Imbalance in the expression of the activating type I and the inhibitory type II interleukin 1 receptors in endometriosis.

BACKGROUND: The ectopic establishment and progression of endometrial tissue is dependent upon its interaction with and responsiveness to the stimuli present in its new environment. Immune cell-derived cytokines, such as interleukin 1 (IL1), may alone or in concert with estrogens enhance the capability of ectopic endometrial cells to implant and develop into the host tissue. The objective of this study was to further evaluate the expression and significance of IL1 receptor type I (IL1R1), the signalling receptor that mediates cell activation by IL1, and IL1 receptor type II (IL1R2), a potent and specific down-regulator of IL1 action, in normal compared to endometriotic/endometrial tissues. METHODS: Techniques included immunohistochemistry, immunofluorescent staining, ELISA, western blotting and endometriotic cell culture transfection. RESULTS: Our study showed an imbalance in the expression of IL1R1 and IL1R2 in eutopic, and particularly in ectopic, endometrial tissues of women with endometriosis. Actually, a decreased IL1R2 expression is predominant in the eutopic and ectopic endometrium of women with endometriosis when compared with normal women, whereas a concomitant increase in IL1R1 expression occurs in ectopic endometrial tissue in comparison to eutopic endometrial tissue of normal or endometriotic women, particularly in the initial and most active implants. Transfection of endometriotic cells with a cDNA coding for IL1R2 resulted in a significant decrease in IL1-induced secretion of vascular endothelial cell growth factor and monocyte chemotactic protein 1. CONCLUSIONS: IL1R1/IL1R2 imbalance may amplify endometrial cell responsiveness to IL1 and represent a key mechanism underlying the ability of these cells to implant and develop into host tissues.

Increased expression of interleukin-1 receptor type 1 in active endometriotic lesions.

The establishment and progression of ectopic endometrial implants are dependent upon their interaction with and responsiveness to the stimuli present in their new environment. According to our and other previous studies, immune cells-derived cytokines, such as IL-1, may alone or in concert with estrogens, enhance the capability of ectopic endometrial cells to implant and develop into the host tissue. In the present study, immunohistochemical and dual immunofluorescence analyses showed that the functional signaling interleukin-1 receptor type 1 (IL-1RI) is expressed in endometriotic tissue, particularly in the glands, and identified endothelial cells, macrophages, and T-lymphocytes as cells having marked expression of IL-1RI. The highest concentrations of IL-1RI protein in endometriotic tissue, as evaluated using histological score (HSCORE) and measured by ELISA, were found in red endometriotic lesions as compared with typical black-blue or white lesions. Western blotting showed a significant increase in the levels of the 50 kDa band, whose apparent molecular weight corresponds to the soluble form of IL-1RI. RT-PCR analysis of IL-1 mRNA levels showed a pattern of expression comparable to that of the protein. Interestingly, IL-1RI expression was more significant in the proliferative than in the secretory phase of the menstrual cycle. Marked expression of IL-1RI, the functional signaling receptor that mediates cell activation by IL-1, in red endometriotic implants, which are highly vascularized and represent the earliest and most active forms of the disease, point to a higher cell receptivity for IL-1 in these lesions, a relationship with the activity of the disease and a possible involvement in the early steps of endometriotic tissue growth and development.

Increased expression of matrix metalloproteinase-9 in the eutopic endometrial tissue of women with endometriosis.

BACKGROUND: Endometriosis is a disease where endometrial tissue implants in ectopic locations. Remodelling of the extracellular matrix (ECM) is a prerequisite for the implantation of this tissue to be possible. METHODS: In this study, we detected immunoreactive matrix metalloproteinase-9 (MMP-9) throughout endometrial tissue and identified von Willebrand factor (vWF)-positive endothelial cells, CD45-positive leukocytes, CD3-positive T lymphocytes and CD68-positive macrophages as cells expressing MMP-9 in the stroma. RESULTS: We found an increased expression of MMP-9 in the uterine endometrial tissue of women with endometriosis, as assessed by zymography and enzyme-linked immunosorbent assay (ELISA) (P < 0.05). However, RT-PCR did not show a statistically significant increase in MMP-9 mRNA expression in these tissues (P = 0.14). There was no significant difference between women with and without endometriosis in the expression of tissue inhibitor of MMPs (TIMP)-1, a known natural inhibitor of the pro- and active forms of MMP-9, whether tested by ELISA or by RT-PCR (P = 0.46 and 0.37, respectively). Interestingly, the ratio of MMP-9/TIMP-1 expression was significantly higher in women with endometriosis than in normal women both at the protein and the mRNA levels (P < 0.05). CONCLUSION: These findings make plausible the involvement of MMP-9/TIMP-1 imbalance in the invasiveness of the endometrial tissue of patients with endometriosis and the ectopic development of the disease.

