNA, Cyclin D1, MMP9 and MAPK signaling pathway. Scratch-wound assay and tube formation assay were used to observe HEMEC migration and tubulogenesis. ResultsThe results demonstrated that XYP pretreatment could improve endometrial receptivity, which leads to high pregnancy rates. In the endometrium, XYP facilitated angiogenesis by promoting tube formation. XYP could enhance HEMEC proliferation and migration induced by VEGF, which were observed by the microscope and Scratch-wound assays. XYP promoted HEMEC proliferation and migration via the p38 and JNK MAPK signaling pathways. ConclusionXYP promotes HEMEC proliferation and migration via the P38 and the JNK MAPK signaling pathways, which contribute to the endometrial angiogenesis mediated by VEGFR-2 that is favorable for endometrial receptivity. We firstly elucidated the molecular mechanisms by which XYP improved endometrial receptivity by promoting angiogenesis.

Drospirenone Effects on the Plasminogen Activator System in Immortalized Human Endometrial Endothelial Cells.

Drospirenone (DRSP) is a fourth-generation progestin that interacts with the progesterone receptor (PR) and androgen receptor (AR) in addition to uniquely interacting to the mineralocorticoid receptor (MR). The known effects of DRSP via the mineralocorticoid receptor (MR) are limited. This study seeks to determine if DRSP alters plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) in human immortalized endometrial endothelial cells (HEEC) and if such changes in the plasminogen activator system (PAS) are mediated through the MR or AR. The in vitro cell culture experiments utilizing an immortalized human endometrial endothelial cell line evaluated two concentrations of DRSP on PAI-1 and tPA levels in the culture media using specific enzyme-linked immunoassays (ELISA). Experiments adding DRSP with an androgen receptor blocker, flutamide, or a mineralocorticoid receptor agonist, aldosterone, were performed to elucidate which receptor(s) mediated the PAS effects. DRSP 10μM significantly decreased both HEEC levels of PAI-1 and tPA to 0.75 ± 0.04 and 0.82 ± 0.05 of control, respectively. These direct effects were blunted by flutamide, an AR antagonist. PAI-1 and tPA were not changed by the MR agonist, aldosterone. DRSP significantly decreased both PAI-1 and tPA in the HEECs via the androgen receptor.

Low molecular weight heparin and aspirin exacerbate human endometrial endothelial cell responses to antiphospholipid antibodies.

Women with antiphospholipid antibodies (aPL) are at risk for pregnancy complications despite treatment with low molecular weight heparin (LMWH) or aspirin (ASA). aPL recognizing beta2 glycoprotein I can target the uterine endothelium, however, little is known about its response to aPL. This study characterized the effect of aPL on human endometrial endothelial cells (HEECs), and the influence of LMWH and ASA. HEECs were exposed to aPL or control IgG, with or without low-dose LMWH and ASA, alone or in combination. Chemokine and angiogenic factor secretion were measured by ELISA. A tube formation assay was used to measure angiogenesis. aPL increased HEEC secretion of pro-angiogenic VEGF and PlGF; increased anti-angiogenic sFlt-1; inhibited basal secretion of the chemokines MCP-1, G-CSF, and GRO-α; and impaired angiogenesis. LMWH and ASA, alone and in combination, exacerbated the aPL-induced changes in the HEEC angiogenic factor and chemokine profile. There was no reversal of the aPL inhibition of HEEC angiogenesis by either single or combination therapy. By aPL inhibiting HEEC chemokine secretion and promoting sFlt-1 release, the uterine endothelium may contribute to impaired placentation and vascular transformation. LMWH and ASA may further contribute to endothelium dysfunction in women with obstetric APS.

Estrogen-dependent regulation of human uterine natural killer cells promotes vascular remodelling via secretion of CCL2.

