Differential expression of T-helper cytokines in the peritoneal fluid of women with normal ovarian cycle compared with women with chronic anovulation

BACKGROUND: Estrogen and progesterone immunoregulate the genital environment by expression of cytokines and growth factors. OBJECTIVE: To investigate the pattern of expression of T-helper cytokines during the ovarian cycle compared with women with chronic anovulation resistant to clomiphene citrate. HYPOTHESIS: Expression of T-helper cytokines in women with chronic anovulation may be different from the pattern in women with a normal ovarian cycle. METHODS: We evaluated 31 infertile women having laparoscopy for evaluation of tubal patency and evidence of ovulation in two groups during (a) the luteal phase (17 women) and (b) the follicular phase (14 women). A third group was composed of 14 women with polycystic ovarian syndrome, but they were resistant to clomiphene citrate for induction of ovulation and had laparoscopic ovarian cautery. Peritoneal fluid was collected during laparoscopy. Estimation of T-helper cytokine interleukin (IL)-2, tumor necrosis factor (TNF)-alpha, IL-4 and IL-6 in serum, peritoneal fluid and culture of the peritoneal mononuclear cells was performed by ELISA. Serum luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin, estradiol and progesterone were evaluated by the Vidas Parametric System. RESULTS: The LH : FSH ratio was significantly higher in the women with polycystic ovaries than in the ovulatory groups. IL-2 and IFN-gamma were more highly expressed in the follicular phase but the T-helper 2 cytokines IL-4 and IL-6 predominated in the luteal phase, serum, peritoneal fluid and culture of the peritoneal mononuclear cells. From the follicular to the mid-luteal phase, IL-6 increased three to fivefold in the serum and peritoneal fluid, but there was low expression with anovulation. CONCLUSIONS: The peritoneal fluid levels of IL-4 and IL-6 are higher in the luteal phase. Low IL-6 levels in chronic anovulation may be a marker of resistance to clomiphene citrate.     

Vascular endothelial growth factor and interleukin-6 in peritoneal fluid of women with endometriosis

OBJECTIVE(S): To determine [1] vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) levels in peritoneal fluid from women with endometriosis and compare them with those from oral contraceptive (OC) users and normal cycling women and [2] any correlation between VEGF and IL-6 concentrations. DESIGN: Controlled clinical study. SETTING: University medical center. PATIENT(S): Patients undergoing laparoscopy for infertility or other benign gynecologic conditions. INTERVENTION(S): Peritoneal fluid samples were collected. MAIN OUTCOME MEASURE(S): Levels of VEGF and IL-6 in peritoneal fluid were determined. RESULT(S): Compared with normal controls or women with less severe endometriosis (implant scores of 5 or less), women with more advanced endometriosis (implant scores of 6 or more) have elevated VEGF and IL-6 levels in peritoneal fluid. Compared with normal controls, markedly suppressed IL-6 but similar VEGF levels were found in peritoneal fluid from OC users. Neither VEGF nor IL-6 varied cyclically in normal women or those with endometriosis. There was no correlation between levels of VEGF and IL-6 in peritoneal fluid. There was no correlation between implant scores and VEGF or IL-6 levels. CONCLUSION: The inflammation associated with endometriosis, through increased levels of peritoneal fluid VEGF, may promote angiogenesis for the progressive growth of endometriosis. Effective treatment of endometriosis by combination estrogen-progestin pills may involve the suppression of such inflammatory responses.     

Cytokine profiles in serum and peritoneal fluid from infertile women with and without endometriosis

