Interleukin 11 is upregulated in uterine lavage and endometrial cancer cells in women with endometrial carcinoma

Background  

Interleukin (IL) 11 is produced by human endometrium and endometrial cancer tissue. It has roles in endometrial epithelial cell adhesion and trophoblast cell invasion, two important processes in cancer progression. This study aimed to determine the levels of IL11 in uterine lavage fluid in women with endometrial cancer and postmenopausal women. It further aimed to determine the levels of IL11 protein and its signaling molecules in human endometrial cancer of varying grades, and endometrium from postmenopausal women and IL11 signalling mechanisms in endometrial cancer cell lines.     

Expression of metalloproteinases and their inhibitors in blood vessels in human endometrium.

Matrix metalloproteinases (MMPs) are zinc-requiring enzymes that can degrade components of the extracellular matrix and that are implicated in tissue remodeling. Their role in the onset of menstruation in vivo has been proven; however, the expression and functions of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in vascular structures are poorly understood. We determined by immunocytochemistry, using characterized monoclonal antibodies, the distribution of MMPs and of their inhibitors TIMP-1 and TIMP-2 in the endometrium during the menstrual cycle. MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 had differing distributions and patterns of expression. In addition to the localization of MMP-9 in the epithelium and of MMP-2, MMP-3, and MMP-1 in the stromal tissue, these MMPs were detected in the vascular structures. MMP-2 (72-kDa gelatinase) and tissue inhibitors TIMP-1 and TIMP-2 were detectable in vessels throughout the cycle. In contrast, MMP-3 (stromelysin-1) was detected only in late-secretory and menstrual endometrial vessels, while MMP-9 (92-kDa gelatinase) was detected in spiral arteries during the secretory phase and in vascular structures during the midfollicular and menstrual phases. The expression of MMP-2 and MMP-9 in endometrial vessels during the proliferative and secretory periods suggests their relationship to vascular growth and angiogenesis. The pronounced expression of MMP-3 (stromelysin-1) in the vessels situated in the superficial endometrial layer during menses suggests that this metalloproteinase initiates damage in the vascular wall during menstrual breakdown. The finding of an intense expression of TIMP-1 and TIMP-2 in the vessels delineating necrotic from non-necrotic areas during menses also suggests that they could limit tissue damage, allowing regeneration of the endometrium after menses. These data indicate that, in addition to expression in epithelial cells and stromal tissue, MMPs are expressed in endometrial vascular cells in a cycle-specific pattern, consistent with regulation by steroid hormones and with specific roles in the vascular remodeling processes occurring in the endometrium during the cycle.     

Effects of substrata on the polarization of bovine endometrial epithelial cells in vitro

Summary Epithelial-cell function requires cellular polarity in which apical membrane surfaces have unique characteristics and cellular organelles are stratified. Physiological investigations of endometrial, epithelial cells would be enhanced greatly by the ability of a method to polarize cells in culture. This study investigates the effects of different substrata on polarization of cultured bovine endometrial epithelial cells. Fetal bovine endometrial epithelial-cell lines were developed from explant outgrowth. Epithelial monolayers were subcultured onto amniotic membranes, Millicell-HA membranes, or Millicell-CM membranes coated with rat-tail collagen, Matrigel, laminin, Vitrogen,or fibronectin. Cultures on these substrata were maintained at the air/liquid interface. Cells grown on either collagen-coated or uncoated Milli-cell membranes also were maintained submerged in medium. Excellent polarized morphology was attained in cultures grown at the air/liquid interface on amniotic membranes and rat-tail collagen-coated membranes. Lectin-binding patterns, to apical membranes of polarized epithelial cell cultures paralleled patterns of binding to bovine endometrial surfaces in vivo. Cultures on rat-tail collagen were maintained for several weeks. These methods provide a valuable system for studying the endometrium in vitro.     

