Jennifer L. Herington

Immune interactions in endometriosis

Endometriosis is a common, complex gynecologic disorder characterized by the presence of endometrial glands and stroma at extrauterine (ectopic) sites. In women who develop this disease, alterations in specific biological processes involving both the endocrine and immune systems have been observed, which may explain the survival and growth of displaced endometrial tissue in affected women. In the past decade, a considerable amount of research has implicated a role for alterations in progesterone action at both eutopic and ectopic sites of endometrial growth which may contribute to the excessive inflammation associated with progression of endometriosis; however, it remains unclear whether these anomalies induce the condition or are simply a consequence of the disease process. In this article, we summarize current knowledge of alterations within the immune system of endometriosis patients and discuss how endometrial cells from women with this disease not only have the capacity to escape immunosurveillance, but also use inflammatory mechanisms to promote their growth within the peritoneal cavity. Finally, we discuss evidence that exposure to an environmental endocrine disruptor, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, can mediate the development of an endometrial phenotype that exhibits both reduced progesterone responsiveness and hypersensitivity to proinflammatory stimuli mimicking the endometriosis phenotype. Future studies in women with endometriosis should consider whether a heightened inflammatory response within the peritoneal microenvironment contributes to the development and persistence of this disease.

Effect of the Conceptus on Uterine Natural Killer Cell Numbers and Function in the Mouse Uterus During Decidualization

Abstract Uterine natural killer (uNK) cells are the most abundant lymphocytes in the uterus during early pregnancy and play a role in spiral arteriole modifications. In the present study, we investigated whether uNK cell populations differed between mouse decidua and deciduoma. Histochemical staining using the Dolichos biflorus agglutinin (DBA) lectin was used to identify uNK cells and classify their stages of maturation. We found differences in the pattern of localization and density of uNK cells between the decidua and deciduoma at Days 2–4 after the onset of decidualization. The cells were more distributed and the densities were significantly greater in the mesometrial region of the decidua than in the deciduoma. Using double-labeling for DBA lectin binding and bromodeoxyuridine incorporation, we found that the higher number of uNK cells in the decidua was not due to an increase in uNK cell proliferation. Western blot analyses revealed that the increase in uNK cell number was accompanied by significant increases in the levels of interferon gamma (IFNG) and prointerleukin 18 when a conceptus was present. Vascular morphometry revealed that modifications of the spiral arterioles occurred in the mesometrial decidua but not in the deciduoma, which could be attributed to the differences observed in uNK cell number and IFNG production. The present study demonstrates that differences exist in uNK cell populations between the decidua and deciduoma, providing evidence that the conceptus generates signals that regulate uNK cell number and function in the uterus during implantation.

Paracrine Signals from the Mouse Conceptus Are Not Required for the Normal Progression of Decidualization

The purpose of this study was to determine whether the conceptus directs the formation of a tight- and adherens-dependent permeability barrier formed by the primary decidual zone and normal progression of decidual cell differentiation during embryo implantation. Four artificial models of decidualization were used, some apparently more physiological than others. The results show that both the formation of the permeability barrier and decidual cell differentiation of three of the artificial models were quite different from that of pregnant uteri. One artificial model of decidualization, namely pseudopregnant animals receiving concanavalin A-coated Sepharose bead transfers on d 2.5 of pseudopregnancy, better recapitulated the decidual changes that occur in the pregnant uterus undergoing decidualization. This included the formation of a primary decidual zone-like permeability barrier and decidual growth. This model also exhibited similar temporal changes of the expression of genes involved in decidualization that are markers of decidual cell differentiation. Overall, the results of this study indicate that some models of inducing decidualization artificially produce responses that are more similar to those occurring in the pregnant uterus, whereas others are quite different. More importantly, the results suggest that concanavalin A-coated Sepharose beads can provide an equivalent stimulus as the trophectoderm to cause the formation of the primary decidual zone permeability barrier.

β-Catenin (CTNNB1) in the Mouse Uterus During Decidualization and the Potential Role of Two Pathways in Regulating Its Degradation

