Endometrial antigens involved in the autoimmunity of endometriosis

Serum and peritoneal fluid from five fertile women without endometriosis and serum (n = 23) and peritoneal fluid (n = 12) from infertile women with endometriosis were tested for the presence of antibodies against endometrial tissue antigens by a Western blot analysis. Antigens with molecular weights (MW) of 19, 31, 38, and 42 kd reacted with antibodies in the serum and peritoneal fluid from both fertile and infertile women. Antibodies in 20 of 23 (87%) sera and all 12 (100%) peritoneal fluid samples from endometriosis patients reacted against endometrial antigens with molecular weights (MW) of 26 kd and/or 34 kd. Serum from 10 patients (43%) and peritoneal fluid from 6 patients (50%) also had antibodies to an endometrial antigen with MW of 21.5 kd. Reactivity to other endometrial antigens with MW 16, 24, 48, and 75 kd was also noted in patients with endometriosis. Antibodies in the serum and peritoneal fluid from fertile women failed to react against these antigens. It is concluded that the humoral and local endometrial autoimmunity detected in patients with endometriosis is primarily directed against antigens with MW of 26 and 34 kd.     

Marked elevation of macrophage migration inhibitory factor in the peritoneal fluid of women with endometriosis

OBJECTIVE: To evaluate the presence of macrophage migration inhibitory factor (MIF) in the peritoneal fluid of normal fertile women and patients with endometriosis and its growth-promoting activity toward human endothelial cells. DESIGN: Retrospective study using ELISA to measure peritoneal fluid MIF, and [3H]-thymidine incorporation into the DNA of human endothelial cells to assess its mitogenic activity. SETTING: Gynecology clinic and human reproduction research laboratory. PATIENT(S): Thirty-six healthy women and 57 women with endometriosis. INTERVENTION(S): Peritoneal fluid samples were obtained at laparoscopy. MAIN OUTCOME MEASURE(S): Macrophage migration inhibitory factor concentrations in the peritoneal fluid samples and [3H]-thymidine incorporation into the DNA of human microvascular endothelial cells to assess proliferation. RESULT(S): This study demonstrated the presence of MIF in the peritoneal fluid and a 238% increase of MIF levels in women with endometriosis as compared with healthy women. Both fertile and infertile women with endometriosis had significantly higher MIF concentrations than did fertile women with normal gynecological status, but the difference was more significant in infertile endometriosis patients. Anti-MIF antibody significantly inhibited proliferation of human microvascular endothelial cells in response to peritoneal fluids from healthy women and women with endometriosis stages I-II and III-IV, as assessed by [3H]-thymidine incorporation. CONCLUSION(S): This study revealed the presence of MIF in the peritoneal fluid and its increased levels in endometriosis and suggests that MIF may be involved in endometriosis-associated infertility and angiogenesis.     

Failure to demonstrate significant antisperm antibodies in peritoneal fluid of patients with endometriosis

Endometriosis, even in mild cases, decreases monthly fecundity. Immunologic disorders have been suggested as the mechanism. In light of possible increases in serum autoimmune antibodies, increased peritoneal macrophages, and increased sperm phagocytosis associated with this disease, we postulated that peritoneal fluid antisperm antibodies would be increased and might be the cause of increased sperm phagocytosis and its associated infertility. Peritoneal fluid, from 18 patients with endometriosis and 10 infertile controls, was tested with the antisperm antibody immunobead test validated for peritoneal fluid. One of 18 patients with endometriosis and none of 10 controls had antisperm antibodies present. Therefore, increased sperm phagocytosis is unlikely a result of peritoneal antisperm antibodies in endometriosis patients.     

Oxidative stress may be a piece in the endometriosis puzzle

OBJECTIVE: To assess the total antioxidant potential of women with endometriosis-associated infertility, women with idiopathic infertility, and fertile controls. DESIGN: Retrospective study. SETTING: University medical hospital. PATIENT(S): Sixty-five women admitted for diagnostic laparoscopy. MAIN OUTCOME MEASURES: Peritoneal fluid samples were analyzed for superoxide dismutase activity and total antioxidant status on spectrophotometry, glutathione peroxidase activity on enzyme-linked immunosorbent assay, and lipid peroxides levels on colorimetry. RESULT(S): Mean activity of superoxide dismutase, glutathione peroxidase, and total antioxidant status was lowest and lipid peroxide level was highest among infertile patients with endometriosis. Women with idiopathic infertility, in contrast, had the highest superoxide dismutase, glutathione peroxidase, and total antioxidant status activity and the lowest lipid peroxide level. CONCLUSION(S): High antioxidant potential is not a contributing factor in women with idiopathic infertility. Low total antioxidant status and low activity of antioxidant enzymes in the peritoneal fluid of infertile women with endometriosis probably do not influence fertility in these women, but these factors may play a role in the development of the disease.     