Tumour necrosis factor-alpha up-regulates macrophage migration inhibitory factor expression in endometrial stromal cells via the nuclear transcription factor NF-kappaB.

BACKGROUND: A series of controlled changes including proliferation, secretion and menstrual shedding occur in the human endometrium during every normal menstrual cycle. Macrophage migration inhibitory factor (MIF), a multifunctional cytokine with numerous proinflammatory, immunomodulatory and angiogenic properties, appears to be expressed in the human endometrium and to follow a regulated cycle phase-dependent expression, but the mechanisms underlying endometrial MIF expression remain to be fully elucidated. METHODS AND RESULTS: Results from enzyme-linked immunosorbent assay (ELISA) demonstrated a significant dose- and time-dependent increase in MIF secretion by human endometrial cells in response to tumour necrosis factor-alpha (TNF-alpha) (0.1-100 ng/ml). This increase was also observed at the mRNA level as shown by reverse transcription (RT)-PCR. Curcumin (10(-8) mol/l), a known nuclear factor (NF)-kappaB inhibitor, inhibited the TNF-alpha-induced pIkappaB phosphorylation as shown by western blotting, NF-kappaB translocation into the nucleus as shown by electrophoretic mobility shift assay, and MIF synthesis and secretion as measured by ELISA and RT-PCR. The expression of a dominant-negative NF-kappaB inhibitor (IkappaB) significantly decreased the TNF-alpha-induced MIF promoter activity as analysed by transient cell transfection. CONCLUSIONS: These results indicate clearly that TNF-alpha up-regulates the expression of MIF in endometrial stromal cells. This took place possibly through NF-kappaB activation, and may play an important role in the physiology of the human endometrium.

Stimulation of macrophage migration inhibitory factor expression in endometrial stromal cells by interleukin 1, beta involving the nuclear transcription factor NFkappaB.

Endometriosis, the ectopic development of endometrial tissue, is, particularly in peritoneal endometriosis, believed to result from tubal reflux of menstrual tissue. The release of cytokines and growth factors by refluxed endometrial cells in response to peritoneal inflammatory stimuli may enhance the capability of endometrial cells to implant and grow into the peritoneal host tissue. Herein we report that interleukin 1 (IL1), a major proinflammatory cytokine that is overproduced by endometriosis women-derived peritoneal macrophages and found in elevated concentrations in the peritoneal fluid of patients with endometriosis, stimulates the synthesis and the secretion of macrophage migration inhibitory factor (MIF) by human endometrial stromal cells. IL1B (0.1-100 ng/ml) exerted dose- and time-dependent effects of MIF protein secretion and mRNA synthesis, as shown by ELISA and reverse transcription-polymerase chain reaction, respectively. IL1B appeared to induce MIF gene transcription via the kappaB nuclear transcription factor (NFkappaB), as shown by electrophoretic mobility shift assay and Western blot analysis of IkappaB phosphorylation. Curcumin (10(-8) M), which is known for inhibiting NFkappaB activation, inhibited IL1B-induced MIF secretion as well as NFkappaB nuclear translocation and DNA binding. Taken together, these findings clearly show that IL1B up-regulates the expression of MIF in endometrial stromal cells in vitro and acts via NFkappaB. This may play an important role in the physiology of the human endometrium and the pathophysiology of endometriosis considering the immunomodulatory properties of MIF as well as its role in cell growth, angiogenesis and tissue remodeling.