Psychological Factors in 155 Patients with Functional Uterine Bleeding

One hundred and fifty-five women with functional uterine bleeding were studied to evaluate the importance of concomitant psychological disorders. Psychological illnesses were diagnosed in 128 patients (82.6%), most of which arose from problems directly related to sexual or reproductive functions. The remaining 27 patients (17.4%) were different in that they were psychologically stable and all but two were at puberty or approaching the menopause.Histological studies of endometrial samples from 135 of these patients indicated little evidence of abnormal sex hormone activity; 77 (57%) showed normal secretory phase endometrium and 32 (23.7%), proliferative phase endometrium. The remaining 26 (19.2%) showed evidence of some endocrine dysfunction, 15 such specimens being obtained from psychologically stable patients.It is probable that psychological disturbances are the principal cause of functional uterine bleeding during the prime reproductive years. The psychological component of the illness is the most important and determines the ultimate prognosis.     

Molecular mechanisms of membrane interaction at implantation

Successful pregnancy is dependent upon the implantation of a competent embryo into a receptive endometrium. Despite major advancement in our understanding of reproductive medicine over the last few decades, implantation failure still occurs in both normal pregnancies and those created artificially by assisted reproductive technology (ART). Consequently, there is significant interest in elucidating the etiology of implantation failure. The complex multistep process of implantation begins when the developing embryo first makes contact with the plasma membrane of epithelial cells within the uterine environment. However, although this biological interaction marks the beginning of a fundamental developmental process, our knowledge of the intricate physiological and molecular processes involved remains sparse. In this synopsis, we aim to provide an overview of our current understanding of the morphological changes which occur to the plasma membrane of the uterine endothelium, and the molecular mechanisms that control communication between the early embryo and the endometrium during implantation. A multitude of molecular factors have been implicated in this complex process, including endometrial integrins, extracellular matrix molecules, adhesion molecules, growth factors, and ion channels. We also explore the development of in vitro models for embryo implantation to help researchers investigate mechanisms which may underlie implantation failure. Understanding the precise molecular pathways associated with implantation failure could help us to generate new prognostic/diagnostic biomarkers, and may identify novel therapeutic targets. Birth Defects Research (Part C) 108:19–32, 2016. © 2016 Wiley Periodicals, Inc.     

Cell-specific expression of estrogen-responsive genes in the uteri of cyclic, early pregnant and ovariectomized ewes

A single physiological dose of estradiol up-regulates estrogen receptor-alpha(ER), progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), c-fos, cyclophilin, and actin mRNAs in the endometrium of ovariectomized ewes. Therefore, we hypothesized that these genes would be up-regulated by the preovulatory surge of estrogen which occurs on the evening of Day 15 in the estrous cycle of sheep. ER and PR mRNA concentrations increased between Day 15 and Day 1 in cyclic ewes in most endometrial epithelial cells, while GAPDH mRNA increased in epithelial and stromal cells in the deep endometrium. Day 15 pregnant ewes had lower expression of ER, PR, GAPDH, cyclophilin and actin genes. For ER and GAPDH mRNAs, the greatest reduction occurred in the superficial endometrium. Ovariectomized ewes demonstrated concentrations of ER, PR, and GAPDH mRNAs that were similar to those in the cyclic ewes. While concentrations of c-fos mRNA did not differ between groups, those of cyclophilin and actin mRNAs were lower in the pregnant and ovariectomized ewes. In conclusion, ER, PR and GAPDH gene expression rose during estrus in endometrial cells with the highest ER gene expression and were repressed in pregnant ewes in superficial endometrial cells with the greatest PR gene expression.     

Regulation of human endometrial function: mechanisms relevant to uterine bleeding

This review focuses on the complex events that occur in the endometrium after progesterone is withdrawn (or blocked) and menstrual bleeding ensues. A detailed understanding of these local mechanisms will enhance our knowledge of disturbed endometrial/uterine function--including problems with excessively heavy menstrual bleeding, endometriosis and breakthrough bleeding with progestin only contraception. The development of novel strategies to manage these clinically significant problems depends on such new understanding as does the development of new contraceptives which avoid the endometrial side effect of breakthrough bleeding.     