β-catenin plays a role in cell adhesion and as a transcriptional coactivator. Its levels are regulated in cells by controlling its degradation through ubiquitination by two different E3 ligase complexes. One complex contains β-transducing repeat containing (BTRC) protein, which binds to β-catenin when phosphorylated on specific (S33 and S37) residues, whereas the other involves calcyclin-binding protein (CACYBP). The aim of this study was to determine the localization and levels of total and active (S33/S37-dephosphorylated) β-catenin in the pregnant mouse uteri and those undergoing artificially stimulated decidualization. These two forms of β-catenin were localized almost exclusively to the endometrial epithelia just prior to the onset of implantation. Although this localization continued after the onset of implantation, there were less epithelial cells present in areas of the uterus undergoing decidualization. Rather, there was a progressive increase in β-catenin localization in endometrial stromal cells undergoing decidualization in the anti-mesometrial and, to a lesser extent, in the mesometrial regions. The presence of a conceptus was not required for the changes in localization seen in the pregnant uterus because similar findings were also seen in uteri undergoing artificially stimulated decidualization. Finally, overall levels of total, active (S33 and S37 dephosphorylated), and phosphorylated (S33/S37/T42) β-catenin protein and the steady-state levels of calcyclin-binding protein mRNA changed in the uterus during decidualization. The result of this study shows the changing localization and levels of β-catenin in the mouse uterus during decidualization. Further, the results suggest potential roles for both the BTRC and CACYBP E3 ligase mechanisms of β-catenin ubiquitination in the uterus during decidualization.

Development and prevention of postsurgical adhesions in a chimeric mouse model of experimental endometriosis

OBJECTIVE To examine the impact of a recent surgery on development of endometriosis-related adhesions in a chimeric model and to determine the therapeutic efficacy of pioglitazone (PIO). DESIGN Human endometrial biopsies were maintained in E(2) with or without PIO for 24 h before intraperitoneal injection into immunocompromised mice also treated with or without PIO at multiple time points after peritoneal surgery. The presence and extent of adhesions were examined in animals relative to the initial establishment of experimental endometriosis. SETTING Medical school research center. PATIENT(S) Endometrial biopsies for experimental studies were provided by normally cycling women without a medical history indicative of endometriosis or adhesions. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Examination of the development of endometriosis-related adhesions in an experimental model. RESULT(S) Without therapeutic intervention, injection of E(2)-treated human endometrial tissue into mice near the time of peritoneal surgery resulted in multiple adhesions and extensive endometriotic-like disease. In contrast, PIO treatment reduced adhesive disease and experimental endometriosis related to surgical injury. CONCLUSION(S) The presence of human endometrial tissue fragments in the peritoneal cavity of mice with a recent surgical injury promoted development of both adhesive disease and experimental endometriosis. Targeting inflammation and angiogenesis with PIO therapy limited the development of postsurgical adhesions associated with ectopic endometrial growth.

Do Molecular Signals from the Conceptus Influence Endometrium Decidualization in Rodents

A critical period in establishing pregnancy occurs after the onset of implantation but before placental development. Evidence strongly suggests that abnormalities occurring during this period can result in pregnancy termination or in pre-eclampsia; the latter may lead to small-for-gestational-weight offspring that are likely to be unhealthy. Clearly, events occurring in the endometrium during the implantation process are crucial for proper fetal development and for optimal offspring health. In several mammalian species bi-directional communication between the conceptus and endometrium during implantation is required for successful pregnancy. Although different implantation and placentation modes occur in different mammalian species, common aspects of this bi-directional signaling may exist. The molecular signals from the trophoblast cells of the conceptus, which direct endometrial changes during implantation progression, are well known in some nonrodent species. Currently, we know little about such signaling in rodents during implantation progression, when the endometrium undergoes decidualization. This review focuses on data that support the hypothesis that paracrine signals from the rodent conceptus influence decidualization. Where possible, these findings are compared and contrasted with information currently known in other species that exhibit different implantation modes.

The conceptus increases secreted phosphoprotein 1 gene expression in the mouse uterus during the progression of decidualization mainly due to its effects on uterine natural killer cells

Within the mouse endometrium, secreted phosphoprotein 1 (SPP1) gene expression is mainly expressed in the luminal epithelium and some macrophages around the onset of implantation. However, during the progression of decidualization, it is expressed mainly in the mesometrial decidua. To date, the precise cell types responsible for the expression in the mesometrial decidua has not been absolutely identified. The goal of the present study was to assess the expression of SPP1 in uteri of pregnant mice (decidua) during the progression of decidualization and compared it with those undergoing artificially induced decidualization (deciduoma). Significantly (P<0.05) greater steady-state levels of SPP1 mRNA were seen in the decidua when compared with deciduoma. Further, in the decidua, the majority of the SPP1 protein was localized within a subpopulation of granulated uterine natural killer (uNK) cells but not co-localized to their granules. However, in addition to being localized to uNK cells, SPP1 protein was also detected in another cell type(s) that were not epidermal growth factor-like containing mucin-like hormone receptor-like sequence 1 protein-positive immune cells that are known to be present in the uterus at this time. Finally, decidual SPP1 expression dramatically decreased in uteri of interleukin-15-deficient mice that lack uNK cells. In conclusion, SPP1 expression is greater in the mouse decidua when compared with the deciduoma after the onset of implantation during the progression of decidualization. Finally, uNK cells were found to be the major source of SPP1 in the pregnant uterus during decidualization. SPP1 might play a key role in uNK killer cell functions in the uterus during decidualization.

