VEGF, TNF-alpha and IL-6 in the peritoneal fluid of women with unexplained infertility

Unexplained infertility is an important problem in diagnosis and therapy in everyday gynecologist's practice. Looking for possible reasons of the unexplained infertility we studied the concentrations of selected cytokines (VEGF, TNF-alpha and IL-6) in the peritoneal fluid of women suffering from the unexplained infertility. We compared the results in the studied group with the control group and with the patients with endometriosis. Immunological disorders of the peritoneal fluid in endometriosis are thought to take part in its pathomechanism. Our results suggest that the levels of one of the main factors of endothelium proliferation (VEGF) in the peritoneal fluid from women with unexplained infertility and women with endometriosis are comparable. Concentration IL-6 and TNF-alpha in the peritoneal fluid in case of unexplained infertility and control group was lower than in the endometriosis patients.     

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.     

Peritoneal fluid prostaglandins in endometriosis, tubal disorders, and unexplained infertility

To elucidate the roles of prostaglandins in peritoneal fluid and sex steroids in patients with endometriosis (N = 29), tubal disorders (N = 15), and unexplained infertility (N = 13), assays were performed using 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) (a metabolite of prostacyclin), thromboxane B2 (a metabolite of thromboxane A2), estradiol, and progesterone. Women with normal pelvic anatomy (N = 25) served as controls. Peritoneal fluid 6-keto-PGF1 alpha concentrations in patients with endometriosis (742 +/- 104 pg/ml, mean +/- SE), tubal disorders (987 +/- 211 pg/ml), and unexplained infertility (1659 +/- 770 pg/ml) were higher than those in the control women (515 +/- 77 pg/ml). The thromboxane B2 levels in the peritoneal fluid in endometriosis (554 +/- 73 pg/ml), tubal disorders (614 +/- 107 pg/ml), and unexplained infertility (668 +/- 161 pg/ml) were higher than the levels in the control subjects (333 +/- 23 pg/ml). There was no relationship between 6-keto-PGF1 alpha/thromboxane B2 in peritoneal fluid and day of menstrual cycle. The concentrations of estradiol and progesterone were normal in all patient groups and were not related to the 6-keto-PGF1 alpha and thromboxane B2 levels. The authors suggest that these prostanoids, which may contribute to infertility, may originate mainly from the peritoneum as a result of irritation by endometriotic implants, tubal adhesions, and scarring.     

子宫内膜异位症不孕妇女腹腔液前列腺素水平

本文测定子宫内膜异位症不孕妇女23例(异位症组)、不明原因不孕妇女11例(不孕对照组)和正常妇女11例(正常对照组)腹腔液前列腺素水平,结果提示:异位症不孕妇女腹腔液中,PGE_2、PGF_(2α)、TXB_2、6-Keto-PGF_(1α)的浓度均高于正常对照组;PGE_2、PGF_(2α)、6-Keto-PGF_(1α)的浓度也高于不孕对照组。四种前列腺素,不孕对照组与正常对照组无区别。表明:腹腔液中前列腺素水平增高可能与异位症引起不孕的机制有关,而不孕对照组的不孕可能是前列腺素以外的其它原因所致。     

Glutathione peroxidase concentration in the peritoneal fluid from infertile women

OBJECTIVES: To assess the concentration of Plasma Glutathione Peroxidase (plGPx) in the peritoneal fluid (PF) of patients with unexplained infertility and infertile women with minimal and mild endometriosis. MATERIALS AND METHODS: 33 women were studied, including 8 infertile women with minimal or mild endometriosis, 15 patients with unexplained infertility and 10 patients with tubal occlusion (a reference group). Concentration of plGPx was measured in the PF using a commercially available ELISA kit (Oxis Inc.). RESULTS: The plGPx concentration was significantly (p = 0.04) lower in PF from women with unexplained infertility (846 +/- 177 ng/ml) compared to the reference group (1023 +/- 238 ng/ml), but did not differ significantly (p = 0.25) between women with endometriosis (918 +/- 81 ng/ml) and patients with tubal infertility. CONCLUSIONS: Our results suggest that low peritoneal plGPx concentration may play a role in the pathogenesis of infertility.     