Increased soluble interleukin-1 receptor type II proteolysis in the endometrium of women with endometriosis.

Numerous functional changes were observed in the intrauterine endometrial tissue of women with endometriosis. Our previous studies revealed a marked decrease in the expression of interleukin-1 receptor type 2 (IL-1RII), a decoy receptor known for its ability to buffer IL-1 effects. The aim of the present study was to assess whether post-translational mechanisms such as proteolysis may contribute to the down-regulation of IL-1RII levels. Our data showed that soluble IL-1RII (sIL-1RII) concentrations released by freshly cultured endometrial tissue were significantly lower in women with endometriosis than in normal women (P < 0.01) and further revealed a statistically significant correlation between increased proteolysis and decreased sIL-1RII levels (P < 0.05; r = -0.47). (125)I-labelled soluble recombinant human IL-1RII ([(125)I]srhIL-1RII) was significantly more degraded in culture supernatant of tissues from women with endometriosis compared to normal women (P < 0.05), and natural tissue inhibitor of matrix metalloproteinase (TIMP)-1 inhibited [(125)I]srhIL-1RII degradation. Incubation of srhIL-1RII with active rhMMP-9 resulted in a dose-dependent degradation of srhIL-1RII as analysed by western blotting. Dual immunofluorescence showed an increased immunostaining for matrix metalloproteinase-9 in situ in the endometrial tissue of women with endometriosis compared to normal women and a decreased immunostaining for IL-1RII. The present study showed a reduced release of sIL-1RII by the endometrial tissue of women with endometriosis and revealed a proteolytic post-translational mechanism which may be involved in the down-regulation of IL-1RII levels. This may enhance IL-1-mediated activation of endometrial cells and contribute to the local immuno-inflammatory process observed in endometriosis patients.

Evidence for an increased release of proteolytic activity by the eutopic endometrial tissue in women with endometriosis and for involvement of matrix metalloproteinase-9.

BACKGROUND: For the implantation of endometrium in ectopic locations, remodelling of the extracellular matrix (ECM) is necessary. Many studies have shown an increased expression of various proteases in the ectopic endometrium of women with endometriosis. Few, however, have addressed possible changes in protease expression in the eutopic endometrium. METHODS AND RESULTS: Herein, we reveal an increased release of proteolytic activity by the eutopic endometrium of women with endometriosis compared with normal women (P < 0.01). Using zymography and western blotting, we identified matrix metalloproteinase (MMP)-2 and MMP-9 in the culture medium, and further found that MMP-9 secretion, as assessed by zymography and enzyme-linked immunosorbent assay (ELISA), was elevated in women with endometriosis compared with normal women (P < 0.05). No statistically significant difference in MMP-2 secretion between women with and without endometriosis was noted. However, a significant difference in the levels of the tissue inhibitor of metalloproteinases (TIMP)-1, a known MMP-9 inhibitor, was found (P < 0.05). CONCLUSION: The endometriosis-associated increase in proteolysis and imbalance between the secretion of MMP-9 and that of its natural inhibitor, TIMP-1, revealed in the culture medium of endometrial tissue may reflect in vivo the enhanced capacity of this tissue to break down the ECM in host tissues, thereby favouring its ectopic implantation and development.

Interleukin-1 receptor accessory protein is constitutively expressed in human endometrium throughout the menstrual cycle.

Interleukin-1 (IL-1) is one of the principal cytokines that participate in endocrine and local regulation of many endometrial and reproductive functions. The cellular response to IL-1 principally implicates receptor type 1 (IL-1R tI) and, according to recent data, an accessory protein (IL-1R-AcP) that seems to play an essential function in signal transduction. In the present study, we examined the expression of IL-1R-AcP in the endometrium of 39 normal fertile women throughout the menstrual cycle. As studied by immunohistochemistry, IL-1R-AcP was detected across endometrial tissue, but more noticeably in the glands and luminal epithelium. The intensity of IL-1R-AcP immunostaining was consistently high throughout the menstrual cycle, and this was confirmed by Western blot analysis of the protein and corroborated by reverse transcription-polymerase chain reaction analysis of the mRNA. To our knowledge, this is the first report showing that IL-1R-AcP is expressed in endometrial tissue, and without any noticeable variation throughout the menstrual cycle. This suggests that the accessory protein, whose co-expression is critical for IL-1R tI-mediated cell activation, is, in contrast to the functional receptor, constitutively expressed and not subject to similar cycle-dependent regulation.