STUDY QUESTIONDoes intrauterine biosynthesis of estrogen play an important role in early pregnancy by altering the function of uterine natural killer (uNK) cells?SUMMARY ANSWEREstrogens directly regulate the function of human uNK cells by increasing uNK cell migration and secretion of uNK cell-derived chemokine (C-C motif) ligand 2 (CCL2) that critically facilitates uNK-mediated angiogenesis.WHAT IS KNOWN ALREADYuNK cells are a phenotypically distinct population of tissue-resident immune cells that regulate vascular remodelling within the endometrium and decidua. Recently we discovered that decidualisation of human endometrial stromal cells results in the generation of an estrogen-rich microenvironment in areas of decidualised endometrium. We hypothesize that intrauterine biosynthesis of estrogens plays an important role in early pregnancy by altering the function of uNK cells.STUDY DESIGN, SIZE, DURATIONThis laboratory-based study used primary human uNK cells which were isolated from first trimester human decidua (n = 32).PARTICIPANTS/MATERIALS, SETTING, METHODSPrimary uNK cells were isolated from first trimester human decidua using magnetic cell sorting. The impact of estrogens on uNK cell function was assessed. Isolated uNK cells were treated with estrone (E1, 10−8 M) or estradiol (E2, 10−8 M) alone or in combination with the anti-estrogen ICI 182 780 (ICI, 10−6 M). uNK cell motility was assessed by transwell migration assay and time-lapse microscopy. Expression of chemokine receptors was assessed by quantitative PCR (qPCR) and immunohistochemistry, and angiogenic factors were assessed by qPCR and cytokine array. Concentrations of CCL2 in supernatants were measured by enzyme-linked immunosorbent assay. Angiogenesis was assessed in a human endometrial endothelial cell network formation assay.MAIN RESULTS AND THE ROLE OF CHANCETreatment with either E1 or E2 increased uNK cell migration (P = 0.0092 and P = 0.0063, respectively) compared with control. Co-administration of the anti-estrogen ICI blocked the effects of E1 and E2 on cell migration. Concentrations of C-X-C chemokine receptor type 4 (CXCR4) mRNA in uNK cells were increased by E2 treatment. The network formation assay revealed that conditioned media from uNK cells treated with E2 significantly increased human endometrial endothelial cell (HEEC) angiogenesis (P = 0.0029 versus control). Analysis of media from uNK cells treated with E2 using an antibody array identified CCL2 as the most abundant cytokine. Validation assays confirmed concentrations of CCL2 mRNA and protein were increased by E2 in uNK cells (P < 0.05 versus controls). Compared with the control, recombinant human CCL2 was found to increase HEEC network formation (P < 0.05) and neutralization of CCL2 in uNK conditioned media significantly decreased E2-dependent uNK-mediated network formation (P = 0.0006).LIMITATIONS, REASONS FOR CAUTIONOur results are based on in vitro responses of primary human cells and we cannot be certain that similar mechanisms occur in vivo in humans. Primary human uNK cells were isolated from first trimester decidua at a range of gestations (8–12 weeks), which may be a source of variation. Primary human uNK cells from non-pregnant endometrium were not assessed and therefore the responses of uNK cells to E2 treatment described in this study may be distinct to uNK cells from first trimester decidua.WIDER IMPLICATIONS OF THE FINDINGSE2 is an essential regulator of reproductive competence. This study demonstrates a critical role for E2 in regulating cellular cross-talk within the endometrium during early pregnancy. We provide the first evidence that E2 directly regulates the function of human uNK cells by altering uNK cell migration and the secretion of uNK-derived angiogenic factors. We describe a novel mechanism of estrogen-dependent secretion of CCL2 which critically mediates uNK-dependent endometrial angiogenesis. Dysregulation of uNK cell function has been implicated in the aetiology of early implantation disorders and disorders of pregnancy. These novel findings provide unique insight into the regulation of uNK cell activity during the establishment of pregnancy in women and highlight key processes which may be targeted in future therapeutic strategies.STUDY FUNDING/COMPETING INTEREST(S)Studies undertaken in the authors' laboratory were supported by MRC Programme Grant G1100356/1 to P.T.K.S. The authors have no conflicts of interest to disclose.

