Hematogenous Dissemination of Mesenchymal Stem Cells from Endometriosis.

Endometriosis is ectopic growth of endometrial tissue traditionally thought to arise through retrograde menstruation. We aimed to determine if cells derived from endometriosis could enter vascular circulation and lead to hematogenous dissemination. Experimental endometriosis was established by transplanting endometrial tissue from DsRed+ mice into the peritoneal cavity of DsRed- mice. Using flow cytometry, we identified DsRed+ cells in blood of animals with endometriosis. The circulating donor cells expressed CXCR4 and mesenchymal stem cell (MSC) biomarkers, but not hematopoietic stem cell markers. Nearly all the circulating endometrial stem cells originated from endometriosis rather than from the uterus. Cells expressing DsRed, CXCR4, and MSCs markers were identified in the peritoneal wall and surrounding vessels of recipient mice, contributing to both endometriosis and angiogenesis. Cells originating in endometriosis lesions migrated and implanted in lung tissue and displayed makers of differentiation, indicating retained multipotency. In vitro these cells demonstrated multipotency and were able to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Endometriosis lesions also expressed high levels of CXCL12, the CXCR4 receptor ligand. Serum CXCL12 levels were greater than in sham control mice. In humans with endometriosis, serum CXCL12 levels were significantly higher than controls, suggesting that the CXCL12/CXCR4 axis is operational in women with spontaneous endometriosis as well. Stem cells, rather than differentiated cells from endometriosis, enter the circulation in response to CXCL12. We identify an endometriosis-derived stem cell population, a potential mechanism of dissemination of this disease and a potential target for treatment of endometriosis. Stem Cells 2018;36:881-890.

Treatment with bazedoxifene and conjugated estrogens results in regression of endometriosis in a murine model.

Bazedoxifene (BZA), a selective estrogen receptor modulator (SERM), inhibits the action of estrogens on endometrial proliferation. Here, we evaluate the effect of a tissue-selective estrogen complex (TSEC) containing BZA and conjugated estrogens (CE) on ectopic endometrial lesions in a mouse model of endometriosis. Experimental endometriosis was created in 60 female CD-1 mice. The mice were randomly divided into 10 groups that received varying doses of either BZA (1, 2, 3, or 5 mg/kg/day), BZA (1, 2, 3, or 5 mg/kg/day) in combination with CE (3 mg/kg/day), CE treatment alone (3 mg/kg/day), or vehicle control for 8 wk. Treatment with BZA alone or the TSEC containing BZA/CE led to a decrease in endometriotic lesion size compared to controls. The mean surface area of the untreated lesions was 19.6 mm(2). Treatment with BZA or BZA/CE resulted in reduced lesion size (to 8.8 and 7.8 mm(2), respectively). No significant difference was found in lesion size between the BZA and BZA/CE treatment groups or between different doses of either treatment. Ovarian cyst formation was not evident in the treated groups. Treatment with the TSEC containing higher BZA dosages (3 and 5 mg/kg/day) led to significantly lower levels of estrogen receptor (Esr1) mRNA expression compared to the control treatment. No differences were observed in expression of progesterone receptor (Pgr). Immunohistochemical analysis also demonstrated a decrease in ESR protein. The combination of CE and BZA may prove to be a novel treatment option for endometriosis.

Bone marrow mesenchymal stem cell-derived exosomes shuttle microRNAs to endometrial stromal fibroblasts that promote tissue proliferation /regeneration/ and inhibit differentiation.