Endometrial contraception: modulation of molecular determinants of uterine receptivity

Modulation of endometrial receptivity is a promising approach for fertility regulation since it allows a contraceptive to act specifically at the endometrium. This was corroborated by our previous observations that treatment with low doses of a pure progesterone antagonist (PA, antiprogestin), onapristone (ZK 98299), in bonnet monkeys inhibited fertility by selectively retarding endometrial development, without affecting the hypophyseal-hypothalamic function. In the present study, further investigations, undertaken to analyze the molecular repertoire of a nonreceptive primate endometrium, determined expression of: steroid hormone receptors, i.e. progesterone receptor (PR) and estrogen receptor (ER); cytokines, i.e. leukemia inhibitory factor (LIF): transforming growth factor beta (TGFbeta) and its receptor (TGFbetaR); and cell adhesion molecules, i.e. integrins (alpha(v)beta(3), alpha(1)beta(1)). These studies were conducted during the different phases of the normal menstrual cycle and following treatment with different doses of onapristone (2.5 mg, 5 mg, or 10 mg every third day for one cycle) in bonnet monkeys. The molecules were analysed collectively to explore the possibility of a correlation between expression of these markers and endometrial receptivity and to investigate whether there exists a regulatory link between expression of these molecules under in vivo conditions. Three types of expression patterns of endometrial factors were observed during the peri-implantation period following onapristone treatment: 1) LIF, alpha(v)beta(3), and alpha(1)beta(1) showed significant (P < 0.02) down regulation in glandular epithelium of endometria in animals treated with all three doses of onapristone as compared to the control group. This was indicative of their critical role in the progesterone-driven cascade leading to implantation. 2) PR, TGFbeta, and TGFbetaR remained unaffected in the endometria from 2.5 mg treated animals and showed down regulation in animals treated with 5 and 10 mg onapristone as compared to the control group, thereby suggesting that the expression of these markers may not truely reflect endometrial receptivity per se. However, their facilitatory role in preparing the endometrium for implantation can not be ruled out since continued perturbation in the expression of these molecules may affect endometrial growth, remodelling, and differentiation, which in turn may render the endometrium nonreceptive; 3) ER remained unaltered in endometria of animals rendered infertile with 2.5, 5, and 10 mg onapristone. This observation indirectly suggests that onapristone-induced endometrial changes are mediated via some specific mechanisms. The present study clearly demonstrates that endometrial non-receptivity induced at low doses of onapristone is associated with changes in the expression pattern of specific molecular markers. However, no direct correlation was observed between in vivo expression of TGFbeta, LIF, and integrins, thereby lending support to the concept that there exists redundancy or multiple pathways which regulate implantation events.     

Extracellular matrix dynamics in scar-free endometrial repair: perspectives from mouse in vivo and human in vitro studies

Repair of the postmenstrual endometrium presents a unique opportunity to examine nonscarring repair in an adult tissue. We aimed to characterize and determine the importance of extracellular matrix (ECM) dynamics in cell migration during endometrial repair. Utilizing an in vivo mouse model of postmenstrual repair and an in vitro model of human endometrial re-epithelialization, we determined the dynamic changes in expression of ECM and related factors in both models by array analysis of repairing areas. We also validated expression of integrins, growth factors, protease inhibitors, basement membrane, and adhesion molecules in vitro and in both mouse and human repairing endometrium by quantitative RT-PCR and immunohistochemical studies. Finally, we determined the functional importance of integrin-fibronectin interactions and matrix metalloprotease (MMP)-facilitated cell movement during re-epithelialization and propose a model for cell locomotion during postmenstrual repair. These data demonstrated the dynamic expression and functional importance of ECM interactions in endometrial repair, which may be important for scar-free repair.     