Goserelin acetate (Zoladex) plus endometrial ablation for dysfunctional uterine bleeding: a 3-year follow-up evaluation.

OBJECTIVE: To report the results of a 3-year follow-up evaluation of a trial comparing goserelin acetate depot injections with sham injections before endometrial ablation for the treatment of dysfunctional uterine bleeding (DUB). DESIGN: Prospective, randomized, double-blind, parallel-group study. SETTING: Thirty-seven centers in 12 countries. PATIENT(S): Three-hundred and fifty-eight premenopausal women aged over 30 years with DUB. INTERVENTION(S): Goserelin acetate (3.6 mg depot) every 28 days for 8 weeks, or sham depot every 28 days for 8 weeks, with endometrial ablation 6 weeks +/- 3 days after the first depot injection (i.e., when the endometrium is at its thinnest). The follow-up continued for 3 years. MAIN OUTCOME MEASURE(S): At the 3-year follow-up, bleeding in the previous 3 months and need for surgical intervention were recorded. RESULT(S): At 3 years, amenorrhea rates were 21% in the goserelin acetate group and 14% in the control group (estimated odds ratio, 1.8; 95% CI, 0.98-3.25; P=.0571). The surgical intervention rate (since the original procedure) was low and did not differ significantly between groups. For hysterectomy, it was 21% for the goserelin acetate group and 15% for the control group. For repeat ablations, it was 5.6% for the goserelin acetate group and 2.1% for the control group. CONCLUSION(S): Prethinning with goserelin acetate before endometrial ablation resulted in higher long-term amenorrhea rates than ablation without prethinning.

Efficacy and safety of a low-dose monophasic combination oral contraceptive containing 100 microg levonorgestrel and 20 microg ethinyl estradiol (Alesse). North american Levonorgestrel Study Group (NALSG).

The efficacy and safety of a low-dose 21-day combination oral contraceptive containing 100 microg levonorgestrel and 20 microg ethinyl estradiol were evaluated in an open-label, multicenter trial. A total of 1708 subjects with regular menstrual cycles (27,011 cycles) were evaluated. The oral contraceptive was administered once a day for 21 days, followed by 7 days of placebo for a complete cycle. During 26,554 cycles evaluated for efficacy, 18 pregnancies occurred (Pearl index of 0.88); 6 of these events were attributable to subject noncompliance. After 30 cycles of exposure the cumulative rate of withdrawal as a result of accidental pregnancy was 1.9%. Breakthrough bleeding (with or without spotting) occurred in 12.9% of the cycles and spotting alone occurred in 10.1% of the cycles. The 2 most common adverse events cited as reasons for discontinuation were headache (2% of subjects) and metrorrhagia (2%). One serious event led to withdrawal of a subject. Overall, the results of this study demonstrate that the monophasic regimen of 100 microg levonorgestrel and 20 microg ethinyl estradiol offers effective contraception, acceptable cycle control, and a good tolerability profile.

Physiological and cytogenetic characterization of immortalized human endometriotic cells containing episomal simian virus 40 DNA.

The study of misplaced endometrial cells, which abnormally implant and grow outside the uterine cavity, is of considerable interest for the understanding of the pathophysiology of endometriosis. However, endometriotic cells, particularly epithelial cells, required for primary cell culture are not easily available. We report here the characterization of an endometriotic cell line immortalized after infection of primary endometriotic cell cultures with simian virus 40. Transformed cells express T-antigen, and blot hybridization analysis showed that the viral genome is present as an episome. Cytogenetic analysis revealed a polyploid karyotype with numerical and structural rearrangements involving mainly the same chromosomes (6, 10, 11, 15, and 17). The cell line has been maintained in culture for over 80 passages and was still proliferating without any noticeable change in the biological properties investigated. Transformed endometriotic cells expressed both progesterone and estradiol receptors and were stimulated by these ovarian hormones to secrete monocyte chemotactic protein-1, a factor that may play an important role in the recruitment and activation of peritoneal macrophages. In addition, this response was enhanced in interleukin-1-treated cells. Taken together, these findings support the view that this cell line may be an interesting tool for the study of the pathophysiology of endometriosis.