OBJECTIVE: To study the serum and peritoneal fluid cytokine profiles in infertile women with minimal/mild active endometriosis. METHODS: Fifty-seven consecutive infertile women undergoing laparoscopy for unexplained infertility had peritoneal fluid and serum samples obtained at the time of laparoscopy. The levels of interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1 beta (IL-1 beta), vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha (TNF-alpha), monocyte chemotatic protein-1 (MCP-1), RANTES, platelet derived growth factor (PDGF), soluble Fas (sFas), and soluble Fas Ligand (sFasL) in peritoneal fluid and serum were measured to compare the concentration in both biological fluids, in women who have minimal/mild red endometriosis using women with no endometriosis as controls. RESULTS: Peritoneal fluid levels of MCP-1, IL-8 and IL-6 were significantly higher in the endometriosis group (P < 0.012, P = 0.003, and P = 0.015, respectively). There was no significant difference in the peritoneal fluid levels of IL-1 beta, TNF-alpha, RANTES, VEGF, PDGF, sFas and sFasL in the two groups. Although serum levels of IL-8 were higher in women with endometriosis, the difference was not significant (P = 0.07). Serum levels of PDGF, IL-6, RANTES, IL-1 beta, TNF-alpha, and sFas, were not significantly different in the two groups. CONCLUSION: The elevated levels of MCP-1, IL-6, and IL-8 in peritoneal fluid but not serum may indicate the importance of local macrophage activating factors in the pathogenesis of endometriosis.     

Altered expression of interleukin-18 in the peritoneal fluid of women with endometriosis

OBJECTIVE: Peritoneal fluid (PF) inflammatory factors may participate in the pathogenesis of endometriosis. The aim of this study was to investigate PF interleukin (IL)-18 levels in women with and without endometriosis. DESIGN: Controlled clinical study. SETTING: Women undergoing laparoscopy at a university hospital. PATIENT(S): Fifty women with previously untreated endometriosis, 8 women on GnRH agonists for endometriosis, and 18 control women with normal pelvic anatomy who were undergoing tubal ligation. INTERVENTION(S): Peritoneal fluid IL-18 levels as measured by ELISA. MAIN OUTCOME MEASURE(S): Peritoneal fluid IL-18 levels. RESULT(S): Peritoneal fluid IL-18 levels were significantly higher in women with previously untreated endometriosis (mean +/- SEM, 91.1 +/- 6.5 pg/mL) than in control women (59.4 +/- 2.0 pg/mL). Interestingly, women with superficial (100.0 +/- 10.2 pg/mL) and deep peritoneal implants (94.0 +/- 10.8 pg/mL) had significantly higher PF IL-18 levels than did women with endometriomas (57.8 +/- 1.8 pg/mL). Similarly, women with stage I-II endometriosis (97.3 +/- 8.0 pg/mL), but not women with stage III-IV endometriosis (74.9 +/- 9.9 pg/mL), had significantly higher PF IL-18 levels than did control women. Peritoneal fluid IL-18 levels were significantly higher in the luteal phase than in the follicular phase but did not discriminate between women with pelvic pain or infertility. CONCLUSION(S): Peritoneal fluid IL-18 is elevated in women with peritoneal, minimal- to mild-stage endometriosis.     

Mouse embryo toxicity of IL-6 in peritoneal fluids from women with or without endometriosis

BACKGROUND: To determine whether there is a factor (or factors) in the peritoneal fluid of endometriosis patients that impairs embryo growth and embryo implantation. METHODS: Growth and development of two-cell mouse embryos which were cultured in media with peritoneal fluid from women with or without endometriosis and interleukin-1-beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) levels in conditioned media were measured. RESULTS: The blastocyst rate in the non-endometriosis group was 46.4 +/- 31.1%, and that of the endometriosis group was 54.6 +/- 28.7%. Logistic regression analysis using the criteria of blastocyst development in 454 embryos, showed that the peritoneal fluid from endometriosis could promote (p=0.015) but IL-6 could arrest embryo growth to blastocyst (p=0.025). IL-1beta and TNF-alpha levels had no significant effect on blastocyst formation. CONCLUSION: Peritoneal fluid from women with endometriosis was not toxic to mouse embryo development. However, IL-6 in the peritoneal fluid deteriorated the growth and development of mouse embryos.     

