RESUMO
BACKGROUND: Evidence suggests that eutopic endometrium from women with endometriosis (US-E) has intrinsic functional anomalies compared with women without endometriosis (US-C). We hypothesized that differences in endometrial haptoglobin (eHp) mRNA and protein levels exist between eutopic endometrium from US-E and US-C and that inflammatory mediators may be involved. METHODS: Endometrial stromal cells and tissue explants from US-E (n = 18) and US-C (n = 18) were cultured (24 h/48 h for cells/explants) with interleukin (IL)-1alpha, -1beta, -6, -8 or tumor necrosis factor-alpha (TNF-alpha) at 0-100 ng/ml. eHp protein in media and mRNA levels were quantified by enzyme-linked immunosorbent assay and quantitative PCR. RESULTS: In eutopic endometrial stromal cells from US-E, IL-1beta, IL-6 and TNF-alpha (10 ng/ml) increased eHp mRNA levels (P = 0.002, P < 0.001 and P < 0.001, respectively) and eHp protein (P = 0.023, 0.031 and 0.006, respectively) versus control. In endometrial tissues from US-E, IL-1beta, IL-6 and TNF-alpha increased eHp mRNA (P < 0.001, P = 0.017 and P < 0.001, respectively) and eHp protein (P < 0.001, P = 0.007 and 0.039, respectively) versus control. IL-1alpha and IL-8 had small or no effects on isolated endometrial cells or tissues. In US-C, IL-1beta, IL-8 and TNF-alpha each reduced eHp mRNA in endometrial stromal cells (all P < 0.001) versus control; IL-1alpha and IL-6 had no effect. eHp mRNA increased in endometrial tissues from US-C in response to IL-1beta (P = 0.008), IL-6 (P = 0.015) and TNF-alpha (P = 0.031) versus control; IL-1alpha or IL-8 had no effect. CONCLUSIONS: Endometrium from US-E differentially responds to specific inflammatory cytokines by production of eHp. We propose that up-regulation of endometrial eHp by inflammatory mediators disrupts normal endometrial function and may facilitate the pathogenesis of endometriosis.
Assuntos
Citocinas/farmacologia , Endometriose/genética , Endometriose/metabolismo , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Haptoglobinas/biossíntese , Haptoglobinas/genética , Citocinas/metabolismo , Endometriose/etiologia , Feminino , Humanos , Técnicas In Vitro , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/farmacologia , Interleucina-1alfa/farmacologia , Interleucina-1beta/farmacologia , Interleucina-6/farmacologia , Interleucina-8/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Based on our previous observation that peritoneal endometriotic (PE) lesions synthesize in vivo substantially more haptoglobin (Hp) than related eutopic tissues, we hypothesized that this increase in Hp production was due to endometrial-peritoneal interactions. As interleukin-6 (IL-6) stimulates Hp in other tissues and is produced by endometrial cells, we tested our hypothesis by evaluating the effects of IL-6 on Hp production by PE cells, normal peritoneal (P) cells, and eutopic endometrial cells from women with (UE-E) and without endometriosis (UE-C) using semiquantitative RT-PCR and enzyme-linked immunoabsorbent assay. Endogenous production of IL-6 was also assessed. Treatment with human recombinant IL-6 and dexamethasone significantly increased Hp production by P or PE cells in a dose- and time-dependent manner (P < 0.05). Hp messenger ribonucleic acid was not detected in UE-E and UE-C cells in the absence or presence of IL-6 and dexamethasone. PE and UE-E cells expressed significantly more IL-6 messenger ribonucleic acid than P and UE-C cells (P < 0.05). Moreover, UE-E cells secreted 6 times more IL-6 protein than UE-C cells (P < 0.05). These findings support our hypothesis and suggest a novel endometrial-peritoneal interaction whereby locally synthesized IL-6 and Hp may participate in the establishment and persistence of peritoneal endometriosis.