BACKGROUND: Human bone marrow-derived stem cells (hBMDSCs) are well characterized mediators of tissue repair and regeneration. An increasing body of evidence indicates that these cells exert their therapeutic effects largely through their paracrine actions rather than clonal expansion and differentiation. Here we studied the role of microRNAs (miRNAs) present in extracellular vesicles (EVs) from hBMDSCs in tissue regeneration and cell differentiation targeting endometrial stromal fibroblasts (eSF). METHODS: Extracellular vesicles (EVs) are isolated from hBMDSCs, characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) techniques. Extracted total RNA from EVs was subjected to RNA seq analysis. Transfection and decidualization studies were carried out in endometrial stromal fibroblasts (eSF). Gene expression was analyzed by qRTPCR. Unpaired t-test with Welch's correction was used for data analysis between two groups. RESULTS: We identified several microRNAs (miRNAs) that were highly expressed, including miR-21-5p, miR-100-5p, miR-143-3p and let7. MiR-21 is associated with several signaling pathways involved in tissue regeneration, quiescence, cellular senescence, and fibrosis. Both miR-100-5p and miR-143-3p promoted cell proliferation. MiR-100-5p specifically promoted regenerative processes by upregulating TGF-ß3, VEGFA, MMP7, and HGF. MiR-100-5p blocked differentiation or decidualization as evidenced by morphologic changes and downregulation of decidualization mediators including HOXA10, IGFBP1, PRL, PR-B, and PR. CONCLUSION: EVs delivered to tissues by hBMDSCs contain specific miRNAs that prevent terminal differentiation and drive repair and regeneration. Delivery of microRNAs is a novel treatment paradigm with the potential to replace BMDSCs in cell-free regenerative therapies.

The immunoconjugate "icon" targets aberrantly expressed endothelial tissue factor causing regression of endometriosis.

Endometriosis is a major cause of chronic pain, infertility, medical and surgical interventions, and health care expenditures. Tissue factor (TF), the primary initiator of coagulation and a modulator of angiogenesis, is not normally expressed by the endothelium; however, prior studies have demonstrated that both blood vessels in solid tumors and choroidal tissue in macular degeneration express endothelial TF. The present study describes the anomalous expression of TF by endothelial cells in endometriotic lesions. The immunoconjugate molecule (Icon), which binds with high affinity and specificity to this aberrant endothelial TF, has been shown to induce a cytolytic immune response that eradicates tumor and choroidal blood vessels. Using an athymic mouse model of endometriosis, we now report that Icon largely destroys endometriotic implants by vascular disruption without apparent toxicity, reduced fertility, or subsequent teratogenic effects. Unlike antiangiogenic treatments that can only target developing angiogenesis, Icon eliminates pre-existing pathological vessels. Thus, Icon could serve as a novel, nontoxic, fertility-preserving, and effective treatment for endometriosis.

Decidual hemostasis, inflammation, and angiogenesis in pre-eclampsia.

Invasion of the decidua by extravillous trophoblasts (EVTs) is accompanied by thrombin generation from decidual cell (DC)-expressed tissue factor (TF). This TF protects against hemorrhage as EVTs breach capillaries and subsequently invade and remodel spiral arteries and arterioles. Pre-eclampsia (P-EC) is the world's leading cause of fetal and maternal morbidity and mortality. It is associated with decidual hemorrhage and maternal thrombophilias, which form excess thrombin from DCs, and with maternal infections and other inflammatory conditions that are associated with excess expression of the proinflammatory cytokines interleukin (IL)-1 ß and tumor necrosis factor (TNF) α. In human first-trimester leukocyte-free DCs, (1) thrombin enhances expression of soluble fms-like tyrosine kinase-1 (sFlt-1), a potent inhibitor of angiogenesis; (2) thrombin, IL-1ß and TNF-α increase monocyte-recruiting chemokine expression leading to a macrophage excess in the pre-eclamptic decidua. The pathogenesis of P-EC likely stems from shallow EVT invasion leading to impaired decidual vascular remodeling. The resulting reduced uteroplacental blood flow is associated with a hypoxic placenta, which appears to secrete excess sFlt-1 into the maternal plasma. A regulatory role for DCs in vascular remodeling is indicated because impaired decidual vascular remodeling could stem from an aberrant local antiangiogenic milieu elicited by excess sFlt-1 and/or macrophage-inhibited EVT decidual invasion.

Novel insights into molecular mechanisms of abruption-induced preterm birth.