Peritoneal fluid and serum levels of hepatocyte growth factor may predict the activity of endometriosis

BACKGROUND: The suitable parameter in PF as well as in serum that may predict the activity of endometriosis is not well described. Therefore, we tried to examine the peritoneal fluid (PF) and serum concentrations of hepatocyte growth factor (HGF) in different revised American Society of Reproductive Medicine (r-ASRM) staging and morphologic appearances of endometriosis in an attempt to determine whether HGF can be clinically useful to predict the activity of pelvic endometriosis. METHODS: Peritoneal fluid was collected from 137 women with endometriosis and 57 women without endometriosis during laparoscopy and blood sampling was collected from 37 women with endometriosis and 21 women without endometriosis before laparoscopy. The concentration of HGF in PF and serum was measured by enzyme-linked immunosorbent assay. The ability of isolated macrophages and stroma to secrete HGF in response to lipopolysaccharide (LPS) was evaluated. RESULTS: A significantly increased concentration of HGF in PF was found in women with endometriosis (1451.75 +/- 90.7 pg/mL) than that in non-endometriosis (1120.5 +/- 77.3 pg/mL, p < 0.01) without any remarkable difference in HGF levels between women with stage I-/II endometriosis and stage III-/IV endometriosis. When we distributed serum and PF levels of HGF according to different color appearances of endometriosis, we found a significantly higher serum and PF levels of HGF in women containing dominant red peritoneal lesions in pelvic cavity (740 +/- 109.3 pg/mL for serum; 1685 +/- 183.4 pg/mL for PF) than those having other pigmented lesions (649 +/- 79.5 pg/mL, p < 0.05 for serum; 1224 +/- 67.8 pg/mL, p < 0.05 for PF) or chocolate cysts (485 +/- 43.1 pg/mL, p < 0.05 for serum; 1118 +/- 83.1 pg/mL, p < 0.01 for PF). Exogenous stimulation with LPS significantly increased the production of HGF in the culture media by macrophages and stroma derived from women with endometriosis than that in women without endometriosis. CONCLUSIONS: These results suggest that women with early or advanced endometriosis as measured by r-ASRM scoring system are not associated with an increase in either serum or PF concentrations of HGF. Rather HGF levels in serum and PF were significantly increased in women harboring blood-filled red peritoneal lesions and may be clinically useful to predict the activity of pelvic endometriosis.     

Peritoneal fluid volume, 17beta-estradiol and progesterone concentrations in women with endometriosis and/or luteinized unruptured follicle syndrome

Data on the presence of an ovulation ostium and the volume and the concentrations of estradiol (17 beta-estradiol) and progesterone In women with endometriosis (n = 80) and women with luteinized unruptured follicle (LUF) syndrome (n = 32) are reported and compared with data obtained from normal ovulatory women, previously reported. in women with endometriosis, less ovulation ostia were observed, the difference being significant in moderate and severe endometriosis. During the luteal phase, no statistical difference was found in the amount of peritoneal fluid of women with endometriosis. Estradiol and progesterone levels in the peritoneal fluid of normal women and women with mild endometriosis were not significantly different. Lower steroid concentrations found in peritoneal fluid of women with moderate (phase days 20-22) and severe endometriosis (phase days 14-19 and 20-22) may explain the high incidence of infertility reported in these women (peritoneal steroids deficiency). During the phases days 14-19 and 20-22, very low peritoneal steroid concentrations were found in women with LUF syndrome. It is suggested that progesterone assay in peritoneal fluid is an aid to diagnose the luteinized unruptured syndrome.     

Role of cytokines in progression of endometriosis

Peritoneal fluid in women with endometriosis contains an increased number of activated macrophages that secrete a variety of cytokines, including interleukin (IL)-6, IL-8, vascular endothelial growth factor, and tumor necrosis factor-alpha (TNF-alpha). Cytokines may be involved in the control of implantation and the growth of endometrial cells outside the uterus. In addition, several cytokines have been implicated in or directly associated with angiogenic activity in endometriosis. There could be a relationship between the levels of cytokines in the peritoneal fluid of patients with endometriosis and the status of the lesions in such patients. Peritoneal endometriosis can be classified as having red, black, or white lesions. Red lesions are known to be an active form of early endometriosis, because vascularization and mitotic activity are shown to be most prominent in these lesions. We found that the peritoneal fluid levels of TNF-alpha and IL-8 were significantly higher in patients with endometriosis, and correlated with the size and number of active lesions. In addition, TNF-alpha and IL-8 stimulated the growth of ectopic endometrial stromal cells. These cytokines with angiogenic activity may therefore have significant roles in the pathogenesis of endometriosis.     