Assuntos
Endometriose/metabolismo , Haptoglobinas/biossíntese , Interleucina-6/farmacologia , Peritônio/metabolismo , Células Cultivadas , Meios de Cultura/farmacologia , Endometriose/patologia , Endometriose/fisiopatologia , Endométrio/patologia , Endométrio/fisiopatologia , Ensaio de Imunoadsorção Enzimática , Feminino , Haptoglobinas/genética , Humanos , Interleucina-6/biossíntese , Peritônio/patologia , Peritônio/fisiopatologia , RNA Mensageiro/metabolismo , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Because so much medical and media attention has been drawn to the alleged benefits of dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS), it is important to evaluate the effects of replacement therapy objectively using double blind, cross-over, randomized research methodology. In this 9-month study, healthy older men (n = 39) received replacement dose DHEA. Lean body mass, blood hematology, chemistry and endocrine values, as well as urological and psychological data were measured. Data showed some mild and temporary, but significant, changes during oral use of 100 mg DHEA for 3 months compared with placebo taken for 3 months. Body composition did not change during the 6 months of treatment, nor did any urological parameters. Concomitant with the endocrine changes, some small but, significant, variations in blood values (blood urea nitrogen, creatinine, uric acid, alanine transaminase, cholesterol, high density lipoprotein, and potassium) were found. After cessation of DHEA and placebo, followed by 3 months of no treatment, all values previously found to be altered returned to entry baseline. Well publicized effects of the drug reported by others, such as a sense of well-being or improved sexual function, were not found in this study.
Assuntos
Sulfato de Desidroepiandrosterona/uso terapêutico , Desidroepiandrosterona/uso terapêutico , Terapia de Reposição Hormonal , Atividades Cotidianas , Idoso , Envelhecimento , Composição Corporal/efeitos dos fármacos , Estudos Cross-Over , Dieta , Método Duplo-Cego , Humanos , Lipídeos/sangue , Masculino , Comportamento Sexual , Equilíbrio Hidroeletrolítico/efeitos dos fármacos , Equilíbrio Hidroeletrolítico/fisiologiaRESUMO
De novo synthesized endometriosis protein-II (ENDO-II; M(r) 28,000 to 32,000; pI 7.0 to 9.0) was partially purified from rat endometriotic tissue culture media using affinity chromatography and two-dimensional SDS-PAGE. The protein was electrophoretically transferred to polyvinyl difluoride membranes which were stained with Coomassie blue R-250. The stained protein corresponding to ENDO-II (M(r) 28,000 to 32,000; pI 7.0 to 9.0) was cut from the membranes for amino acid sequencing. Partial amino acid sequence was determined by automated Edman degradation using a gas phase sequencer and phenylthiohydantoin analyzer. Sequence analysis of ENDO-II yielded 25 residues: C S C A P T H P Q T A F C N S D L V I R K F M G. Comparison to sequence databanks demonstrated significant homology with rat (100%) and human (84%) tissue inhibitor of metalloproteinases-1 (TIMP-1). Western blot analysis using a TIMP-1 antibody confirmed amino acid sequence analysis. In conclusion, ENDO-II shares sequence homology with TIMP-1 and cross-reactivity with TIMP-1 antibody and subsequently identifies production of TIMP-1 by endometriotic tissues. The synthesis and secretion of TIMP-1 by endometriosis may derange the normal proteolytic milieu of the peritoneal cavity and contribute to the etiology and underlying physiological sequelae associated with the disease endometriosis.
Assuntos
Glicoproteínas/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Sequência de Aminoácidos , Animais , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Dados de Sequência Molecular , Ratos , Homologia de Sequência de Aminoácidos , Inibidores Teciduais de MetaloproteinasesRESUMO
Endometriotic lesions are defined by extrauterine growth of endometrial glands and stroma. Retrograde menstruation with subsequent attachment, invasion, and neovascularization are believed to give rise to the endometriotic lesions. As most women exhibit some degree of retrograde menstruation, some other unidentified factor(s) must render certain women susceptible to attachment and growth of ectopic endometrial tissue. A variety of theories have been proposed to account for this susceptibility, including genetic predisposition, aberrant immunological response, and an altered peritoneal environment. Ectopic endometriotic lesions are histologically similar to their putative eutopic precursors, yet significant biochemical differences exist between these two tissues. Less information is available regarding differences between eutopic endometrium from women with or without endometriosis. This report describes anomalies in structure, proliferation, immune components, adhesion molecules, proteolytic enzymes and inhibitors, steroid and cytokine production and responsiveness, and gene expression and protein production that have been identified in eutopic endometrium from women with endometriosis.