Characterization of leukocyte subpopulations in the peritoneal fluid of women with endometriosis

Monoclonal antibodies identifying leukocytes subpopulations were applied to smears of laparoscopically collected peritoneal fluid leukocytes and parallel samples of peripheral blood leukocytes from women with endometriosis (n = 33), those with unexplained infertility (n = 9), and fertile controls (n = 8). Peripheral blood leukocyte profiles in all groups were indistinguishable from reported normal values. Peritoneal fluid leukocyte profiles were observed to be different between groups. The most significant elevations in total leukocytes, macrophages, helper T lymphocytes and natural-killer cells were observed in women with stage I and II endometriosis. Significantly elevated levels of total leukocytes, macrophages, and T lymphocytes were also observed in peritoneal fluid from women with unexplained infertility. The results from this study indicate that the peritoneal environment is immunologically dynamic and suggest that cellular immune mechanisms may contribute to reproductive failure in women with endometriosis and unexplained infertility.     

Total antioxidant status of peritoneal fluid in infertile women

OBJECTIVE: To determine whether impairment of the antioxidant systems of peritoneal fluid might be a factor responsible for infertility. STUDY DESIGN: Total antioxidant status was measured in peritoneal fluid obtained from 18 infertile women suffering from minimal or mild endometriosis, 23 patients with unexplained infertility, 12 women with tubal infertility and 13 fertile women. RESULTS: Total antioxidant status was significantly lower in peritoneal fluid from women with unexplained infertility (0.49+/-0.21 mmol/l) compared to both fertile patients (0.67+/-0.24 mmol/l, P=0.02) and women with tubal infertility (0.76+/-0.26 mmol/l, P=0.001). Peritoneal fluid total antioxidant status did not differ significantly between patients with endometriosis (0.61+/-0.2 mmol/l), tubal infertility and the fertile group (P>0.05). CONCLUSIONS: Our results suggest that low antioxidant status in peritoneal fluid may play a role in the pathogenesis of infertility.     

Lymphocyte activity in the presence of peritoneal fluid from fertile women and infertile women with and without endometriosis

Peritoneal fluid from women with endometriosis, unexplained infertility, and fertile controls were compared to one another and to normal human serum for effects on lymphocyte proliferation in vitro. Peritoneal fluid samples were also assayed for both interleukin-1 and interleukin-2. All peritoneal fluid samples significantly enhanced lymphocyte proliferation in both mitogen-stimulated and unstimulated cultures compared with serum controls. Mitogen-induced leukocyte proliferation was higher in the presence of peritoneal fluid from women with endometriosis compared with other samples. Five out of 23 samples from endometriosis patients contained elevated levels of interleukin-1 and three out of 23 contained elevated levels of interleukin-2. Six out of eight peritoneal fluid samples from unexplained infertility patients also had elevated levels of interleukin-2; samples from fertile women did not contain elevated levels of either cytokine. Our data indicate that peritoneal fluid from women with endometriosis and unexplained infertility support the activation and proliferation of lymphocytes. Leukocyte products may locally affect the progression of disease and fertility.     

Inducible nitric oxide synthase expression by peritoneal macrophages in endometriosis-associated infertility.