Characteristics related to the prevalence of minimal or mild endometriosis in infertile women. Canadian Collaborative Group on Endometriosis.

The objective of this case-control study is to identify factors associated with the prevalence of minimal or mild endometriosis among infertile women. Cases (N = 329) were women diagnosed by laparoscopy with minimal or mild endometriosis and without any other factors explaining their infertility. Controls (N = 262) were women in whom the infertility remained unexplained after a diagnostic laparoscopy. Selected characteristics were documented by means of a face-to-face interview before the laparoscopy. The prevalence of minimal or mild endometriosis was higher in women age 25 years or older, in those who reported menarche at the age of 13 years [prevalence odds ratio (POR) = 1.63; 95% confidence interval (CI) = 1.02-2.60] or older (POR = 1.73; 95% CI = 1.07-2.78), menstrual cycles of 27 days or less (POR = 1.63; 95% CI = 1.02-2.60), or caffeine intake of 300 mg per day or more (POR = 1.33; 95% CI = 0.91-1.94). The prevalence of minimal or mild endometriosis was inversely related to body mass index. Parous women were less likely to have endometriosis (POR = 0.61; 95% CI = 0.39-0.96) than were nulliparous women. Education, duration of infertility, and smoking status were not related to the presence of endometriosis.

Fecundity of infertile women with minimal or mild endometriosis and women with unexplained infertility. The Canadian Collaborative Group on Endometriosis.

OBJECTIVE: To assess whether infertile women with minimal or mild endometriosis have lower fecundity than women with unexplained infertility. DESIGN: Prospective cohort study. SETTING: Twenty-three infertility clinics across Canada. PATIENT(S): Three hundred thirty-one infertile women aged 20-39 years. INTERVENTION(S): Diagnostic laparoscopy for infertility. Infertile women with minimal or mild endometriosis (n = 168) were compared with women with unexplained infertility (n = 263). Both groups were managed expectantly. The women were followed up for 36 weeks after the laparoscopy or, for those who became pregnant, for up to 20 weeks of the pregnancy. MAIN OUTCOME MEASURE(S): Fecundity refers to the probability of becoming pregnant in the first 36 weeks after laparoscopy and carrying the pregnancy for > or = 20 weeks. The fecundity rate is the number of pregnancies per 100 person-months. RESULT(S): Fecundity was 18.2% in infertile women with minimal or mild endometriosis and 23.7% in women without endometriosis (log-rank test). The fecundity rate was 2.52 per 100 person-months in women with endometriosis and 3.48 per 100 person-months in women with unexplained infertility. The crude and adjusted fecundity rate ratios were 0.72 and 0.83 (95% confidence interval = 0.53-1.32), respectively. CONCLUSION(S): The fecundity of infertile women with minimal or mild endometriosis is not significantly lower than that of women with unexplained infertility.

Immunoblot detection of decreased antibodies to haptoglobin-like protein in the serum of infertile women with or without endometriosis.

The purpose of this study was to find out differences in the levels of antibodies to distinct antigens in the serum of fertile versus infertile patients with and without endometriosis and to identify these antigens. Blood was collected from 61 patients undergoing laparoscopy for pelvic pain, infertility, or tubal ligation. Serum antibodies against serum antigens with apparent molecular masses of 22 kDa and 18 kDa were assessed by immunoblot analysis. Gel filtration, HPLC DEAE ion-exchange chromatography, NH2-terminal sequencing, and double immunodiffusion were used to characterize and identify these proteins. The relative amount of antibodies reacting with 22- and 18-kDa proteins detected in a standard preparation of antigens was significantly lower in the serum of infertile patients with endometriosis (0.20 +/- 0.05 and 0.57 +/- 0.10) and without endometriosis (0.21 +/- 0.06 and 0.53 +/- 0.08) compared to that of control fertile women without endometriosis (0.53 +/- 0.08 and 1.09 +/- 0.13). After purification by chromatography, the NH2-terminal amino acid sequence of the proteins in the 22- and 18-kDa range was identical through 20 amino acids with the alpha chain of the human haptoglobin. Double immunodiffusion implied immunochemical identity between commercial human haptoglobin and the purified proteins. We conclude that infertile patients with and without endometriosis show reduced serum levels of antibodies against a haptoglobin-like protein. These results would indicate an alteration of the immune system or changes in the levels of these antigens in infertility and/or endometriosis.