Preterm birth (PTB) complicates more than 12% of all deliveries. Despite significant research, the aetiology of most cases of PTB remains elusive. Two major antecedents of PTB, intra-amniotic infection and decidual haemorrhage (abruption), can exhibit dissimilar demographic and genetic predispositions, despite sharing common molecular and cellular pathways. The use of high-throughput, high-dimensional technologies reveals substantial crosstalk between the coagulation and inflammation pathways. Tissue factor, thrombin and cytokines are key mediators of this crosstalk. Abruptions are associated with excess thrombin generated from decidual-cell-expressed tissue factor. Although thrombin is a primary mediator of the coagulation cascade, it can also promote inflammation-associated PTB by enhancing expression of matrix metalloproteinase and neutrophil-chemoattracting and -activating chemokines. Here, we provide novel insights into the molecular mechanisms and pathways leading to PTB in the setting of placental abruption.

Preimplantation factor promotes first trimester trophoblast invasion.

OBJECTIVE: Preimplantation factor is a novel embryo-derived peptide that influences key processes in early pregnancy implantation, including immunity, adhesion, remodeling, and apoptosis. Herein, we explore the effects of synthetic preimplantation factor on trophoblast invasion. STUDY DESIGN: Invasion patterns of immortalized cultured HTR-8 trophoblast cells were analyzed through Matrigel extracellular matrix ± synthetic preimplantation factor (25-100 nM) in a transwell assay. Effects were compared with epidermal growth factor 10 µg/mL, scrambled aminoacid sequence of preimplantation factor, or media alone as controls. RESULTS: Synthetic preimplantation factor enhances trophoblast invasion at physiologic doses (at 50 nM, 260%; 95% confidence interval [CI], 174-346%; P = .05; 100 nM ,178%; 95% CI, 170-184%; P < .02), compared with scrambled amnioacid sequence preimplantation factor or control media. Epidermal growth factor added to synthetic preimplantation factor does not further enhance trophoblast invasion (synthetic preimplantation factor 50 nM + epidermal growth factor, 238%; 95% CI, 237-239%; P < .03; synthetic preimplantation factor 100 nM + epidermal growth factor 269%; 95% CI, 265-273%; P < .04). CONCLUSION: Preimplantation factor should be further investigated as it shows a potential preventative or therapeutic role for pregnancy complications associated with inadequate trophoblast invasion.

A genomic and proteomic investigation of the impact of preimplantation factor on human decidual cells.

OBJECTIVE: Preimplantation factor (PIF) is a novel, 15 amino acid peptide, secreted by viable embryos. This study aims to elucidate PIF's effects in human endometrial stromal cells (HESC) decidualized by estrogen and progestin, which mimics the preimplantation milieu, and in first-trimester decidua cultures (FTDC). STUDY DESIGN: HESC or FTDC were incubated with 100 nmol/L synthetic PIF or vehicle control. Global gene expression was analyzed using microarray and pathway analysis. Proteins were analyzed using quantitative mass spectrometry, and PIF binding by protein array. RESULTS: Gene and proteomic analysis demonstrate that PIF affects immune, adhesion, and apoptotic pathways. Significant up-regulation in HESC (fold change) include: nuclear factor-k-beta activation via interleukin-1 receptor-associated kinase binding protein 1 (53); Toll-like receptor 5 (9); FK506 binding protein 15, 133kDa protein (2.3); and Down syndrome cell adhesion molecule like 1 (16). B-cell lymphoma protein 2 was down-regulated in HESC (21.1) and FTDC (27.1). Protein array demonstrates PIF interaction with intracellular targets insulin-degrading enzyme and beta-K+ channels. CONCLUSION: PIF displays essential multitargeted effects, of regulating immunity, promoting embryo-decidual adhesion, and regulating adaptive apoptotic processes.

Preeclampsia-related inflammatory cytokines regulate interleukin-6 expression in human decidual cells.