OBJECTIVE: Determine whether peritoneal macrophages from women with endometriosis-associated infertility express more inducible nitric oxide synthase (NOS2) and produce more NO than fertile controls. DESIGN: Unblinded clinical study. PATIENT(S): Nine infertile women with endometriosis and nine normal fertile women undergoing laparoscopy.Intervention(s): Peritoneal fluid and macrophages were collected. Cells were also cultured with the NOS2 inducers interferon-alpha (IFN-alpha) or IFN-gamma plus lipopolysaccharide (LPS). MAIN OUTCOME MEASURE(S): Peritoneal fluid NO levels, peritoneal macrophage NOS activity, and peritoneal macrophage NOS2 protein expression. RESULT(S): NOS enzyme activity was higher in peritoneal macrophages from endometriosis patients. Immunoblots demonstrated NOS2 protein only in peritoneal macrophages from women with endometriosis. Peritoneal fluid NO concentration was similar in the two groups, but total peritoneal fluid NO content was higher in endometriosis patients. After 3 days' culture, peritoneal macrophages from women with endometriosis produced more NO in response to IFN-alpha or IFN-gamma plus LPS than controls. CONCLUSION(S): Peritoneal macrophages from women with endometriosis-associated infertility express higher levels of NOS2, have higher NOS enzyme activity, and produce more NO in response to immune stimulation in vitro. As high levels of NO adversely affect sperm, embryos, implantation, and oviductal function, reducing peritoneal fluid NO production or blocking NO effects may improve fertility in women with endometriosis.     

Identifying the presence of antibodies against endometrial antigens. A preliminary study.

Serum and peritoneal fluid from women with and without evidence of endometriosis were tested for the presence of antibodies against endometrial tissue antigens with Western blot analysis. Serum antibodies against endometrial cytosolic antigens of molecular weight 45, 52, 58, 62 and 66 kd were present in samples obtained from women both with and without endometriosis. The patients with endometriosis had serum antibodies against 34-kd endometrial cytosolic antigen, which was not present in serum from fertile women without endometriosis. The peritoneal fluid from patients with endometriosis also reacted with 34-kd endometrial antigen but not the peritoneal fluid from control fertile women. There was no difference in the antigens detected with serum antibody when endometrium from fertile women without evidence of endometriosis and from women with endometriosis was used as a source of antigen. The presence of serum antibody against 34-kd endometrial antigen is specific to endometriosis. However, this antigen is expressed by endometrium of women both with or without endometriosis. Isolation and identification of this antigen may lead to development of a noninvasive aid for the diagnosis of endometriosis.     

Antisperm antibodies in women: variability in antibody levels in serum, mucus, and peritoneal fluid.

OBJECTIVE: To look for patterns of antisperm antibody expression in women by exploring the levels of antisperm antibodies in different body fluids. This was achieved by studying sequential serum samples from individual patients and by comparing the levels of antisperm antibodies in serum from a number of patients with the levels of antisperm antibodies in cervical mucus or peritoneal fluid (PF). DESIGN: Prospective studies were performed on sequential serum samples within a menstrual cycle. Retrospective studies were done to compare antisperm antibodies in serum and mucus or PF. The immunobead assay was used to measure antisperm antibodies in these fluids. SETTING: Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire. PATIENTS: A random sample of patients undergoing evaluation for infertility. RESULTS: The levels of antisperm antibodies in sera drawn from patients at different points in a menstrual cycle stimulated by the presence of exogenous hormones did not change during the follicular phase of the menstrual cycle. Also, in many samples, the antisperm antibody level in serum did not correlate with the antisperm antibody levels in mucus or PF. CONCLUSIONS: The data suggest that measurement of antisperm antibodies at a single point in time or from a single fluid is not sufficient when evaluating a woman for immunological infertility. The data also suggest that numerous and complex factors contribute to the expression of antisperm antibodies in women.     

Importance portance of reactive oxygen species in the peritoneal fluid of women with endometriosis or idiopathic infertility

Objective: To determine whether reactive oxygen species in peritoneal fluid might be a factor in infertility.Design: Prospective study.Setting: Andrology laboratory and gynecology clinic at a tertiary care facility.Patient(s): Women with endometriosis (n = 15) or idiopathic infertility (n = 11) who under-went laparoscopy for infertility. Patients undergoing tubal ligation served as controls (n = 13).Intervention(s): Aspiration of peritoneal fluid.Main Outcome Measure(s): Reactive oxygen species levels, presence of polymorphonuclear granulocytes, and leukocyte distribution in peritoneal fluid.Result(s): Reactive oxygen species were present in the peritoneal fluid of patients with endometriosis, idiopathic infertility, and tubal ligation. Levels of reactive oxygen species did not show a statistically significant difference between patients with endometriosis and the control group in either unprocessed or processed (cell-free) peritoneal fluid, but did differ significantly between patients with idiopathic infertility and controls in process peritooneal fluid. Polymorphonuclear granulocytes (> 1 × 10⁸/mL) were not present in the peritoneal fluid of any patient. Macrophage concentrations of peritoneal fluid did not differ significantly between controls and patients with endometriosis or idiopathic infertility.Conclusion(s): Reactive oxygen species in the peritoneal fluid may not affect fertility directly in women with endometriosis; however, they may have a role in patients with idiopathic infertility.     