Assuntos
Endometriose/patologia , Endométrio/patologia , Apoptose/fisiologia , Divisão Celular/fisiologia , Endometriose/imunologia , Endometriose/metabolismo , Endométrio/imunologia , Endométrio/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Biossíntese de ProteínasRESUMO
OBJECTIVES: To evaluate the effectiveness of GnRH agonist (GnRH-a) therapy on adhesion formation and reformation in established rat models for surgically induced adhesion formation and endometriosis. DESIGN: Before surgery, female Sprague-Dawley rats were injected with GnRH-a or control diluent. Six days later, rats were assigned to one of four surgical groups: [1] endometriosis, [2] endometriosis sham, [3] adhesion model, or [4] adhesion sham. Three weeks after surgery, a second-look laparotomy was performed, adhesions were scored (0 = no adhesions to 3 = severe adhesions) and mechanically disrupted, and rats received a second GnRH-a or diluent injection either analogous to their initial injection or in a crossover design. Three weeks after the second injection, rats were killed and adhesion reformation was scored. Data were evaluated using nonparameteric tests including Mann-Whitney, Kruskal-Wallis, and Friedman's tests comparing GnRH-a treatments with diluent controls. RESULTS: Preoperative GnRH-a therapy reduced adhesion scores in rats with surgically induced endometriosis (mean +/- SEM; GnRH-a 1.1 +/- 0.2 versus diluent 2.2 +/- 0.2) and adhesions (GnRH-a 0.3 +/- 0.1 versus diluent 0.6 +/- 0.1). Pretreatment GnRH-a therapy did not affect adhesion scores in the endometriosis sham procedure. Combined preoperative and postoperative GnRHa therapy (GnRH-a-GnRH-a) but not postoperative GnRH-a therapy alone (diluent-GnRH-a) reduced adhesion reformation after adhesiolysis in the endometriosis model (GnRH-a-GnRH-a 1.1 +/- 0.3, diluent-GnRH-a 1.6 +/- 0.7), the endometriosis sham (GnRH-a-GnRH-a 0.7 +/- 0.2, diluent-GnRHa 1.8 +/- 0.1), and the adhesion model (GnRH-a-GnRH-a 0.3 +/- 0.2, diluent-GnRHa 1.0 +/- 0.5). No adhesions were observed in the adhesion sham group. CONCLUSIONS: Gonadotropin-releasing hormone agonist therapy was successful in reducing adhesion formation and reformation. These studies suggest that GnRH-a therapy for adhesion prevention in women should be explored.
Assuntos
Leuprolida/uso terapêutico , Aderências Teciduais/prevenção & controle , Animais , Estradiol/sangue , Feminino , Complicações Pós-Operatórias , Progesterona/sangue , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To establish tissue inhibitor of metalloproteinase-1 (TIMP-1) concentrations in peritoneal fluid (PF) and sera of women with endometriosis and compare them to disease-free controls. DESIGN: Prospective randomized study. SETTING: Academic medical center. PATIENT(S): Women with laparoscopically documented endometriosis and disease-free women of reproductive age. INTERVENTION(S): Peritoneal fluid and sera were collected, and some women received gonadotropin-releasing hormone agonist (GnRH-a) therapy for endometriosis. MAIN OUTCOME MEASURE(S): Peritoneal fluid and sera TIMP-1 concentrations were measured with a specific RIA. RESULT(S): The TIMP-1 concentrations were significantly lower in PF and sera of women with endometriosis compared with disease-free women. The GnRH-a therapy restored serum TIMP-1 concentrations. CONCLUSION(S): Aberrant expression and localization of TIMP-1 may derange the proteolytic milieu of the peritoneal cavity and contribute to the etiology and underlying physiologic sequelae associated with endometriosis. Measurement of TIMP-1 in serum may aid in diagnosing endometriosis and assist with monitoring treatment efficacy in women with this disease.