Chinese herbal medicine for miscarriage affects decidual micro-environment and fetal growth

IntroductionIntrauterine growth restriction complicates 5–10% of pregnancies. This study aims to test the hypothesis that Chinese herbal formula, JLFC01, affects pregnancy and fetal development by modulating the pro-inflammatory decidual micro-environment. MethodsHuman decidua from gestational age-matched elective terminations or incomplete/missed abortion was immunostained using anti-CD68 + anti-CD86 or anti-CD163 antibodies. qRT-PCR and Luminex assay measured the effects of JLFC01 on IL-1β- or TNF-α-induced cytokine expression in first trimester decidual cells and on an established spontaneous abortion/intrauterine growth restriction (SA/IUGR)-prone mouse placentae. The effect of JLFC01 on human endometrial endothelial cell angiogenesis was evaluated by average area, length and numbers of branching points of tube formation. Food intake, litter size, fetal weight, placental weight and resorption rate were recorded in SA/IUGR-prone mouse treated with JLFC01. qRT-PCR, Western blot and immunohistochemistry assessed the expression of mouse placental IGF-I and IGF-IR. ResultsIn spontaneous abortion, numbers of decidual macrophages expressing CD86 and CD163 are increased and decreased, respectively. JLFC01 reduces IL-1β- or TNF-α-induced GM-CSF, M-CSF, C–C motif ligand 2 (CCL2), interferon-γ-inducible protein-10 (IP-10), CCL5 and IL-8 production in first trimester decidual cells. JLFC01 suppresses the activity of IL-1β- or TNF-α-treated first trimester decidual cells in enhancing macrophage-inhibited angiogenesis. In SA/IUGR-prone mice, JLFC01 increases maternal food intake, litter size, fetal and placental weight, and reduces fetal resorption rate. JLFC01 induces IGF-I and IGF-IR expression and inhibits M-CSF, CCL2, CCL5, CCL11, CCL3 and G-CSF expression in the placentae. DiscussionJLFC01 improves gestation by inhibiting decidual inflammation, enhancing angiogenesis and promoting fetal growth.

Bisphenol A affects human endometrial endothelial cell angiogenic activity in vitro

The widespread Bisphenol A (BPA) is classified as an endocrine-disrupting chemical (EDC) with estrogenic properties. Human endometrial endothelial cells (HEECs) play a key role in the endometrial angiogenesis that is under the control of estradiol. The hypothesis was that BPA may affect endometrial angiogenesis by disturbing some functional properties of the HEEC.To study this, primary HEECs were exposed to environmentally relevant doses of BPA. The HEECs were co-cultured with primary endometrial stromal cells to create conditions as similar to the in vivo situation as possible. The effects of BPA were evaluated by proliferation and viability assays, tube-formation assays, quantitative PCRs, Western blots and ELISAs.BPA slightly increased HEEC tube formation and VEGF-D protein expression compared with vehicle, without affecting HEEC viability or proliferation.Bisphenol A thus caused changes in HEEC activities in vitro, and may therefore have disturbing effects on endometrial angiogenesis.

23: Synergistic effect of thrombin and bacterial LPS on human endometrial endothelial cell inflammatory cytokine response

23: Synergistic effect of thrombin and bacterial LPS on human endometrial endothelial cell inflammatory cytokine response

Human Endometrial Endothelial Cells Generate Distinct Inflammatory and Antiviral Responses to the TLR3 agonist, Poly(I:C) and the TLR8 agonist, viral ssRNA