Decreased peritoneal concentrations of interleukin-15 in women with advanced stage endometriosis

OBJECTIVE: To assess the concentrations of interleukin-15 (IL-15) in peritoneal fluid from women with endometriosis and fertile disease-free controls. STUDY DESIGN: Peritoneal fluid samples were obtained from 50 women with endometriosis and 29 fertile women having tubal ligation. Concentrations of IL-15 were measured. RESULTS: The mean (S.D.s) concentration of IL-15 in peritoneal fluid was 11.17 pg/mL (3.89) for women with endometriosis, and 12.59 pg/mL (4.11) for fertile disease-free controls. The difference of peritoneal IL-15 concentrations between endometriosis and control women was not statistically significant. However, peritoneal IL-15 concentrations were significantly lower in women with moderate/severe endometriosis when compared with those in women with minimal/mild endometriosis, and in controls (P<0.05). In addition, peritoneal IL-15 concentrations did not correlate with the phase of menstrual cycle in endometriosis or control women. CONCLUSIONS: Our results suggest that the decreased peritoneal IL-15 concentrations in women with moderate/severe endometriosis imply a role of IL-15 in the pathogenesis of advanced endometriosis as compared to those with minimal/mild endometriosis and fertile disease-free controls.     

Higher activity by opaque endometriotic lesions than nonopaque lesions

BACKGROUND: Higher activity by early endometriosis than advanced endometriosis has been reported. However, the pattern of activity in individual colored endometriotic lesions in pelvic cavity is unknown. We investigated the variation in activity of the different colored morphologic lesions as proposed by the current revised American Society of Reproductive Medicine (ASRM) classification in women with endometriosis. METHODS: Peritoneal fluid (PF) and biopsies were collected during laparoscopy from 45 infertile women with pelvic endometriosis and 15 women without endometriosis. PF concentrations of hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) were measured by enzyme-linked immunosorbent assay (ELISA). The immunohistochemical staining of HGF and proliferating cell nuclear antigen (PCNA) in opaque and nonopaque endometriotic lesions was examined using respective antibodies, and a computer-analyzed modified quantitative-histogram score (Q-H score) was used to quantify the immunoreaction. RESULTS: Opaque red lesions were defined as nontransparent lesions, including polypoid excrescence, blood bleb or ecchymosis, and nonopaque lesions were defined as translucent or transparent lesions, including vesicular bleb or clear papule. The women with endometriosis containing dominant opaque red lesions showed significantly higher PF concentrations of HGF, VEGF, IL-6 or MCP-1 than those with either nonopaque lesions or black lesions or control women. There was no difference in any of these cytokine or chemokine levels between women harboring nonopaque lesions and black lesions. Interestingly, PF concentrations of all these markers were significantly reduced in women containing true black lesions and true white lesions after excluding the presence of coexisting red spot from dominant black or white lesions. In a parallel fashion, the immunoexpressions of HGF and PCNA in intact tissue as shown by the Q-H score were significantly stronger in the opaque red lesions than in the nonopaque lesions or black lesions. CONCLUSIONS: Opaque red lesions display a higher activity than with nonopaque red lesions in both PF and intact tissue. This indicates different histogenesis in their generation in the pelvic cavity.     

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.     

Effect of ovarian involvement on peritoneal fluid cytokine concentrations in endometriosis patients

Peritoneal fluid cytokines are important for initiation and progression of endometriosis. The objective of this study was to compare a group of five cytokines (interleukins IL-1 beta, IL-6, IL-8, IL-13 and tumour necrosis factor alpha; TNFalpha) in peritoneal fluid of endometriosis patients with ovarian involvement (Group I, n = 17) to those in patients without ovarian involvement (Group II, n = 33) and to a reference group without endometriosis (Group III, n = 25). All three groups were comparable regarding age, parity and body mass index. IL-8 concentrations were significantly higher in groups I and II compared with the reference group (P = 0.01 and 0.02, respectively). Similarly, TNFalpha concentrations were significantly higher in groups I and II compared with the reference group (P < 0.0001 and 0.0004, respectively). All other cytokines were comparable in the three groups. No significant differences were found between groups I and II with respect to the cytokines measured. In conclusion, peritoneal fluid IL-8 and TNFalpha concentrations are significantly higher in endometriosis. Ovarian involvement does not alter the pattern of cytokines. It appears that the inflammatory mediators of endometriosis are similar with and without ovarian involvement.     