Assuntos
Líquido Ascítico/metabolismo , Endometriose/tratamento farmacológico , Endometriose/metabolismo , Hormônio Liberador de Gonadotropina/agonistas , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Administração por Inalação , Adulto , Feminino , Humanos , Concentração Osmolar , Valores de Referência , Inibidor Tecidual de Metaloproteinase-1/sangueRESUMO
OBJECTIVE: To evaluate the effects of a gonadotropin-releasing hormone agonist (GnRH-a) on plasminogen activator (PA), matrix metalloproteinase (MMP), plasminogen activator inhibitor (PAI) and matrix metalloproteinase inhibitor (MMPI) activities in peritoneal fluid relative to GnRH-a-induced reduction of adhesion formation. DESIGN: Continuation of prospective randomized study using surgical models for adhesion formation. SETTING: Department of Obstetrics and Gynecology research laboratory at the University of Missouri School of Medicine. PATIENT(S): Forty reproductively cycling female Sprague-Dawley rats. INTERVENTION(S): Female rats were injected with depot GnRH-a or diluent and randomly assigned to adhesion and endometriosis surgeries. Peritoneal fluid was collected prior to (time 1) and 7 weeks from (time 2) initial surgery. MAIN OUTCOME MEASURE(S): Peritoneal fluid was analyzed for PA, PAI, MMP, and MMPI activities. RESULT(S): At time 1, MMP and MMPI activities were similar in all rats; however, PA and PAI activities were less in rats pretreated with GnRH-a than with diluent. Between time 1 and time 2, GnRH-a-treated rats showed an increase in PAI and MMPI activities without significant changes in PA or MMP activities, whereas rats receiving diluent showed a significant increase in PAI and MMP activities but no significant changes in PA or MMPI activities. At time 2, rats receiving GnRH-a had less PA and MMP activities than those receiving diluent. Adhesion scores showed a positive correlation with MMP activity. CONCLUSION(S): In the absence of GnRH-a therapy, surgical tissue manipulation increased peritoneal fluid MMP and PAI activity. Gonadotropin-releasing hormone agonist therapy decreased PA and MMP activities and also increased PAI and MMPI activities. This GnRH-a-induced shift to a less invasive phenotype may alter fibrinolysis and extracellular matrix remodeling and thereby play a role in the mechanism of GnRH-a-induced reduction in adhesion formation.
Assuntos
Endometriose/metabolismo , Leuprolida/uso terapêutico , Metaloendopeptidases/análise , Ativadores de Plasminogênio/análise , Inativadores de Plasminogênio/análise , Aderências Teciduais/prevenção & controle , Animais , Modelos Animais de Doenças , Feminino , Metaloendopeptidases/antagonistas & inibidores , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To investigate in vivo expression of matrix metalloproteinase enzymes (MMP), MMP-3 and MMP-7, by the baboon endometrium in relation to the process of tissue remodeling that accompanies menstruation in the primate. METHODS: Endometrial tissues and uterine fluids obtained from reproductively intact cycling baboons, cycling baboons treated with an anti-progestin, and ovariectomized steroid-treated baboons were evaluated for MMP mRNA expression and protein production by using multiple analytic techniques. RESULTS: The pattern of MMP protein production matched the pattern of MMP mRNA expression. In intact cycling baboons, MMP mRNA expression and protein production were specific to cell type and stage of the menstrual cycle. Endometrial stroma expressed minimal amounts of MMP-3 during the proliferative and secretory stages, whereas endometrial epithelia expressed MMP-7 during the proliferative stage. Expression of stromal MMP-3 and epithelial MMP-7 increased during early menses. Administration of anti-progestin beginning on the day of the LH surge increased MMP-7 but not MMP-3 expression at the mid secretory phase. Expression of MMP-3 and MMP-7 in the ovariectomized steroid-treated animals paralleled that of the intact cycling animals. CONCLUSIONS: These results highlight the similarity of human and baboon endometrial MMP-3 and MMP-7 production, providing further evidence for use of the baboon as a model for studying events associated with hormonally regulated changes during the menstrual cycle. These results also demonstrate that endometrial epithelial MMP-7 is suppressed by progesterone in the baboon endometrium, but endometrial stromal MMP-3 is regulated by a different mechanism.