Human endometrial endothelial cell (HEEC) innate immunity remains poorly characterized. Based on their direct contact with the circulation, HEECs are uniquely positioned to be exposed to viral infections. This study evaluated the innate immune response generated by HEECs after exposure to the TLR3 agonist, Poly(I:C) and the TLR8 agonist, viral ssRNA. HEECs were treated with or without Poly(I:C) or ssRNA. Culture supernatants were measured for cytokines by multiplex analysis. RNA was analyzed by qRT-PCR for type I interferons and antiviral factors. Treatment of HEECs with Poly(I:C) rapidly upregulated the secretion of IL-2, IL-6, IL-8, IFN-γ, G-CSF, GM-CSF, MCP-1, MIP-1β, RANTES, and GRO-α after 12hr, while ssRNA treatment induced the slower secretion of IL-6, IL-8, IFN-γ, G-CSF, VEGF, and GRO-α after 24hr. Both viral components induced HEEC IFN-α and IFN-β expression. While treatment with Poly(I:C) induced APOBEC3G and OAS expression, treatment with ssRNA upregulated APOBEC3G and M×A mRNA. Our findings demonstrate that HEECs can differentially sense and respond to viral components by generating distinct inflammatory and antiviral immune responses, indicating that these cells likely play an active role in the immune protection of the uterus toward viral infections.

Lipopolysaccharide Appears to Activate Human Endometrial Endothelial Cells Through TLR‐4‐Dependent and TLR‐4‐Independent Mechanisms

Uterine innate immunity remains poorly characterized, and while endometrial endothelial cells are known to express Toll-like receptors (TLRs), little is known about their function in these cells. The present study evaluated the effect of Gram-negative bacterial lipopolysaccharide (LPS) on human endometrial endothelial cell (HEECs) cytokine secretion and tissue factor expression, and the role of TLR-4 in these responses. Human endometrial endothelial cells were treated with or without LPS ± LPS-RS, a TLR-4 antagonist, via the binding of MD-2. After 24 hr, cell-free supernatants were evaluated for cytokines by multiplex analysis and cell lysates were analyzed for tissue factor expression by Western blot. Treatment of HEECs with LPS significantly upregulated the secretion of IL-6, IL-8, and G-CSF, and this was prevented by LPS-RS. LPS also induced tissue factor expression by the HEECs; however, this was unaffected by LPS-RS. These findings suggest that TLR-4 is functional in HEECs and its activation by bacterial LPS induces a specific cytokine/chemokine response. However, bacterial LPS also induced tissue factor expression in what seemed to be a TLR-4-independent fashion, suggesting that this bacterial component can act on the HEECs through TLR-4-dependent and TLR-4-independent pathways. These findings indicate that endometrial endothelial cells may play an active role in uterine innate immunity.

Effect of Low Molecular Weight Heparins (LMWHs) on antiphospholipid Antibodies (aPL)-mediated inhibition of endometrial angiogenesis.

Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by vascular thrombosis and/or pregnancy morbidity in the presence of circulating antiphospholipid antibodies (aPL). Different pathogenic mechanisms for aPL-mediated pregnancy failure have been proposed. In particular a direct effect of aPL on both maternal and fetal side of the placental tissue has been reported, since their reactivity with β2-glycoprotein I (β2GPI) makes them adhere to trophoblast and human endometrial endothelial cell (HEEC) membranes. β2GPI can be recognized by aPL that, once bound, interfere with both trophoblast functions and with the HEEC differentiation.APS patients can be successfully treated with Low Molecular Weight Heparin (LMWH). Recent reports suggest that LMWH acts through mechanisms alternative to its well known anticoagulant effect, because of its ability to bind β2GPI. In our previous studies, we showed that LMWH is able to reduce the aPL binding to trophoblasts and restore cell invasiveness and differentiation. So far, however, no study has described its effects on endometrial angiogenesis.The aim of our research was to evaluate whether two LMWHs, tinzaparin and enoxaparin, have an effect on the aPL-inhibited endometrial angiogenesis. This prompted us to investigate: (i) in vitro HEEC angiogenesis through a Matrigel assay; (ii) VEGF secretion by ELISA; (iii) matrix metalloproteinase-2 (MMP-2) activity by gelatin zymography; (iv) Nuclear Factor-κB (NF-κB) DNA binding activity by colorimetric assay; (v) STAT-3 activation by a sandwich-ELISA kit. Furthermore, using an in vivo murine model we investigated the LMWHs effects on angiogenesis.We demonstrated that the addition of LMWHs prevents aPL-inhibited HEEC angiogenesis, both in vitro and in vivo, and is able to restore the aPL inhibited NF-κB and/or STAT-3 activity, the VEGF secretion and the MMPs activity.The demonstration of a beneficial role for LMWHs on the aPL-inhibited HEEC angiogenesis might provide additional mechanisms whereby this treatment protects early pregnancy in APS.