Dilated thin-walled blood and lymphatic vessels in human endometrium: a potential role for VEGF-D in progestin-induced break-through bleeding

Progestins provide safe, effective and cheap options for contraception as well as the treatment of a variety of gynaecological disorders. Episodes of irregular endometrial bleeding or breakthrough bleeding (BTB) are a major unwanted side effect of progestin treatment, such that BTB is the leading cause for discontinued use of an otherwise effective and popular medication. The cellular mechanisms leading to BTB are poorly understood. In this study, we make the novel finding that the large, dilated, thin walled vessels characteristic of human progestin-treated endometrium include both blood and lymphatic vessels. Increased blood and lymphatic vessel diameter are features of VEGF-D action in other tissues and we show by immunolocalisation and Western blotting that stromal cell decidualisation results in a significant increase in VEGF-D protein production, particularly of the proteolytically processed 21 kD form. Using a NOD/scid mouse model with xenografted human endometrium we were able to show that progestin treatment causes decidualisation, VEGF-D production and endometrial vessel dilation. Our results lead to a novel hypothesis to explain BTB, with stromal cell decidualisation rather than progestin treatment per se being the proposed causative event, and VEGF-D being the proposed effector agent.     

Ultrastructural analysis of the pathogenesis of Neisseria gonorrhoeae endometrial infection

We have studied gonococcal infection in human endometrium organ culture and in human primary endometrial epithelial cells using various microscopic techniques including scanning electron microscopy, transmission electron microscopy, bright field light microscopy and laser scanning confocal microscopy. Here we describe the interactions between Neisseria gonorrhoeae and human endometrial luminal epithelial cells at the ultrastructural levels. N. gonorrhoeae attached to cilia but were not observed associated with the plasma membrane of ciliated epithelial cells or internalized into ciliated epithelial cells. N. gonorrhoeae could be found in intracellular vacuoles in secretory epithelial cells. N. gonorrhoeae have diverse interactions with endometrial epithelium. These include intimate association and colocalization with asialoglycoprotein receptor (ASGP-R) and CEACAM, lamellipodia and ruffle formation and colocalization with CR3, and microvillus engagement. These studies indicate that N. gonorrhoeae utilize multiple mechanisms to associate with endometrial epithelial cells and can associate with both ciliated and secretory cells. This diversity is consistent with a role of the endometrium as a transition zone between frequently asymptomatic cervical gonorrhoea and symptomatic pelvic inflammatory disease.     

New insights into the normal menstrual cycle-regulatory molecules

Although the growth and regression of the endometrium is primarily a function of the ovarian hormones, recent studies indicate a potential autocrine/paracrine role for regulatory molecules. Thus, growth factors, angiogenesis stimulating factors and proliferating cell markers are high in the proliferative phase endometrium contributing to its regeneration. At the same time, other proteins promote endometrial cell survival by preventing extracellular matrix degradation and apoptosis. As glandular proliferation persists in the early secretory phase of the menstrual cycle, the activity of some proteins stimulating growth remains unchanged, but declines significantly thereafter, shifting the balance between proliferation and apoptosis in favour of apoptosis. During this period, several other regulatory substances are expressed at high levels, suggesting a role in endometrial maturation. If, however, implantation of a fertilized ovum fails to take place, menstruation occurs probably as the result of matrix metalloprotinases which antagononizes the anti-degradation factors (inhibitors of metalloproteinases). This review examines the changing endometrial patterns of a normal menstrual cycle in relation to these regulatory molecules.     

Stromal-to-Epithelial Transition during Postpartum Endometrial Regeneration

Endometrium is the inner lining of the uterus which is composed of epithelial and stromal tissue compartments enclosed by the two smooth muscle layers of the myometrium. In women, much of the endometrium is shed and regenerated each month during the menstrual cycle. Endometrial regeneration also occurs after parturition. The cellular mechanisms that regulate endometrial regeneration are still poorly understood. Using genetic fate-mapping in the mouse, we found that the epithelial compartment of the endometrium maintains its epithelial identity during the estrous cycle and postpartum regeneration. However, whereas the stromal compartment maintains its identity during homeostatic cycling, after parturition a subset of stromal cells differentiates into epithelium that is subsequently maintained. These findings identify potential progenitor cells within the endometrial stromal compartment that produce long-term epithelial tissue during postpartum endometrial regeneration.     