Assuntos
Endométrio/enzimologia , Regulação Enzimológica da Expressão Gênica , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 7 da Matriz/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Western Blotting , Líquidos Corporais/enzimologia , Feminino , Metaloproteinase 3 da Matriz/análise , Metaloproteinase 3 da Matriz/química , Metaloproteinase 7 da Matriz/análise , Metaloproteinase 7 da Matriz/química , Ciclo Menstrual , Papio , RNA Mensageiro/análise , Útero/enzimologiaRESUMO
A more thorough understanding of the mechanisms associated with the cause and pathophysiology of endometriosis may help in the development of new diagnostic and therapeutic methods for the management of endometriosis. Research has begun to enhance our understanding of endometriosis by demonstrating the differences and similarites between eutopic and ectopic endometrium, and by characterizing the peritoneal environment. Animal models have been developed and validated to conduct studies that are ethically impossible in women. Recently, cell culture models, using purified populations of cells from endometriotic lesions, have provided an appropriate in vitro endometriosis model to study the language by which cells communicate; to evaluate the biochemical effects of steroids, growth factors, pharmacological agents and immunomodulatory agents on the cells; and to study the effects of endometriosis on reproduction.
Assuntos
Endometriose/fisiopatologia , Animais , Líquido Ascítico/citologia , Antígeno Ca-125/metabolismo , Células Cultivadas , Fatores Estimuladores de Colônias/fisiologia , Modelos Animais de Doenças , Endometriose/etiologia , Endometriose/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Glicoproteínas de Membrana/biossíntese , PesquisaRESUMO
OBJECTIVE: To investigate what effect natural killer (NK) cells have on the implantation of heterologous endometrial scrapings. STUDY DESIGN: Anti-asialo GM1 (AA-GM1) anti-sera have been shown to eliminate NK cell activity in various strains of rats and mice. Either AA-GM1 antibodies (+) or rabbit antiglobulin (-) was administered to beige mice (NK cell deficient) or beige control mice (not NK cell deficient of the same strain). The heterologous endometrial scrapings were prepared by scraping seven pairs of uterine horns from normal mice of the same strain. Beige and normal mice were then injected intraperitoneally every 3 days with the heterologous endometrial scraping and antibodies for a period of 50 days. The four experimental groups (n = 10 per group) can be summarized as being beige (+), beige (-), normal (+) and normal (-). RESULTS: There was no evidence of ectopic endometrial tissue in any of the four test groups by histologic examination or by using immunohistochemical staining techniques. Histologic evidence of an impaired immune response was clearly demonstrated in the beige mice receiving AA-GM1 antibodies. CONCLUSION: Using this model, a deficiency of NK cell activity did not appear to enhance the implantation of endometrial tissue on the abdominal peritoneum of mice.
Assuntos
Endométrio/transplante , Células Matadoras Naturais/imunologia , Serpinas , Animais , Anticorpos/farmacologia , Modelos Animais de Doenças , Endometriose/etiologia , Endometriose/imunologia , Endometriose/patologia , Endométrio/química , Endométrio/patologia , Feminino , Gangliosídeo G(M1)/imunologia , Glicoproteínas/análise , Humanos , Imuno-Histoquímica , Terapia de Imunossupressão , Proteínas de Filamentos Intermediários/análise , Células Matadoras Naturais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Peritônio , Transplante HeterotópicoRESUMO
BACKGROUND: HLA-G is a major histocompatability antigen with documented immune-regulatory function. Various epithelial cancers and tissue allografts have been noted to express HLA-G, which is postulated to aid in their escape from immunosurveillance. We evaluated peritoneal endometriosis and eutopic endometrium for the expression of HLA-G protein and gene transcript. METHODS: Two experiments were performed: (i) archived tissue blocks from peritoneal endometriotic lesions (n = 15) and eutopic endometrium (n = 12) were evaluated for extent of protein immunostaining, and (ii) eutopic endometrial biopsies from women without (n = 17) and with (n = 24) endometriosis, and peritoneal endometriotic lesions (n = 14) were evaluated for presence of RNA transcript by in situ hybridization. RESULTS: HLA-G protein localized in the glandular epithelium of 14 of 15 (93.3%) peritoneal endometriotic lesions, but not in stromal cells. HLA-G protein staining was absent in endometrial biopsies (n = 12). HLA-G gene transcript localized to the glandular epithelium in 13 of 14 (92.8%) peritoneal endometriotic lesions. HLA-G transcript was never observed in eutopic endometrium, regardless of cycle stage or whether from women with (n = 24) or without (n = 18) endometriosis. CONCLUSIONS: HLA-G is expressed by endometriotic glandular epithelium but not by eutopic endometrium under normal conditions. Differential expression of HLA-G suggests that peritoneal inflammation or cellular stress may up-regulate mechanisms to promote ectopic endometrial survival.