Laparoscopic surgery in infertile women with minimal or mild endometriosis. Canadian Collaborative Group on Endometriosis.

BACKGROUND: Minimal or mild endometriosis is frequently diagnosed in infertile women. It is often treated by resection or ablation of the lesions, but whether this improves fertility has not been established. We carried out a randomized, controlled trial to determine whether laparoscopic surgery enhanced fecundity in infertile women with minimal or mild endometriosis. METHODS: We studied 341 infertile women 20 to 39 years of age with minimal or mild endometriosis. During diagnostic laparoscopy the women were randomly assigned to undergo resection or ablation of visible endometriosis or diagnostic laparoscopy only. They were followed for 36 weeks after the laparoscopy or, for those who became pregnant during that interval, for up to 20 weeks of pregnancy. RESULTS: Among the 172 women who had resection or ablation of endometriosis, 50 became pregnant and had pregnancies that continued for 20 weeks or longer, as compared with 29 of the 169 women in the diagnostic-laparoscopy group (cumulative probabilities, 30.7 percent and 17.7 percent, respectively; P=0.006 by the log-rank test). The corresponding rates of fecundity were 4.7 and 2.4 per 100 person-months (rate ratio, 1.9; 95 percent confidence interval, 1.2 to 3.1). Fetal losses occurred in 20.6 percent of all the recognized pregnancies in the laparoscopic-surgery group and in 21.6 percent of all those in the diagnostic-laparoscopy group (P=0.91). Four minor operative complications (intestinal contusion, slight tear of the tubal serosa, difficult pneumoperitoneum, and vascular trauma) were reported (three in the surgery group and one in the control group). CONCLUSIONS: Laparoscopic resection or ablation of minimal and mild endometriosis enhances fecundity in infertile women.

A new low-dose monophasic combination oral contraceptive (Alesse) with levonorgestrel 100 micrograms and ethinyl estradiol 20 micrograms. North American Levonorgestrel Study Group (NALSG).

The efficacy and safety of a new, low-dose, 21-day combination oral contraceptive containing 100 micrograms of levonorgestrel and 20 micrograms of ethinyl estradiol were evaluated in an open-label, multicenter trial. A total of 1477 subjects were enrolled and had 7870 cycles of exposure as of the data cutoff of this interim report. Of these, 792 subjects had completed six cycles of treatment. A total of five pregnancies occurred during treatment during 7720 efficacy cycles, for a Pearl index of 0.84. To date, 415 (28%) subjects have been withdrawn from the study for any reason, including 131 (9%) due to adverse events. The cumulative life table pregnancy rate was 0.0041 per woman entering the sixth cycle. Breakthrough bleeding alone occurred in 4.3% of the cycles and breakthrough bleeding and spotting occurred together during 11% of the cycles. Of the cycles evaluable, 2.6% were amenorrheic. The most commonly reported adverse events in this trial considered at least possibly drug related were headache (14%) and metrorrhagia (8%). This formulation provides contraceptive efficacy similar to higher-dose oral contraceptives, while maintaining a safety and common OC side effect profile that is consistent with prior years of reported use with levonorgestrel-containing products.

Increased monocyte chemotactic protein-1 level and activity in the peripheral blood of women with endometriosis. Le Groupe d'Investigation en Gynécologie.