Proteomic Analysis of Stromal and Epithelial Cell Communications in Human Endometrial Cancer Using a Unique 3D Co‐Culture Model

Epithelial and stromal communications are essential for normal uterine functions and their dysregulation contributes to the pathogenesis of many diseases including infertility, endometriosis, and cancer. Although many studies have highlighted the advantages of culturing cells in 3D compared to the conventional 2D culture system, one of the major limitations of these systems is the lack of incorporation of cells from non‐epithelial lineages. In an effort to develop a culture system incorporating both stromal and epithelial cells, 3D endometrial cancer spheroids are developed by co‐culturing endometrial stromal cells with cancerous epithelial cells. The spheroids developed by this method are phenotypically comparable to in vivo endometrial cancer tissue. Proteomic analysis of the co‐culture spheroids comparable to human endometrial tissue revealed 591 common proteins and canonical pathways that are closely related to endometrium biology. To determine the feasibility of using this model for drug screening, the efficacy of tamoxifen and everolimus is tested. In summary, a unique 3D model system of human endometrial cancer is developed that will serve as the foundation for the further development of 3D culture systems incorporating different cell types of the human uterus for deciphering the contributions of non‐epithelial cells present in cancer microenvironment.     

Human endometrial epithelial cell lines for studying steroid and cytokine actions

Summary Recent studies suggest that the proliferation and expression of HLA-DR molecules in endometrial epithelium may be regulated by systemic steroids and local cytokines. To test the interacting influences of cytokines and steroids on the expression of HLA-DR and proliferation of epithelial cells, an endometrial cell model is required that is sensitive to both signals. In this study, we characterize cells of carcinoma cell lines of endometrial lineage for their responsiveness to cytokines and steroids. Independently developed for its response to steroid hormones from a well-differentiated adenocarcinoma of human endometrium, EnCa101AE cell line is further cloned for the expression of progesterone receptor. Immunohistochemical localization using monoclonal antibodies demonstrates that both EnCa101AE cell line and cloned ECC1 cells are purely epithelial, as evidenced by the expression of cytokeratin and epithelial membrane antigen, express estrogen receptors, and concomitantly exhibit IFN-gamma receptor. Experiments using radioiodinated IL-1 reveal that these cell lines also possess high affinity receptors for IL-1. As indicated by the induction of HLA-DR molecules, and alterations in morphologic characteristics, these cell lines are sensitive to both IFN-gamma and IL-1 action. The class II molecules (HLA-DR, HLA-DP, and HLA-DQ) are differentially induced by IFN-gamma treatment in carcinoma cell lines, with HLA-DR being the prevailing induced molecule. IFN-gamma inhibits and estradiol-17β promotes growth of ECC1 cells in a dose-and time-dependent manner. These findings indicate that the interacting effect(s) of the cytokines and steroid hormones on endometrial epithelium may be studied in these unique steroid-and cytokine-sensitive epithelial cell lines.     

Long-term label retaining cells localize to distinct regions within the female reproductive epithelium

The uterus is an extremely plastic organ that undergoes cyclical remodeling including endometrial regeneration during the menstrual cycle. Endometrial remodeling and regeneration also occur during pregnancy and following parturition, particularly in hemochorial implanting species. The mechanisms of endometrial regeneration are not well understood. Endometrial stem/progenitor cells are proposed to contribute to endometrial regeneration in both humans and mice. BrdU label retention has been used to identify potential stem/progenitor cells in mouse endometrium. However, methods are not available to isolate BrdU label-retaining cells (LRC) for functional analyses. Therefore, we employed a transgenic mouse model to identify H2B-GFP LRCs throughout the female reproductive tract with particular interest on the endometrium. We hypothesized that the female reproductive tract contains a population of long-term LRCs that persist even following pregnancy and endometrial regeneration. Endometrial cells were labeled (pulsed) either transplacentally/translactationally or peripubertally. When mice were pulsed transplacentally/translactationally, the label was not retained in the uterus. However, LRCs were concentrated to the distal oviduct and endocervical transition zone (TZ) following natural (i.e., pregnancy/parturition induced) and mechanically induced endometrial regeneration. LRCs in the distal oviduct and endocervical TZ expressed stem cell markers and did not express ERα or PGR, implying the undifferentiated phenotype of these cells. Oviduct and endocervical TZ LRCs did not proliferate during endometrial re-epithelialization, suggesting that they do not contribute to the endometrium in a stem/progenitor cell capacity. In contrast, when mice were pulsed peripubertally long-term LRCs were identified in the endometrial glandular compartment in mice as far out as 9 months post-pulse. These findings suggest that epithelial tissue of the female reproductive tract contains 3 distinct populations of epithelial cells that exhibit stem/progenitor cell qualities. Distinct stem/progenitor-like cells localize to the oviduct, endometrium, and cervix.     