Pregnancy-specific Glycoprotein 1 Induces Endothelial Tubulogenesis through Interaction with Cell Surface Proteoglycans

Pregnancy-specific β1 glycoproteins (PSGs) are the most abundant fetal proteins in the maternal bloodstream in late pregnancy. They are secreted by the syncytiotrophoblast and are detected around day 14 postfertilization. There are 11 human PSG genes, which encode a family of proteins exhibiting significant conservation at the amino acid level. We and others have proposed that PSGs have an immune modulatory function. In addition, we recently postulated that they are proangiogenic due to their ability to induce the secretion of VEGF-A and the formation of tubes by endothelial cells. The cellular receptor(s) for human PSGs remain unknown. Therefore, we conducted these studies to identify the receptor for PSG1, the highest expressed member of the family. We show that removal of cell surface glycosaminoglycans (GAGs) by enzymatic or chemical treatment of cells or competition with heparin completely inhibited binding of PSG1. In addition, PSG1 did not bind to cells lacking heparan or chondroitin sulfate on their surface, and binding was restored upon transfection with all four syndecans and glypican-1. Importantly, the presence of GAGs on the surface of endothelial cells was required for the ability of PSG1 to induce tube formation. This finding indicates that the PSG1-GAG interaction mediates at least some of the PSG1 proposed functions.

O746 Phagocytosis of apoptotic trophblast cell by human endometrial endothelial cell induce proinflammatory cytokine production

International Journal of Gynecology & ObstetricsVolume 107, Issue S2 p. S306-S306 Free communication (oral) presentations O746 Phagocytosis of apoptotic trophblast cell by human endometrial endothelial cell induce proinflammatory cytokine production B. Peng, B. PengSearch for more papers by this authorK. Koga, K. KogaSearch for more papers by this authorG. Mor, G. MorSearch for more papers by this author B. Peng, B. PengSearch for more papers by this authorK. Koga, K. KogaSearch for more papers by this authorG. Mor, G. MorSearch for more papers by this author First published: 06 October 2011 https://doi.org/10.1016/S0020-7292(09)61119-XAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume107, IssueS2Abstracts of XIX FIGO World Congress of Gynecology and ObstetricsOctober 2009Pages S306-S306 RelatedInformation

Effect of vascular endothelial growth factor (VEGF) withdrawal on apoptosis in an immortalized human endometrial endothelial cell (HEEC) line

Progestin only contraception (POC) is both a safe and effective means of birth control. However, POC is associated with a high rate of breakthrough bleeding. Breakthrough bleeding is a major reason why women chose to discontinue the use of POC. The mechanisms leading to breakthrough bleeding are largely unknown. VEGF levels by immunohistochemistry are variable in the endometrium of progestin only contraceptors. VEGF has been shown to act as an endothelial cell survival factor. Endothelial apoptosis and the resultant capillary collapse could be one of the etiologies leading to breakthrough bleeding.

A Novel Three‐Dimensional In Vitro System to Study Trophoblast–Endothelium Cell Interactions

Pregnancy complications have been linked to improper trophoblast migration and failure of spiral artery transformation. Endothelial cells play an essential role in directing trophoblast migration and transformation, although by an unknown mechanism. We describe a novel in vitro model to evaluate endothelial-trophoblast interaction and signaling in a three-dimensional system. Immortalized human endometrial endothelial cell line and first trimester trophoblast cells were co-cultured. Endothelial transformation into vessel-like structures occurred in Matrigel(TM) OpenLab Image Analysis software was used to monitor labeled trophoblast migration and endothelium transformation. Cytokine/chemokine production was determined using Multiplex. Trophoblast migrates toward endothelial cells in Matrigel, aligns on top of the endothelium within 4-8 hr and achieves complete replacement of the endothelium by 72-96 hr. Lipopolysaccharide treatment damages the endothelium and disrupts endothelium-trophoblast interaction. We report a novel three-dimensional in vitro and in vivo system of trophoblast-endothelium cell interaction. Significant changes in endothelial cells' phenotype are observed upon differentiation in Matrigel. These changes may be necessary for endothelium to direct trophoblast migration and transformation.