Assuntos
Endometriose/imunologia , Endométrio/imunologia , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Doenças Peritoneais/imunologia , Biópsia , Endometriose/patologia , Endométrio/patologia , Epitélio/imunologia , Epitélio/patologia , Feminino , Antígenos HLA/genética , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imuno-Histoquímica , Hibridização In Situ , Doenças Peritoneais/patologia , RNA/metabolismoRESUMO
A unique glycoprotein, called endometriosis protein-I (ENDO-I), has previously been shown to be synthesized and secreted by rat and human endometriotic explants. Furthermore, the N-terminal amino acid sequence analysis showed that ENDO-I shares homology with haptoglobin. The present study was designed to determine this sequence of ENDO-I cDNA from human peritoneal endometriotic lesions and to determine the relative expression of the ENDO-I gene in several human tissues. Poly A-enriched RNA was isolated and reverse-transcribed. To determine the sequence of ENDO-I cDNA, a polymerase chain reaction was performed on cDNA from human endometriotic lesions and a gene-specific primer based on the human haptoglobin sequence. A similar procedure was followed to assess the relative expression of the ENDO-I gene in various tissues. Glyceraldehyde-3-phosphate-dehydrogenase was used as an internal control. ENDO-I gene expression was quantified by densitometry. Sequence analysis of ENDO-I cDNA identified 873 nucleotides that displayed 99.4% homology with the human haptoglobin beta-chain. Relative expression of ENDO-I mRNA by peritoneal endometriotic lesions was 19-fold greater than peritoneum, 28-fold greater than endometrioma and 37-fold greater than eutopic endometrium (P<0.01). Haptoglobin-like ENDO-I may be associated with localized angiogenesis and altered immune response involved with the aetiology/pathophysiology of endometriosis.
Assuntos
Endometriose/genética , Glicoproteínas/genética , Haptoglobinas/genética , Doenças Peritoneais/genética , Adolescente , Adulto , Animais , Sequência de Bases , Estudos de Casos e Controles , Endométrio/metabolismo , Endométrio/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Fígado/fisiologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Ratos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
In vivo levels of mRNA and the specificity of the extrauterine environment on matrix metalloproteinase (MMP)-3, MMP-2, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 were evaluated in eutopic and ectopic endometrial tissue during the establishment of endometriosis in a rat model. Uteri and endometriotic implants were collected and frozen at 36 h, 2 wk, and 4 wk postsurgery to study in vivo mRNA levels. Intact uteri, uterine tissues implanted in the peritoneum or under the skin, and peritoneal adipose implants were collected at 2 wk, halved, and either frozen or cultured. Gene-specific reverse transcriptase-polymerase chain reaction was performed to detect and quantify MMP-2, MMP-3, and TIMP-1 mRNA levels. The peritoneal endometriotic implants progressed from avascularized implants, to vascularized red lesions, to well-established encapsulated cysts. In vivo, MMP-3 mRNA was detectable at all times in ectopic tissues but not in eutopic uterine tissues, whereas MMP-2 and TIMP-1 were ubiquitously expressed at all times in both tissues. In vitro, only MMP-3 mRNA levels were elevated in endometrial tissues collected from the intact uterine and from under the skin, at levels similar to in vivo endometriotic implant MMP-3. In conclusion, ectopic endometrial MMP-3 may participate in the process of invasion and tissue remodeling that is hypothesized to occur in the pathogenesis of endometriosis.