Preeclampsia, a common pregnancy disorder associated with an increase in systemic inflammation, is the leading cause of maternal and fetal morbidity and mortality throughout the world. It is associated with shallow extravillous trophoblast invasion of the decidua, leading to uteroplacental blood flow that is inadequate for the developing fetal-placental unit. In preeclamptic women, interleukin-6 (IL-6) levels in plasma, but not placenta, are elevated, prompting evaluation of the decidua as a potential source of this excess, circulating IL-6. The current study found significantly higher immunohistochemical staining for IL-6 in decidual cells from preeclamptic versus preterm, gestational age-matched control placentas. Pro-inflammatory cytokines associated with the genesis of preeclampsia (i.e., tumor necrosis factor-alpha and interleukin-1beta) enhanced IL-6 mRNA levels and increased secreted IL-6 levels in first trimester leukocyte-free decidual cell incubations, as measured by real time quantitative RT-PCR, ELISA, and Western blotting. Therefore, decidual cell-derived IL-6 may contribute to excess circulating IL-6 levels that can promote both endothelial cell dysfunction (and subsequent vascular dysfunction) and the pathogenesis of preeclampsia whereas locally elevated IL-6 levels may contribute to an excess of decidual macrophages implicated in shallow extravillous trophoblast invasion of the decidua.

Micrometastasis of endometriosis to distant organs in a murine model.

Endometriosis is an inflammatory gynecological disorder among reproductive-aged women caused by the engraftment and proliferation of endometrial cells outside the uterus, most commonly in the pelvis. It is thought that the disease arises primarily from retrograde menstruation where cells from the endometrium travel through the fallopian tubes to the peritoneal cavity. However, migration of endometriosis-derived cells to distant organs outside of the peritoneal cavity have not been explored. In the present study, we developed and validated a mouse model of disseminated endometriosis using syngeneic DsRed endometrial tissue introduced into the peritoneum of immunocompetent mice. Flow cytometry and immunofluorescence analysis, demonstrated the presence of endometriosis-derived cells in multiple organs (including lung, spleen, liver and brain) in the murine endometriosis model. Immunostaining revealed the presence of DsRed+/CD45- cells in brain, liver and lung. Engraftment occurred in all experimental animals examined. Cells from endometriotic lesions are capable of migration to and engraftment of multiple organs outside of the peritoneal cavity. Micrometastasis of endometriosis is a novel and frequent phenomenon. These data suggest that widespread dissemination of endometriosis may be common, clinically unrecognized and contribute to the diffuse clinical manifestations of this disease.

Endometriosis and tissue factor.

Tissue factor (TF), is a cellular receptor that binds the ligand factor VII/VIIa to initiate the blood coagulation cascade. In addition to its role as the initiator of the hemostatic cascade, TF is known to be involved in angiogenesis via an interaction with factor VIIa and protease-activated receptor-2 (PAR-2). In this article we review previous studies from our laboratory demonstrating that the pattern and level of TF expression is altered in multiple cell types derived from eutopic and ectopic endometrium from women with endometriosis compared with normal endometrium. We posit that the inflammatory environment that occurs in ectopic and eutopic endometrium from patients with disease results in high TF expression that in turn, signals via PAR-2 to further produce inflammatory cytokine or chemokine production and macrophage recruitment. Thus, our studies suggest that TF might be an ideal target for therapeutic intervention in endometriosis.

Decidual cell-expressed tissue factor in human pregnancy and its involvement in hemostasis and preeclampsia-related angiogenesis.

During extravascular trophoblast (EVT) invasion of the decidua, thrombin generated from decidual cell-expressed tissue factor (TF) forms a "hemostatic envelope" that protects against hemorrhage during the initial breaching of capillaries by EVTs and subsequent invasion and remodeling of the spiral arteries and arterioles. Preeclampsia, the world's leading cause of fetal and maternal morbidity and mortality, stems from shallow trophoblast invasion leading to incomplete vascular remodeling that impairs uteroplacental blood flow. A considerable subset of cases of preeclampsia is associated with decidual hemorrhage and maternal thrombophilias, which form excess thrombin from decidual cell-expressed TF. Thrombin affects several cell functions by binding to protease-activated receptors. In first-trimester decidual cells, thrombin enhances expression of sFlt-1, which can block the angiogenic effects of vascular endothelial growth factor (VEGF) and placental growth factor. By contrast, thrombin does not affect decidual cell VEGF expression. Thrombin-enhanced sFlt-1 expression by decidual cells, the predominant cell type encountered by invading cytotrophoblasts, could promote preeclampsia by interfering with angiogenesis-dependent vascular remodeling to reduce uteroplacental blood flow.