Low serum and peritoneal fluid concentration of interferon-gamma-induced protein-10 (CXCL10) in women with endometriosis

OBJECTIVE: To evaluate serum and peritoneal fluid concentrations of interferon-gamma-inducible protein-10 (CXCL10), a chemokine involved in local immune function, in women with endometriosis. DESIGN: Prospective study. SETTING: Division of Obstetrics and Gynecology, University of Siena. PATIENT(S): A total of 147 women were divided in two groups: women with (n = 77) and without (n = 70) endometriosis. INTERVENTION(S): Serum and peritoneal fluid were collected from all patients undergoing laparoscopy. MAIN OUTCOME MEASURE(S): CXCL10 concentrations were measured by a specific ELISA. RESULT(S): Serum CXCL10 concentrations in women with endometriosis were significantly lower than in those without endometriosis. No statistically significant difference between women with early endometriosis and those with advanced endometriosis was found. CXCL10 concentrations in peritoneal fluid of women with advanced endometriosis were significantly lower than in that of women with an early stage of, or without, endometriosis. CONCLUSION(S): The decreased concentrations of CXCL10 in serum and peritoneal fluid of women with endometriosis indicate an impaired immune activity in women with endometriosis.     

Elevated soluble Fas ligand levels may suggest a role for apoptosis in women with endometriosis

OBJECTIVE: To evaluate soluble Fas ligand concentrations in serum and peritoneal fluid from women with endometriosis and from fertile controls without endometriosis, and to study levels of soluble Fas ligand in conditioned media of cultured endometrial stromal cells. DESIGN: Prospective, experimental trial. SETTING: Two academic IVF centers. PATIENT(S): Twenty-nine fertile women without endometriosis and 57 infertile women with endometriosis (32 with stage I or II disease and 25 with stage III or IV disease). MAIN OUTCOME MEASURE(S): Enzyme-linked immunosorbent assay was used to measure soluble Fas ligand concentrations in paired samples of serum and peritoneal fluid from women with and without endometriosis. Concentrations were also measured in conditioned media of cultured endometrial stromal cells at basal conditions and after stimulation with interleukin-8 (0.001-10 ng/mL) and tumor necrosis factor-alpha (1-10 ng/mL). RESULT(S): Compared with fertile controls and women with early-stage of endometriosis, women with moderate to severe endometriosis had elevated serum (87.2 +/- 6.4, 88.2 +/- 6.9, and 162.3 +/- 7.8 pg/mL, respectively) and peritoneal fluid (81.0 +/- 6.0, 80.5 +/- 6.8, and 166.2 +/- 10.3 pg/mL, respectively) concentrations of soluble Fas ligand. Serum levels of soluble Fas ligand positively correlated with levels in peritoneal fluid. Comparison of patients in the same menstrual cycle in each group revealed that increased levels of soluble Fas ligand in patients with advanced endometriosis were not attributable to the difference in cycle phases. Soluble Fas ligand was not detected in conditioned media of endometrial stromal cells under baseline conditions or after stimulation. CONCLUSION(S): Serum and peritoneal fluid of women with moderate to severe endometriosis contain elevated concentrations of soluble Fas ligand compared to women with minimal or mild endometriosis and women without endometriosis. These findings suggest a role for apoptotic dysregulation in the pathophysiology of endometriosis.     