OBJECTIVE: Our purpose was to evaluate monocyte chemotactic protein-1 in the peripheral blood of women with and without endometriosis. STUDY DESIGN: Fifty-seven patients with endometriosis at laparoscopy done for infertility and pelvic pain were compared with 44 fertile women with no evidence of endometriosis at tubal ligation by laparoscopy. Monocyte chemotactic protein-1 concentration in the plasma was determined by enzyme-linked immunosorbent assay and its biologic activity was evaluated by measuring monocyte chemotaxis with use of a human histiocytic cell line (U937). RESULTS: Monocyte chemotactic protein-1 concentrations (median and range of values) found in the plasma were higher in patients with endometriosis (163, 0 to 788 pg/ml) than in normal controls (0, 0 to 355 pg/ml). This elevation was significant only in the minimal stage of endometriosis (revised American Fertility Society stage I). However, increased chemotactic activity (mean number of migrating cells/mm2 +/- SEM) was found in the stages I (1240 +/- 141), II (519 +/- 30), and III-IV (523 +/- 23) of the disease compared with normal controls (205 +/- 20). A total of 35% to 44% of this activity was inhibited in the presence of an antibody specific to monocyte chemotactic protein-1. CONCLUSION: Endometriosis is associated with increased level and activity of monocyte chemotactic protein-1 in the peripheral blood. The elevation and activation of this cytokine could play a relevant role in the immunoinflammatory process associated with the disease.

Secretion of interleukin-6 by human endometriotic cells and regulation by proinflammatory cytokines and sex steroids.

Endometriosis is generally associated with an immunoinflammatory process that takes place in the peritoneal cavity of patients. Interleukin (IL)-6, a multifunctional cytokine involved in numerous immunological and proliferative processes, has been found at high concentrations in the peritoneal fluid of endometriosis patients. The purpose of this study was to investigate the ability of endometriotic cells to produce IL-y and to assess the regulation of its secretion by proinflammatory cytokines and sex steroids. Cultures of human endometriotic cells were exposed to different concentrations of cytokines and sex steroid hormones for varying periods of time. IL-6 secretion was measured using an enzyme-linked immunosorbent assay. Endometriotic cells spontaneously released IL-6 in culture. IL-1 beta and tumour necrosis factor (TNF)-alpha (0.1-100.0 ng/ ml) potentiated IL-y secretion in a time- and dose-dependent manner. Interferon-gamma (0.4-400 ng/ml) induced a dose-related increase in IL-6 secretion and showed a synergistic effect on that secretion in combination with TNF-alpha (10 ng/ ml). Either spontaneous or cytokine-induced IL-6 secretion was inhibited by progesterone (10(-8)-10(-5) M) and danazol (10(-6) M), whereas oestradiol (10(-8)-10(-5) M) had a limited inhibitory effect. The antiprogestin RU486 (10(-8)-10(-4) M) antagonized the inhibitory effects of progesterone and danazol, but showed agonist action when used alone. These findings indicate that endometriotic tissue may actively contribute to the biological changes observed in the peritoneal fluid of endometriosis patients. They also provide new insights into the mechanisms of action of progesterone and those of danazol and RU486 used in the treatment of endometriosis.

Human endometrial cells cultured in a type I collagen gel.

OBJECTIVE: To elaborate in vitro conditions that enable epithelial and stromal cells of human endometrium to grow within a gel of collagen. STUDY DESIGN: Primary cultures of epithelial cells derived from human endometrial biopsies were dissociated and mixed with a collagen solution, and the gel was allowed to form at physiologic pH. Control cultures were grown in plastic dishes. DNA replication was assessed by 3H-thymidine incorporation, morphology by histology and cell characterization by monoclonal antibodies to cytokeratins. RESULTS: Cells grown on plastic dishes exhibited a typical monolayer arrangement, and replication was increased 1.6-fold by the addition of stromal cell-conditioned medium (50% vol/vol). After a two- to three-week period of culture within the collagen gel in the presence of either stromal cells or stromal cell-conditioned medium, epithelial cells formed circular arrangements of cuboidal to columnar cells with open lumina resembling glands. These glandlike structures were cytokeratin positive as assessed by immunohistochemistry, thereby confirming their epithelial nature. CONCLUSION: The development of differentiated epithelial structures in a three-dimensional gel provides a promising method of studying various biochemical and cellular interactions of eutopic and ectopic endometrium.