Peritoneal fluid from patients with unexplained infertility inhibits growth of 2-cell mouse embryos.

A significantly higher DHEAS concentration was measured in the peritoneal fluid of unexplained infertility patients (1171.4 +/- 155 ng/mL) in comparison to normal controls (667.6 +/- 82 ng/mL). Since the androgenic male serum does not promote blastocyst formation in the mouse embryo assay system, the potential of growth impairment by peritoneal fluid (PF) obtained from 22 women with unexplained infertility and 10 fertile controls was assessed. Where peritoneal fluid and serum from unexplained infertile (UI) patients were used as media supplement in mouse embryo culture, a significant inhibition of growth was observed in dishes containing PF but not serum. When DHEAS was added in varying concentrations to the culture media, a dose-dependent inhibition of embryo growth was observed. These findings show that the elevated DHEAS concentrations in the PF of UI patients adversely effect embryo growth and further suggest that increased DHEAS levels in the cul-de-sac fluid may be a causative factor for infertility.     

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.     

Antizona and antisperm antibodies in women with endometriosis and/or infertility

OBJECTIVE: To measure the levels of antigamete antibodies in serum and peritoneal fluid of women with endometriosis and/or infertility. DESIGN: Antibody activity against human sperm and porcine oocytes was analyzed in selected subgroups of women. SETTING: Clinic of reproduction. PATIENT(S): Women with endometriosis and/or infertility. INTERVENTION(S): No treatment was implemented before peritoneal fluid and blood sample collection. MAIN OUTCOME MEASURE(S): Quantitative ELISA. RESULT(S): Four groups of women (n = 98) were analyzed for the presence of antizona and antisperm antibodies: infertile with endometriosis (n = 30), idiopathic infertility (n = 28), fertile with endometriosis (n = 20), and healthy fertile controls (n = 20). Antibodies were analyzed simultaneously in serum and peritoneal fluid. No statistically significant differences in antibody levels were detected in serum samples among the analyzed groups. The median values for antizona and antisperm antibodies in peritoneal fluid were significantly higher in women with idiopathic infertility than in the control group. In women with unexplained infertility, a high degree of correlation (Spearman) was found between the presence of antizona antibodies in peritoneal fluid and serum (r = 0.579). A positive predictive value of 80% was calculated for the presence of antizona antibodies (>5 ng/oocyte) in the peritoneal fluid of patients with infertility. CONCLUSION(S): Antizona antibodies locally produced in the peritoneal fluid have diagnostic value for infertility status; however, they cannot be treated as a marker or prognostic factor for minimal endometriosis and/or its treatment.     

Cytotoxic activity of NK cells in the presence of the peritoneal fluid fro women with unexplained infertility and with endometriosis related infertility

We examined peritoneal fluid obtained during laparoscopy in the aspect of its influence on cytotoxic activity of NK cells. We showed significant suppression of this activity in women with unexplained infertility. The suppression of NK activity was also present in women fertile and infertile both, with severe endometriosis. These results may indicate the immunological disorders in unexplained infertility and in endometriosis-related infertility.     

Prostanoids in peritoneal fluid of infertile women with pelvic endometriosis and PID

Peritoneal fluid collected at celioscopy in infertile subjects was assayed for steroids and several prostanoids (PGE2, PGF2, TXB2, LTB4) as part of a study into pathophysiology of the female reproductive tract. Prostaglandins, produced massively in the pelvis, might interfere with fertility through various mechanisms (alterations in the egg implantation, follicle genesis, luteinization as well as tubal disorders). Our study of 54 patients showed a marked increase only of TXB2 out of the prostanoids assayed in overall endometriosis. In pelvic flogosis peritoneal LTB4 (and TXB2) were considerably increased if related to controls. This would suggest their role in the ethiopathogenesis of unexplained infertility (in relation to these pathologic patterns).     