Dietary fish oil supplementation inhibits formation of endometriosis-associated adhesions in a chimeric mouse model

OBJECTIVE To examine whether dietary fish oil supplementation reduces development of spontaneous endometriosis-associated adhesions using an established model. DESIGN Laboratory-based study. SETTING Medical center research laboratory. PATIENT(S)/ANIMAL(S): Disease-free women of reproductive age and nude mice. INTERVENTION(S) Women were not provided any intervention. Mice were randomized to receive fish oil supplementation or control diet. MAIN OUTCOME MEASURE(S) Experimental endometriosis was established in mice via injection of human endometrial tissue within 16 hours of ovariectomy. Mice were provided standard or menhaden fish oil-supplemented diets for ≥ 2 weeks before initiation of experimental endometriosis and until killing them 1 week later. At necropsy, mice were examined for the presence and extent of adhesions and endometriotic-like lesions. Tissues were excised and morphologically characterized. RESULT(S) Adhesions/lesions were reduced in mice provided with dietary fish oil compared with control animals. Leukocytes were more numerous within the adhesions/lesions of the mice maintained on the standard diet compared with animals provided with fish oil. As indicated by staining intensity, collagen deposition was greater at adhesion sites within control mice compared with fish oil-supplemented animals. CONCLUSION(S) Wound-healing associated with surgery created an inflammatory peritoneal microenvironment that promoted the development of both experimental endometriosis and adhesions in a murine model. Targeting excessive inflammation with fish oil may be an effective adjuvant therapy to reduce the development of postsurgical adhesions related to endometriosis.

High-Throughput Screening of Myometrial Calcium-Mobilization to Identify Modulators of Uterine Contractility.

The uterine myometrium (UT-myo) is a therapeutic target for preterm labor, labor induction, and postpartum hemorrhage. Stimulation of intracellular Ca2+-release in UT-myo cells by oxytocin is a final pathway controlling myometrial contractions. The goal of this study was to develop a dual-addition assay for high-throughput screening of small molecular compounds, which could regulate Ca2+-mobilization in UT-myo cells, and hence, myometrial contractions. Primary murine UT-myo cells in 384-well plates were loaded with a Ca2+-sensitive fluorescent probe, and then screened for inducers of Ca2+-mobilization and inhibitors of oxytocin-induced Ca2+-mobilization. The assay exhibited robust screening statistics (Z´ = 0.73), DMSO-tolerance, and was validated for high-throughput screening against 2,727 small molecules from the Spectrum, NIH Clinical I and II collections of well-annotated compounds. The screen revealed a hit-rate of 1.80% for agonist and 1.39% for antagonist compounds. Concentration-dependent responses of hit-compounds demonstrated an EC50 less than 10μM for 21 hit-antagonist compounds, compared to only 7 hit-agonist compounds. Subsequent studies focused on hit-antagonist compounds. Based on the percent inhibition and functional annotation analyses, we selected 4 confirmed hit-antagonist compounds (benzbromarone, dipyridamole, fenoterol hydrobromide and nisoldipine) for further analysis. Using an ex vivo isometric contractility assay, each compound significantly inhibited uterine contractility, at different potencies (IC50). Overall, these results demonstrate for the first time that high-throughput small-molecules screening of myometrial Ca2+-mobilization is an ideal primary approach for discovering modulators of uterine contractility.

In vivo Raman spectral analysis of impaired cervical remodeling in a mouse model of delayed parturition

Monitoring cervical structure and composition during pregnancy has high potential for prediction of preterm birth (PTB), a problem affecting 15 million newborns annually. We use in vivo Raman spectroscopy, a label-free, light-based method that provides a molecular fingerprint to non-invasively investigate normal and impaired cervical remodeling. Prostaglandins stimulate uterine contractions and are clinically used for cervical ripening during pregnancy. Deletion of cyclooxygenase-1 (Cox-1), an enzyme involved in production of these prostaglandins, results in delayed parturition in mice. Contrary to expectation, Cox-1 null mice displayed normal uterine contractility; therefore, this study sought to determine whether cervical changes could explain the parturition differences in Cox-1 null mice and gestation-matched wild type (WT) controls. Raman spectral changes related to extracellular matrix proteins, lipids, and nucleic acids were tracked over pregnancy and found to be significantly delayed in Cox-1 null mice at term. A cervical basis for the parturition delay was confirmed by other ex vivo tests including decreased tissue distensibility, hydration, and elevated progesterone levels in the Cox-1 null mice at term. In conclusion, in vivo Raman spectroscopy non-invasively detected abnormal remodeling in the Cox-1 null mouse, and clearly demonstrated that the cervix plays a key role in their delayed parturition.