The placental syncytium and the pathophysiology of preeclampsia and intrauterine growth restriction: a novel assay to assess syncytial protein expression.

Preeclampsia is associated with an increased release of factors from the placental syncytium into maternal blood, including the antiangiogenic factors soluble fms-like tyrosine kinase-1 and soluble endoglin, the antifibrinolytic factor plasminogen activator inhibitor-1, prostanoids, lipoperoxides, cytokines, and microparticles. These factors are suggested to promote maternal endothelium dysfunction and are associated with placental damage in pregnancies also complicated with intrauterine growth restriction (IUGR). In this report, we briefly describe the interaction of syncytial factors with hypoxia, reactive oxygen species, and apoptosis in the pathophysiology of preeclampsia and IUGR. Given the critical role of the syncytium in these complications of pregnancy, we also present a novel methodology in which laser capture microdissection followed by Western blotting is used to assess levels of syncytial Fas ligand, a key protein in the apoptotic cascade.

The CXL12/CXCR4/CXCR7 axis in female reproductive tract disease: Review.

Initial studies on the chemokine stromal derived factor 1 (now referred to as CXCL12) were proposed to be enhanced in several diseases including those which affect the female reproductive tract. These include endometriosis, Asherman's syndrome, endometrial cancers, and ovarian cancers. Additionally, recent studies from our laboratory suggest that CXCL12 signaling is involved in leiomyomas (fibroids). These diseases present an inflammatory/hypoxic environment which further promotes pathology. At first, studies focused on signaling by CXCL12 via its well-known receptor, CXCR4. However, the discovery of CXCR7 as another receptor for CXCL12 with rather high binding affinity and recent reports about its involvement in endometrial disease and cancer progression has questioned the potential of "selective blockade"' of CXCR4 to treat these ailments. This review will focus on the signaling and effects of the potent chemokine CXCL12, and its long-known G protein-coupled receptor CXCR4, as well as the alternate receptor CXCR7 on the female reproductive tract and related diseases such as endometriosis, Asherman's syndrome, leiomyomas, endometrial cancer, and ovarian cancer. Although several other mechanisms are inherent to these diseases such as gene mutations, differential expression of miRNAs and epigenetics, for this review, we will focus on the CXCL12/CXCR4/CXCR7 axis as a novel target.

Icon immunoconjugate treatment results in regression of red lesions in a non-human primate (Papio anubis) model of endometriosis.

Endometriosis is a common condition in reproductive-aged women characterized by ectopic endometrial lesions of varied appearance, including red, white, blue, black or powder burn coloration, which contribute to chronic pain and infertility. The immunoconjugate molecule (Icon) targets Tissue Factor, a transmembrane receptor for Factor VII/VIIa that is aberrantly expressed in the endothelium supporting ectopic endometrial tissue. Icon has been shown to cause regression of endometriosis in a murine model of disease but prior to this study had not been tested in non-human primates. This study evaluated Icon as a novel treatment for endometriosis in non-human primates (Papio anubis) using an adenoviral vector (AdIcon) delivery system. Female baboons (n = 15) underwent surgical induction of endometriosis. After laparoscopic confirmation of endometriosis lesions 6-weeks post-surgery, the treatment group (n = 7) received weekly intraperitoneal injections of viral particles carrying the sequence for Icon, resulting in expression of the protein, while the control group (n = 8) received no treatment. Icon preferentially reduced the number and volume of red vascularized lesions. Icon may present a novel treatment for endometriosis by degrading red vascularized lesions, likely by targeting tissue factor aberrantly expressed in the lesion vasculature.

Differential cell-specific modulation of HOXA10 by estrogen and specificity protein 1 response elements.