Assuntos
Endometriose/enzimologia , Endométrio/enzimologia , Regulação da Expressão Gênica , Metaloproteinase 3 da Matriz/genética , Animais , Endometriose/patologia , Endométrio/transplante , Feminino , Metaloproteinase 2 da Matriz/genética , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-1/genética , Útero/enzimologiaRESUMO
A variety of models have been developed to study endometrial receptivity which involves normal, appropriately timed endometrial development and remodeling for blastocyst attachment and trophoblast invasion during the luteal phase of the menstrual cycle. Due to species differences, the human is by far the best model per se by which to study human endometrial receptivity. Techniques have evolved to obtain in vivo data on endometrial receptivity using hysteroscopy, ultrasonography or magnetic resonance imaging. Despite species differences, comparative studies of mammalian models and tissue- and cell culture models using endometrial tissue or cells harvested at particular phases of the reproductive cycle, or following experimental manipulation, have been used productively to study endometrial function. Differences as well as similarities have proven to be instructive. Such models have been used to study a variety of entities, such as homotypic and heterotypic cell-cell interaction, the role of steroids, cytokines, growth factors, immunomodulatory agents and pharmacological substances. These models have also been used to study cellular, biochemical and molecular mechanisms involved with uterine receptivity. This chapter was designed to provide a critical review of contemporary literature relating to in vivo models and laboratory strategies and paradigms for the study of uterine receptivity for blastocyst implantation.
Assuntos
Blastocisto/fisiologia , Implantação do Embrião , Modelos Biológicos , Útero/fisiologia , Células Epiteliais/fisiologia , Feminino , Glicoproteínas , Humanos , Gravidez , Células Estromais/fisiologia , Útero/citologiaRESUMO
Endometriosis protein-I (ENDO-I) mRNA expression and protein localization were evaluated using in-situ hybridization and immunohistochemistry in endometriotic lesions and eutopic endometrium from women with endometriosis, and in eutopic endometrium from women without endometriosis (controls). When present, ENDO-I mRNA and protein were observed in the functionalis zone of endometrial stroma and the stroma of endometriotic lesions. Expression and localization differences were scored and statistically analysed. During the secretory stage, ENDO-I mRNA expression by endometriotic lesions and eutopic endometrium from women with disease was significantly greater than ENDO-I mRNA expression by proliferative stage eutopic endometrium from women with disease or eutopic endometrium from controls, regardless of cycle stage (P < 0.001). More ENDO-I protein was localized in endometriotic lesions and eutopic endometrium from women with disease than in eutopic endometrium from controls, regardless of cycle stage (P < 0.001). Differential expression and localization of ENDO-I may help develop minimally invasive diagnostic strategies for endometriosis. Further, as ENDO-I shares nucleotide sequence and amino acid sequence with hepatic haptoglobin-which in certain disease states is immunosuppressive and angiogenic-differences in ENDO-I expression and localization in the peritoneal cavity may contribute to the pathogenesis of endometriosis and/or facilitate development of unprecedented diagnostic or therapeutic approaches for management of this enigmatic disease.
Assuntos
Endometriose/metabolismo , Endométrio/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Endometriose/genética , Endometriose/patologia , Feminino , Humanos , Modelos Estatísticos , RNA Mensageiro/análise , RNA Mensageiro/metabolismoRESUMO
OBJECTIVE: Murine endometrial granulocyte-macrophage colony-stimulating factor has been related to macrophage recruitment and activation and postulated to mediate reproductive events. This study was designed to determine whether granulocyte-macrophage colony-stimulating factor is present in normal human endometrium or endometriosis. STUDY DESIGN: Granulocyte-macrophage colony-stimulating factor was immunohistochemically evaluated in matched endometrial and endometriosis biopsy specimens (n = 19) and endometrial biopsy specimens from disease-free patients (n = 8). Staining differences were determined with McNemar's, Fisher's, and Wilcoxon's tests. RESULTS: Granulocyte-macrophage colony-stimulating factor was primarily localized in endometrial and endometriotic epithelial cells. Expression (p = 0.71) and staining intensity (p = 0.37) was similar in matched proliferative-phase endometrium and endometriosis. Matched secretory-phase endometrium and endometriosis also expressed granulocyte-macrophage colony-stimulating factor in similar proportions (p = 0.12), but staining intensity was enhanced in secretory endometriosis compared with secretory endometrium (p = 0.05). Endometrial granulocyte-macrophage colony-stimulating factor did not vary throughout the menstrual cycle, but endometriotic expression (p = 0.013) and staining intensity (p = 0.008) were significantly greater in the secretory phase. CONCLUSIONS: Granulocyte-macrophage colony-stimulating factor is localized in endometrial and endometriotic epithelial cells with increased expression in secretory-phase endometriosis. Granulocyte-macrophage colony-stimulating factor may elicit migration, proliferation, and activation of endometrial and peritoneal macrophages.