Leptin is not involved in the pathophysiology of endometriosis-related infertility

OBJECTIVE: Changes in peritoneal fluid (PF) composition may affect fertilization as well as early embryonic development. Leptin, an adipocyte hormone, has been shown to act as a link between adipose tissue and the reproductive system. Therefore, we decided to assess peritoneal and serum leptin levels in infertile endometriotic patients. PATIENTS: Seventy-two women were studied, including 30 fertile and 18 infertile women with ovarian endometriotic cysts and, as a reference group, 24 patients with unexplained infertility. RESULTS: No significant difference in the peritoneal and leptin levels was found between the studied groups. Significantly higher PF leptin concentration was observed in patients with stages III and IV of endometriosis as compared to those with minimal stage of the disease. In fertile patients with endometriosis a positive correlation has been found between PF and serum leptin concentrations. CONCLUSIONS: No differences in peritoneal or serum leptin levels between infertile and fertile women with endometriosis suggest that this cytokine is not involved in pathophysiology of endometriosis-related infertility.     

Increased nitric oxide in peritoneal fluid from women with idiopathic infertility and endometriosis.

OBJECTIVE: To verify whether nitric oxide in peritoneal fluid is associated with endometriosis and infertility. STUDY DESIGN: Twenty-five women with idiopathic infertility and 38 with endometriosis were recruited, and 18 cases of uterine myomata and 2 cases of ovarian cyst served as controls. Peritoneal fluid samples were aspirated from the pouch of Douglas during laparoscopy or laparotomy. Metabolites of nitric oxide (nitrite and nitrate) in peritoneal fluid were determined by a method using nitrate reductase and the Griess reaction. RESULTS: Peritoneal concentrations of nitrate/nitrite in both infertile women (42.02 +/- 12.98 mmol/L) and patients with endometriosis (41.75 +/- 16.42 mmol/L) were significantly higher than that in controls (33.96 +/- 13.07, P < .05 for both). No significant difference in peritoneal nitrate/nitrite level was found between infertile women and patients with endometriosis (P > .5). Peritoneal levels of nitrate/nitrite were comparable among patients with endometriosis at different stages (P > .5). Patients with endometriosis had more peritoneal fluid than controls and idiopathic infertile women, while controls and idiopathic infertile women had comparable amounts of peritoneal fluid. CONCLUSION: An increased peritoneal level of nitric oxide is a common alteration in endometriosis, endometriosis-associated infertility and idiopathic infertility and may be associated with the pathogenesis of these diseases.     

Cytokine profiles in autologous peritoneal fluid and peripheral blood of women with deep and superficial endometriosis

In a preliminary study the hypothesis was tested that cytokine profiles in peripheral blood were higher in women with deep infiltrating endometriosis and cytokine profiles in peritoneal fluid were higher in women with superficial endometriosis. Thirteen women of reproductive age having laparoscopy for infertility (n=9), pain (n=3) or combined pain and infertility (n=1). Peripheral blood and peritoneal fluid were obtained and analyzed for Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-α), Interleukin-10 (IL-10), Transforming Growth Factor-betal (TGFβ1), and Interferon-gamma (IFN-γ). No significant cytokine differences were observed in either peritoneal fluid or peripheral blood between IL-6, TGFβ1, IFNγ, TNF-alpha and IL-10 of women with superficial endometriosis (n=7) and women with deeply infiltrating endometriosis (n=6). The results of this preliminary study do not show significant differences in peripheral blood and peritoneal fluid cytokine levels between women with deep infiltrating endometriosis compared to women with superficial disease. Future studies with increased sample size are required to either confirm or refute these preliminary findings. Received: 5 January 2000 / Accepted: 14 August 2000     

Effect of peritoneal fluid from endometriosis patients on sperm motion characteristics and acrosome reaction

OBJECTIVE: To determine whether peritoneal fluid from women with endometriosis contributes to infertility by impairing sperm motion and functional characteristics. METHODS: Women with endometriosis (n = 20) underwent laparoscopy for infertility or pelvic pain. Patients undergoing tubal ligation served as controls (n = 14). Peritoneal fluid was aspirated from women with endometriosis, or from women undergoing laparoscopic tubal ligation. Sperm motility, motion characteristics and acrosome reaction were assessed following incubation with peritoneal fluid. RESULTS: Sperm motility, motion characteristics, and acrosome reaction did not differ significantly between the two groups after 3, 5, or 24 hours of incubation with peritoneal fluid. CONCLUSIONS: Sperm motion or functional characteristics showed no significant impairment when sperm from normal donors were incubated with peritoneal fluid from patients with endometriosis. It is unlikely that peritoneal fluid in these patients contributes to infertility.     