CONTEXT: HOX genes are highly evolutionarily conserved regulators of embryonic development. HOXA10 also regulates differentiation of the adult reproductive tract and mammary gland in response to sex steroids. OBJECTIVE: We recently identified two HOXA10 estrogen response elements (EREs). Here we demonstrate that estrogen-responsive HOXA10 expression is cell type specific. DESIGN AND SETTING: We conducted an in vitro study at an academic medical center. MAIN OUTCOME MEASURE: Reporter assay, gel shift assays (electrophoretic mobility shift assay), and immunohistochemistry were done. RESULTS: The HOXA10 EREs and a specificity protein 1 (Sp1) binding site differentially drive the cell-type-specific E2 response. In electrophoretic mobility shift assays, both estrogen receptor-alpha and -beta bound both EREs but not the Sp1 site. In reporter assays, both EREs and the Sp1 site demonstrated estrogen responsiveness and tissue specificity; transiently transfected uterine Ishikawa cells or breast MCF-7 cells showed differential responses to E2 treatment. Each response element (Sp1, ERE1, and ERE2) drove distinct differential expression in each cell type. Sp1 protein was expressed in a menstrual-cycle stage-specific expression pattern in endometrium, first expressed in perivascular cells. CONCLUSIONS: Tissue specificity inherent to a regulatory element as well as differential cellular expression of transcription factors imparts differential tissue-specific estrogen responsiveness.

Thrombin activation of endometrial endothelial cells: a possible role in intrauterine growth restriction.

Preeclampsia (PE), intrauterine growth restriction (IUGR) and abruption with or without fetal loss are associated with reduced uteroplacental blood flow, decidual vasculopathy, endothelial cell dysfunction, thrombosis, inflammation and hemorrhage. Our hypothesis is that reduced uteroplacental blood flow causes focal decidual hypoxia that generates vascular endothelial growth factor (VEGF). The latter acts directly on decidual endothelial cells to induce aberrant expression of tissue factor (TF), the primary initiator of coagulation. This in turn generates thrombin that induces: i) further TF expression; and ii) inflammatory cytokines. Both VEGF and TF induce aberrant angiogenesis-vessel maintenance reflected by endothelial cell fenestrations and induction of a prothrombotic surface causing both the decidual hemorrhage (i.e. abruption) and thrombosis (i.e. uteroplacental vascular insufficiency) observed in these adverse pregnancy outcomes. This novel hypothesis is supported by our finding of TF expression in decidual endothelium of pregnancies complicated by IUGR and/or fetal loss. Moreover, treatment of cultured endometrial endothelial cells with VEGF or thrombin induces TF protein and mRNA expression. Quantitative RT-PCR analysis indicates that thrombin enhances (>10-fold) the output of diverse inflammatory cytokines in these cultures. The greatest effect (>2-log) was seen on macrophage inflammatory protein 3alpha (MIP3alpha). In vitro, thrombin results in endometrial endothelial cell aggregations and changes in the apoptotic pathway. Thus, we postulate that reductions in uteroplacental flow initiate a cascade of molecular effects leading to hypoxia, thrombosis, inflammation, and endothelial cell dysfunction resulting in untoward pregnancy outcomes.

Thrombin regulates monocyte chemoattractant protein-1 expression in human first trimester and term decidual cells.

OBJECTIVE: Excess decidual macrophage infiltration has been linked to preeclampsia and a failure of endovascular trophoblast invasion. Severe preeclampsia with shallow placentation has also been linked to acquired and inherited maternal thrombophilias and recurrent decidual hemorrhage, which generates thrombin from decidual cell-expressed tissue factor. Therefore, the current study evaluated whether thrombin affects monocyte chemoattractant protein-1 (MCP-1) expression in stromal cells that are derived from cycling and gestational endometrium. STUDY DESIGN: Stromal cells that are isolated from cycling endometrium and first trimester and term decidua were grown to confluence, treated for 7 days with 10(-8) mol/L estradiol (E2) + 10(-7) mol/L medroxyprogesterone acetate (MPA), then switched to a serum-free medium that contained the corresponding steroids +/- thrombin. MCP-1 protein release was measured by enzyme-linked immunosorbent assay and Western blot; MCP-1 messenger RNA levels were assessed by real-time quantitative reverse transcription-polymerase chain reaction. RESULTS: Secreted MCP-1 levels were not significantly different in stromal or decidual cell cultures that were incubated with E2 or with E2 + MPA. Thrombin increased immunoreactive MCP-1 expression in a dose-response fashion in first trimester and term decidual cells but not in endometrial stromal cells. Thrombin-induced MCP-1 protein output was unaffected by MPA but was abrogated by incubation with the thrombin inhibitor, hirudin. Unexpectedly, thrombin-enhanced MCP-1 protein expression was unaccompanied by corresponding changes in steady state MCP-1 messenger RNA levels, which suggests its effects were posttranslational. CONCLUSION: MCP-1 protein expression is up regulated by thrombin in decidual cells across gestation, but not in stromal cells from predecidualized cycling endometrium.