1141249460 A novel three‐dimensional in vitro system of trophoblast–endothelium cell interaction

Introduction: Preeclampsia and IUGR have been linked to improper trophoblast migration that impairs maternal spiral artery transformation minimizing vessel dilation and therefore restricting blood flow to the developing fetus. Endothelial cells are thought to play an essential role in directing trophoblast migration and transformation, although the mechanism by which this occurs is not fully understood. We have developed a novel in vitro system that evaluates endothelial‐trophoblast interaction and signaling in a three‐dimensional environment.Methods: An immortalized human endometrial endothelial cell line (HEEC) (Krikun et al., 2004) and first trimester trophoblast cell lines HTR‐8 and Swan.71 were used. Endothelial transformation into vessel‐like structures occurred in Matrigel Basement Membrane Matrix. Cell membrane linker dye and OpenLab Image Analysis software were used to monitor trophoblast migration and transformation. Cytokine and chemokine production was determined using a multiplex bead assay.Results: Upon differentiation in matrigel, endothelial cells undergo a shift in cytokine production characterized by an increased secretion of chemokines. First trimester trophoblast cells migrate toward the differentiated endothelium, and reach it within 4–8 hr. Complete replacement of the endothelium is seen by 48 hr. The transformed tubes maintain structure and remain functional for several weeks allowing for further co‐culture studies.Conclusion: We report the characterization of a novel three‐dimensional in vitro system of trophoblast–endothelium cell interaction. We found significant changes in the phenotype (cytokine expression and LPS responses) of endothelial cells upon differentiation in matrigel as compared to the monolayer culture. These changes may be necessary for the endothelium to direct trophoblast migration. Furthermore, this model represents an ideal co‐culture system to evaluate the role of additional cell types, e.g. immune cells, involved in the process of spiral artery transformation.

Regulation of Angiogenic Activity of Human Endometrial Endothelial Cells in Culture by Ovarian Steroids

Blood vessel growth and regression in human endometrium are regulated throughout the menstrual cycle. We sought a direct role of ovarian steroids on human endometrial endothelial cell (HEEC) proliferation and vascularization. To investigate the HEEC angiogenicity of sex steroids, we developed a reliable method for the isolation of HEEC, which allowed us to investigate the angiogenic effects of sex steroids using immunohistochemistry, immunocytochemistry, Western blot, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt proliferation, and vascular tube formation analyses. We were able to obtain 95-99% pure HEEC with our isolation technique. HEEC expressed predominantly estrogen receptor beta, minimally expressed estrogen receptor alpha, and but did not express progesterone (P(4)) receptors A and B in vivo and in vitro. Estradiol (E(2); 10(-10)-10(-8) m) and P(4) (10(-12)-10(-8) m), alone or in combination, induced HEEC proliferation compared with control values after 48 h of treatment (P < 0.05). Furthermore, after 8 d of treatment, there were significantly more angiogenic patterns in E(2) (10(-8) m), P(4) (10(-10) m), and E(2) plus P(4) (10(-8) and 10(-10) m) treatment groups compared with the control group (angiogenic scores, 2.95 +/- 0.16, 3.26 +/- 0.16, 3.06 +/- 0.17, and 1.93 +/- 0.15, respectively; P < 0.01). In conclusion, our results suggest that there are direct effects of E(2) and P(4) on HEEC and provide a new understanding of the physiological role of sex steroids in the regulation of endometrial events such as angiogenesis.