Altered tenascin expression during spontaneous endometrial carcinogenesis in the bdii/han rat

The BDII/Han rat develops spontaneous endometrial adenocarcinoma, which appears virtually identical histologically to human endometrial adenocarcinoma. The incidence rate of cancer formation in the rat is 90% and the mean lifetime of the animals is 22 months. This animal model therefore, is useful in the study of molecular aspects of spontaneous transformation as well as mammalian neoplastic progression. In this study we address the in-situ expression of tenascin, an extracellular matrix glycoprotein, during normal cyclic growth, during development of proliferative states, and during malignant transformation of the endometrium. Trace amounts of immunocytochemically detectable tenascin were found in 10% of young BDII/Han rats with a normal estrus cycle. In these inbred animals no tenascin was detectable in uteri without neoplastic progressive alterations of the endometrium. Tenascin immunoreactivity first appeared during proliferation in one of three uteri with cystic glandular hyperplasia. Prominent tenascin expression was detectable in all adenomatous hyperplasia, but restricted to the stromal mesenchyme, that surrounded the glands. In all endometrial adenocarcinomas tested, essentially the entire extracellular space of the stromal mesenchyme was immunoreactive with anti-tenascin antibodies while the epithelial glands themselves were negative. This staining pattern was observed independent of the degree of tumor differentiation or extent of myometrial invasion. The tenascin staining pattern was not significantly altered in tumors transplanted into the soft tissues of the neck of female BDII/Han rats. From our studies we conclude that tenascin may be a marker for the early detection of proliferative endometrial states. Further, previous investigation by us showing nearly identical findings in human endometrium reinforces the value of this animal model system in the study of human epithelial hyperplastic conditions including those associated with malignancies of the endometrium.     

Human Endometrial Extracellular Vesicles Functionally Prepare Human Trophectoderm Model for Implantation: Understanding Bidirectional Maternal‐Embryo Communication

Embryo implantation into maternal endometrium is critical for initiation and establishment of pregnancy, requiring developmental synchrony between endometrium and blastocyst. However, factors regulating human endometrial–embryo cross talk and facilitate implantation remain largely unknown. Extracellular vesicles (EVs) are emerging as important mediators of this process. Here, a trophectoderm spheroid‐based in vitro model mimicking the pre‐implantation human embryo is used to recapitulate important functional aspects of blastocyst implantation. Functionally, human endometrial EVs, derived from hormonally treated cells synchronous with implantation, are readily internalized by trophectoderm cells, regulating adhesive and invasive capacity of human trophectoderm spheroids. To gain molecular insights into mechanisms underpinning endometrial EV‐mediated enhancement of implantation, quantitative proteomics reveal critical alterations in trophectoderm cellular adhesion networks (cell adhesion molecule binding, cell–cell adhesion mediator activity, and cell adherens junctions) and metabolic and gene expression networks, and the soluble secretome from human trophectodermal spheroids. Importantly, transfer of endometrial EV cargo proteins to trophectoderm to mediate changes in trophectoderm function is demonstrated. This is highlighted by correlation among endometrial EVs, the trophectodermal proteome following EV uptake, and EV‐mediated trophectodermal cellular proteome, important for implantation. This work provides an understanding into molecular mechanisms of endometrial EV‐mediated regulation of human trophectoderm functions—fundamental in understanding human endometrium–embryo signaling during implantation.