The concentration of 13,14-dihydro-15-keto prostaglandin F2 alpha and prostaglandin E2 in peritoneal fluid of infertile patients with and without endometriosis

Prostaglandin F2 alpha (PGF2 alpha) metabolite (13,14-dihydro-15-keto PGF2 alpha) and prostaglandin E2 (PGE2) were assayed in the cul-de-sac fluid aspirated from 15 patients with endometriosis, in saline peritoneal washings of 5 patients with unexplained infertility, and in 5 control subjects. The fluid from the cul-de-sac of patients with endometriosis showed wide variations in the concentrations of PGF2 alpha metabolite and PGE2. There was no correlation between the concentration of these prostaglandins (PGs) and the stage of the cycle. Saline peritoneal washings from patients with unexplained infertility had significantly higher concentrations of PGF2 alpha metabolite than the control subjects. The concentration of PGE2 in the saline peritoneal washings was higher in the group with unexplained infertility than in the control group; however, the difference was not significant. The concentration of PGF2 alpha metabolite and PGE2 in the peritoneal saline washings from patients with unexplained infertility were no different from the concentrations of these PGs in the peritoneal fluid (PF) from patients with endometriosis. PG concentration in PF of infertile patients with or without endometriosis is a new variable for evaluation in these patients.     

Identification of myeloid dendritic cells in peritoneal fluid by flow cytometry

The myeloid lineage-derived DCs are the most potent antigen-presenting cells (APC), which are highly specialized in capturing antigens and triggering adaptive immune responses. The purpose of our study was to identify of the myeloid DCs in peritoneal fluid of women with nonmalignant ovarian tumors. With use of flow cytometry we detected myeloid dendritic cells in peritoneal fluid of the studied patients. Myeloid DCs were identified as a discrete population of mononuclear cells expressing high levels of HLA-DR and CD33, but without expression of CD14 and CD16. The number of DCs was shown as a percentage of mononuclear cells. Dendritic cells comprised 3.02% (0.35%-8.41%) in patients with ovarian tumors and 1.72% (0.51%-6.2%) of peritoneal fluid mononuclear cells in women with unexplained infertility. The percentage of DCs in peritoneal fluid was significantly higher than in peripheral blood.     

Peritoneal fluid leptin concentration in infertile patients.

Leptin may play a role in the regulation of menstrual cycle acting either directly on ovaries or at the level of the hypothalamic-pituitary axis. Peritoneal fluid is a biologically active environment that influences ovarian function but, on the other hand, concentration of many substances in peritoneal fluid can reflect the functional status of ovaries. In our study, we estimated leptin concentration in peritoneal fluid in relation to serum leptin concentration in infertile patients. A study group consisted of 31 infertile patients that underwent laparoscopy. In 15 patients, laparoscopy did not solve the problem and they were grouped as unexplained infertility, in eight patients we found endometriosis and eight patients were diagnosed as polycystic ovary syndrome (PCOS). We found significantly higher peritoneal fluid leptin concentrations in patients with unexplained infertility and endometriosis compared to those with PCOS. The plasma leptin concentration did not differ between the groups studied. There was no correlation between peritoneal fluid and plasma leptin concentrations in any patient group. Taking into account our results, the role of leptin in reproduction should be emphasized but further investigation is needed to determine its mechanism of action.     