Assuntos
Endometriose/metabolismo , Endométrio/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Epitélio/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Ciclo Menstrual , Variações Dependentes do ObservadorRESUMO
OBJECTIVE: Progesterone-induced uterine protein-1, a product of secretory endometrial stromal cells (relative molecular mass 70,000, isoelectric point 5.7), was immunolocalized in endometrium and placenta. STUDY DESIGN: Biopsies were performed to obtain human endometrium and placenta throughout the menstrual cycle and gestation. Formalin-fixed, paraffin-embedded tissues (n = 74) were sectioned and immunohistochemically stained for progesterone-induced uterine protein-1 by the avidin-biotin peroxidase procedure. Isolated endometrial cells were also stained for progesterone-induced uterine protein-1. RESULTS: Progesterone-induced uterine protein-1 localized in proliferative endometrial stroma and in early to midsecretory stroma and ciliated epithelia and vanished from nonpregnant, late-secretory endometrium yet localized in the decidua, syncytiotrophoblast, and intermediate cytotrophoblast during pregnancy. Isolated, cultured endometrial stromal but not epithelial cells displayed progesterone-induced uterine protein-1 staining. CONCLUSION: Endometrial progesterone-induced uterine protein-1 localization shifts from stromal to epithelial, coinciding with the time of ovulation, fertilization, and implantation. This observation, combined with the disappearance of progesterone-induced uterine protein-1 in late-secretory, nonpregnant endometrium and its presence in decidua and trophoblast, suggests that progesterone-induced uterine protein-1 may play a role in decidualization, endometrial or embryo cross-talk, or placental physiologic features.
Assuntos
Endométrio/metabolismo , Ciclo Menstrual/metabolismo , Placenta/metabolismo , Proteínas da Gravidez/análise , Gravidez/metabolismo , Anticorpos , Biópsia , Células Cultivadas , Endométrio/citologia , Células Epiteliais , Epitélio/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Placenta/citologiaRESUMO
To explore the identity and possible function of endometriosis protein-I (ENDO-I), which is an acidic glycoprotein synthesized and secreted by endometriotic lesions, partial amino acid sequence and cDNA sequence were determined. Partially purified, de novo-synthesized rat endometriosis glycoproteins were separated by two-dimensional SDS-PAGE, transferred to polyvinyl difluoride membranes, and stained with Coomassie blue. Protein corresponding to the size and pI of ENDO-I was cut from the membranes and analyzed by automated Edman degradation. ENDO-I amino acid sequence analysis identified 15 residues that shared significant homology with the beta-chain of rat, mouse, and human haptoglobin (Hp) and human Hp-related protein. Western blot analyses using anti-Hp antibody demonstrated cross-reactivity with de novo-synthesized ENDO-I protein in endometriosis culture media. For nucleotide sequence analysis, poly A-enriched mRNA was isolated from rat endometriotic tissues. A gene-specific oligonucleotide primer was designed and used for 3' rapid amplification of cDNA ends (RACE). Automated sequencing of RACE cDNA fragments identified 859 base pairs, of which 858 were identical to rat Hp. Reverse transcription-polymerase chain reaction was used to demonstrate that ENDO-I transcripts are differentially expressed by endometriosis but not by uterine tissues. In the human, distinct subtypes of Hp as well as proteins sharing epitopes with Hp have been used to diagnose a variety of diseases; therefore, Hp-like ENDO-I may prove to be a nonsurgical diagnostic tool to assess endometriosis. Hepatic Hp, induced by acute-phase stimuli, modulates macrophage function and angiogenic activity. If ENDO-I possesses similar activities, it may be involved with anomalies of the immune system or the etiology and pathophysiology of endometriosis.