Lipid peroxidation in the peritoneal fluid of infertile women with peritoneal endometriosis

OBJECTIVE: To assess the level of lipid peroxidation in the peritoneal fluid of infertile women with peritoneal endometriosis and of fertile disease-free controls. STUDY DESIGN: Level of lipid peroxidation (malondialdeyde, malondialdeyde with copper addition, and cholest-3,5-dien-7-one) was measured in the peritoneal fluid obtained from 21 women with endometriosis-related infertility and from 21 fertile women having tubal ligation. RESULTS:: The level of lipid peroxidation did not differ significantly (P > 0.05) according to the stage of endometriosis. The level of lipid peroxidation (malondialdeyde, malondialdeyde with the addition of copper, and cholest-3,5-dien-7-one) did not differ significantly (P > 0.05) between patients with endometriosis-related infertility (0.07 nmol/ml, 0.34 nmol/ml, 0.24 microg/ml, respectively) and disease-free controls (0.04 nmol/ml, 0.21 nmol/ml, 0.25 microg/ml, respectively). CONCLUSION: The level of lipid peroxidation did not differ between women with endometriosis-related infertility and fertile disease-free controls, suggesting that increased reactive oxygen species may not be one of the factors responsible for compromised fertility in patients with endometriosis.     

Detection of interleukin-6 in human follicular fluid.

OBJECTIVE: To determine if interleukin-6 (IL-6) is a normal constituent of human follicular fluid (FF) after ovarian hyperstimulation and to assess whether IL-6 levels differ in conditions associated with immunological causes of infertility. DESIGN: After ovarian hyperstimulation for an in vitro fertilization (IVF) treatment cycle, FF samples were obtained at the time of oocyte retrieval. SETTING: Referral center at a tertiary care hospital. PATIENTS: Thirty women referred for IVF, including 10 patients with significant titers (greater than 40%) of antisperm antibodies and 10 with pelvic endometriosis. Ten patients with tubal infertility without antisperm antibodies or endometriosis served as controls. MAIN OUTCOME MEASURES: Analysis of FF levels for IL-6 using both bioassay and immunoassay. RESULTS: Bioactive (range 0.32 to 32.2 U/mL) and immunoreactive (range 0.34 to 13.6 ng/mL) IL-6 levels were detected in FF of all subjects after ovarian hyperstimulation. Follicular fluid IL-6 levels were substantially higher (3 to 30-fold) than that reported in serum. There was no difference in the mean concentrations of IL-6 levels between patients with antisperm antibodies, endometriosis, or tubal infertility. CONCLUSIONS: Bioactive and immunoreactive IL-6 are present in human FF after ovarian hyperstimulation, supporting a potential autocrine or paracrine role within the follicular microenvironment.     

Ethnicity and mode of delivery in 'low-risk' first-time mothers, East London, 1988-1997

OBJECTIVE: To test whether serum monocyte chemotactic protein-1 (MCP-1) chemokine levels correlate with endometriosis in infertile women. STUDY DESIGN: A group of women with endometriosis (n = 18, infertile) was compared with patients with uterine leiomyoma (n = 16, fertile), unexplained infertility (n = 5, infertile), and healthy women (n = 16, fertile). MCP-1 expression levels were evaluated by ELISA assay. The data obtained were statistically analyzed using the Mann-Whitney test. P-Values <0.05 were considered as significant. RESULTS: MCP-1 concentrations (median; range of values) in serum were as follows: women with endometriosis (221; 101-635 pg/ml), women with unexplained infertility (167, 114-234 pg/ml), women with uterine leiomyoma (137; 88-200 pg/ml), and healthy donors (123; 98-194 pg/ml). Significant differences were observed in the women with endometriosis compared with those with uterine leiomyoma (p = 0.02) and healthy donors (p = 0.002). Among the women with endometriosis, the level of significance in MCP-1 level at rAFS stages III-IV was higher than that at rAFS stages I-II compared with healthy donors and women with leiomyoma (p = 0.002 and p = 0.02, respectively). CONCLUSIONS: These data show that an increased level of MCP-1 can characterize infertile women with endometriosis. However, further studies are needed to be able to determine whether increased MCP-1 chemokine expression can be related to infertility or is a result of endometriosis progress.