Recovery of Chlamydia trachomatis from the endometria of women with unexplained infertility

Fifty-two women with unexplained infertility and 55 controls with recently proved fertility were screened for Chlamydia trachomatis (CT) infection in the blood and genital tract. Serum antibody titration was performed with indirect fluorescence. Cell cultures were performed to screen for CT in urethral and endocervical swabs, in endometrial samples taken without endocervical contamination and in salpingeal and/or peritoneal fluid samples. Anti-CT serum antibodies were detected in 36.5% of the patients; CT was isolated in urethral cultures in 26.9%, endocervical cultures in 23.1%, endometrial cultures in 25% and endosalpingeal and/or peritoneal fluid cultures in 1.9%. Comparison of the results in the patients and controls showed a significant difference in the incidence of CT infection in endometrial, urethral and endocervical cultures. Chlamydial endometritis could have been the direct cause of infertility in the patients studied or merely might have indicated endosalpingitis that was not detectable at laparoscopy.     

Tumor necrosis factor alpha and interferon gamma levels in peritoneal fluid of infertile women

Changes in the peritoneal fluid environment have been implicated in the pathogenesis of endometriosis as well as in the decrease of fertility. Tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) play a critical role in the generation of the immune response, leading to the enhanced proinflammatory cytokines and free radicals production. MATERIALS AND METHODS: 59 women were studied, including 24 patients with unexplained infertility, 10 infertile women with endometriosis (Io or IIo rAFS), 11 patients with PCOS and 14 patients with tubal occlusion. TNF alpha and IFN gamma concentrations were measured in the PF using commercially available ELISA kits. RESULTS: Peritoneal fluid IFN gamma concentrations did not differ significantly between the studied groups. TNF alpha levels were significantly (p = 0.02) higher in the PF of endometriotic patients compared to women with tubal infertility. The positive correlation (R = 0.83; p < 0.01) has been found between TNF alpha and IFN gamma levels. CONCLUSIONS: Increased PF TNF alpha levels observed in patients with endometriosis may play a role in activation of peritoneal macrophages. Positive correlation between TNF alpha and IFN gamma suggests their synergistic stimulatory effect on the immunocompetent PF cells.     

Early endometriosis invades the extracellular matrix.

OBJECTIVES: To investigate whether the aminoterminal propeptides of type III procollagen are increased in patients with early endometriosis and to demonstrate that the subtle lesion of endometriosis is an active stage of the disease. DESIGN: Aminoterminal propeptide of type III procollagen was determined in serum and peritoneal fluid (PF) of 100 consecutive patients undergoing laparoscopy. SETTING: Academisch Ziekenhuis Maastricht, The Netherlands, a tertiary care center. RESULTS: Aminoterminal propeptide PF levels were significantly higher in women with early lesions of endometriosis compared with levels in two groups of controls, i.e., fertile, cycling, women without the disease (P = 0.019) and women on oral contraceptives without the disease (P = 0.036). No difference was found in aminoterminal propeptide PF levels when comparing patients with early lesions of endometriosis and patients with unexplained infertility, the third control group. Aminoterminal propeptide PF levels of patients with endometriosis without early lesions were not different from PF levels in controls. CONCLUSION: The early lesion is an active stage of endometriosis, invading the extracellular matrix. In women with unexplained infertility active, microscopic endometriosis may be present.     

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.     

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.     

Total antioxidant status and activity of an extracellular superoxide dismutase in peritoneal fluid and plasma from women with unexplained infertility

OBJECTIVES: To evaluate the activity of an extracellular superoxide dismutase (EC SOD) and total antioxidant status in peritoneal fluid (PF) and plasma from women with unexplained infertility (UI). MATERIALS AND METHODS: PF and plasma samples were collected from 10 women with UI and 10 patients with tubal infertility (reference group). TAS was measured using Randox diagnostic reagent system. The activity of EC SOD was estimated using Calbiochem assay kit. RESULTS: TAS was significantly lower in PF from women with UI. Plasma TAS did not differ significantly between the groups. Peritoneal TAS was significantly lower compared to plasma TAS and there was a positive correlation between PF and plasma total antioxidant status. Activity of EC SOD did not differ significantly between the groups in either PF and plasma samples. CONCLUSIONS: These data demonstrate that low antioxidant status of PF but not that of blood may be responsible for idiopathic infertility. The activity of EC SOD doesn't appear to play a role in UI.