Intercellular adhesion molecule-1 expression in human endometrium: implications for long term progestin only contraception.

BACKGROUND: Neutrophils infiltrate the endometrium pre-menstrually and after long-term progestin only-contraceptive (LTPOC) treatment. Trafficking of neutrophils involves endothelial cell-expressed intercellular adhesion molecule (ICAM-1). Previous studies observed that ICAM-1 was immunolocalized to the endothelium of endometrial specimens across the menstrual cycle, but disagreed as to whether extra-endothelial cell types express ICAM-1 and whether ICAM-1 expression varies across the menstrual cycle. METHODS: Endometrial biopsies were obtained from women across the menstrual cycle and from those on LTPOC treatment (either Mirena or Norplant). The biopsies were formalin-fixed and paraffin-embedded with subsequent immunohistochemical staining for ICAM-1. RESULTS: The current study found prominent ICAM-1 staining in the endometrial endothelium that was of equivalent intensity in different blood vessel types irrespective of the steroidal or inflammatory endometrial milieu across the menstrual cycle and during LTPOC therapy. Unlike the endothelial cells, the glands were negative and the stromal cells were weakly positive for ICAM immunostaining. CONCLUSION: The results of the current study suggest that altered expression of ICAM-1 by endothelial cells does not account for the influx of neutrophils into the premenstrual and LTPOC-derived endometrium. Such neutrophil infiltration may depend on altered expression of neutrophil chemoattractants.

Tibolone exerts progestational inhibition of matrix metalloproteinase expression in human endometrial stromal cells.

Tibolone and its metabolites were evaluated on matrix metalloproteinase (MMP) expression in human endometrial stromal cells (HESCs) under the hypothesis that these steroids would act as progestins on MMP-1, -2, and -3 expression. After 7 days of priming and 24h experimental incubation of confluent cultured HESCs, 10(-7) M medroxyprogesterone acetate (P) reduced MMP-1 to 49+/-34% (p<0.05) and MMP-3 to 33+/-22% of basal levels (mean+/-S.E.M., p<0.05, n=5). Although HESCs were unaffected by 10(-8) M estradiol (E), E+P reduced MMP-1 and MMP-3 levels an additional 2.5-fold from P alone. Tibolone and Delta-4 tibolone were equivalent to E+P in inhibiting MMP-1 and MMP-3 output, whereas 10(-6)M of 3alpha-OH or 3beta-OH tibolone was required to elicit significant inhibition of both MMPs (p<0.05). By contrast, none of the treatments affected HESC-secreted MMP-2 output. The ELISA results were confirmed by Western blotting and by substrate gel zymography. Quantitative RT-PCR demonstrated corresponding changes in MMP-1 and MMP-3 mRNA levels. Inhibition of MMP-1 and MMP-3 expression by tibolone and Delta-4 tibolone is consistent with the metabolism of tibolone to Delta-4 tibolone, and subsequent binding of Delta-4 tibolone to the progesterone receptor. Since 3alpha-OH and 3beta-OH tibolone bind exclusively to the estrogen receptor, their inhibition of MMP-1 and MMP-3 suggests metabolism by HESCs to Delta-4 tibolone. These observations help to explain the paradox that the endometrium becomes atrophic after tibolone administration despite the persistence in the circulation of 3alpha-OH and 3beta-OH tibolone, but not tibolone or Delta-4 tibolone.