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.     

High concentrations of activin A in the peritoneal fluid of women with epithelial ovarian cancer

OBJECTIVE: The aim of the present study was to evaluate the concentrations of activin A in the peritoneal fluid of women with epithelial (serous) ovarian cancer. METHODS: A group of 160 women was studied and divided in four subgroups as follows: 1) serous ovarian carcinoma (n = 32); 2) serous ovarian cystadenoma (n = 20); 3) endometriosis (n = 53); and 4) healthy controls (n = 55), including both fertile (n = 32) and postmenopausal women (n = 23). Specimens of peritoneal fluid were collected during surgical interventions, and activin A was quantified using a specific two-site enzyme immunoassay. RESULTS: Peritoneal fluid activin A concentrations in women with ovarian carcinoma were about five-fold higher than those found in the control group (median [interquartile range] = 7.60 [2.85-10.15] and 1.50 [1.00-2.50] ng/mL, respectively, P <.001). In contrast, the women with benign serous cystadenoma had peritoneal fluid activin A concentrations (1.50 [1.0-2.70] ng/mL) similar to those of the control group. High peritoneal fluid activin A levels (>2 multiples of the mean) distinguished carcinoma from cystadenoma with a sensitivity of 72% and a specificity of 80%. The follow-up of nine patients with stage IIIc ovarian cancer showed no apparent relationship between the peritoneal fluid activin A levels and overall survival. No significant difference in peritoneal fluid activin A concentrations between patients with endometriosis and control women was observed. CONCLUSION: Most women with serous ovarian carcinoma had high concentrations of activin A in the peritoneal fluid, supporting a possible role of this growth factor in ovarian cancer.     

Elevated interleukin-10 and sex steroid levels in peritoneal fluid of patients with ovarian hyperstimulation syndrome.

BACKGROUND: The ovarian hyperstimulation syndrome (OHSS) following ovulation induction is characterized by a cystic enlargement of the ovaries with an acute third space fluid sequestration. Inflammatory cytokines mediate the inflammatory response (IL-1, IL-2, IL-6, IL-8, TNFalpha) and play a crucial role in the pathogenesis of OHSS. OBJECTIVE: To determine the role of the anti-inflammatory cytokine interleukin-10 (IL-10) in OHSS and to examine its correlation with 17beta-estradiol and progesterone. STUDY DESIGN: Peritoneal fluid and serum samples were collected from 9 patients with severe OHSS after ovulation induction by administration of GnRH-analogues followed by hMG (n=5) or recombinant FSH (n=4). Patients (n=19) without pathological findings at laparoscopy served as non-pregnant controls and pregnant women (n=14) between 7 and 16 weeks of gestation served as positive controls. Samples were assayed for IL-10 by commercially available ELISA and for for 17beta-estradiol and progesterone by RIA. Statistical analysis was performed by non-parametric Mann-Whitney U-test and results are presented as the median and range. RESULTS: OHSS patients had significantly higher peritoneal fluid IL-10, 17beta-estradiol and progesterone levels than patients during early pregnancy and than the control group. No correlation was found between peritoneal fluid or serum IL-10 and 17beta-estradiol or progesterone in the different groups. Serum 17beta-estradiol and progesterone, but not serum IL-10 levels were elevated in OHSS and during early pregnancy. CONCLUSIONS: High concentrations of IL-10 in peritoneal fluid suggest a role of this anti-inflammatory cytokine during OHSS. 17beta-estradiol and progesterone were elevated in peritoneal fluid and serum during OHSS but no correlation with IL-10 concentrations was found. Therefore, we assume that IL-10 has a role in OHSS as a local mediator of inflammation, however, it presents different aspects of the OHSS than the sex steroids 17beta-estradiol and progesterone.     

Peritoneal fluid evaluation in infertility

Cul-de-sac fluid collected at laparoscopy from 58 women during various stages of the menstrual cycle was evaluated by volume and by estradiol and progesterone content. Volumes were consistently higher in the third cycle week, especially with evidence of a fresh corpus hemorrhagicum. Peritoneal hormonal values were equal to serum values before ovulation, but were significantly elevated after ovulation. In the present study, maximal fluid volume was 78 ml, maximal peritoneal estradiol exceeded 3000 pg/ml, and maximal peritoneal progesterone exceeded 265 ng/ml. Luteal phase peritoneal fluid volumes and hormonal values were not significantly different in women with or without endometriosis. Aspiration of peritoneal fluid at laparoscopy in the infertile woman may provide significant information regarding ovulation.     

Different concentrations of interleukins in the peritoneal fluid of women with endometriosis: relationships with lymphocyte subsets.

The present study explored the possible relationships between immune cell subsets and interleukin (IL)-12 or IL-13 levels in the peritoneal fluid of patients with and without endometriosis. Peritoneal fluid samples were obtained from 80 women while they were undergoing laparoscopy for pain, infertility, tubal ligation or re-anastomosis. The American Fertility Society scoring system was used to determine the extension of endometriosis. The peritoneal fluid mononuclear cells were analyzed for immunophenotyping using cytometry, whereas peritoneal fluid concentrations of interleukins were measured using two ultrasensitive commercially available enzyme-linked imnunosorbent assay kits. Significantly higher peritoneal fluid IL-12 levels were found in women with moderate or severe endometriosis (stages III and IV) than in healthy controls (p < 0.01). Conversely, subjects with endometriosis showed remarkably lower peritoneal fluid IL-13 concentrations than controls, independent of the severity of the disease (p < 0.05). Considering immune system effectors, patients with endometriosis presented a significantly higher peritoneal fluid CD8+/CD4+ ratio when compared with healthy controls. Moreover, the number of peritoneal fluid CD8+ and CD4+ activated T cells was significantly lower in the former than in the latter group, independent of the endometriosis stage. Connections were observed between peritoneal fluid interleukins and peritoneal fluid T cells: both patients with endometriosis and controls presented an inverse correlation between peritoneal fluid activated T cells and IL-13 levels, and a direct correlation between peritoneal fluid T cells and IL-12 concentrations. These data seem to suggest that a reciprocal modulation exists between peritoneal fluid cytokines and T lymphocyte subsets in patients with endometriosis.     

Peritoneal fluid interleukin-6 in women with chronic pelvic pain

OBJECTIVE: To determine the relationship between peritoneal fluid concentrations of interleukin-6 (IL-6) and chronic pelvic pain symptomatology in women with adhesions, endometriosis, or no obvious intraperitoneal pathology. DESIGN: Clinical research study. SETTING: Healthy volunteers in an academic research environment. PATIENT(S): Reproductive-aged women undergoing laparoscopy for the diagnosis of pelvic pain, infertility, or sterilization were selected. INTERVENTION(S): Peritoneal fluid was collected at the time of the laparoscopy and later assayed for IL-6. Subjects completed a pelvic pain questionnaire, and operative reports were used to obtain the underlying diagnosis. MAIN OUTCOME MEASURE(S): Interleukin-6 concentrations. RESULT(S): No correlation between the presence or absence of pelvic pain, findings of adhesions or endometriosis, and the concentration of peritoneal fluid IL-6 was observed. Conclusion(s): The cytokine IL-6 does not seem to play a role in the genesis of chronic pelvic pain in women with adhesions or endometriosis.     

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.     

Association of leptin with inflammatory cytokines and lymphocyte subpopulations in peritoneal fluid of patients with endometriosis

INTRODUCTION: Endometriosis is a common, complex and chronic disease related to ectopic implantation and growth of endometrial tissue that may manifest by pelvic inflammatory reactions, chronic pelvic pain and subfertility. Endometriosis may be associated with increased peritoneal fluid leptin levels. Leptin is known to exert immunomodulatory effects; however, an association between leptin and inflammatory reactions in endometriosis has not been documented. Therefore, the aim of this study was to investigate a relationship between leptin concentrations in peritoneal fluid and the levels of peritoneal fluid inflammatory cytokines and mononuclear leukocyte subpopulations. MATERIALS AND METHODS: Peritoneal fluid was aspirated by laparoscopy from 46 women in whom endometriosis had been confirmed by clinical and histopathological examinations and from 10 control women qualified for ART in whom pelvic pathology has been excluded. Concentrations of leptin and inflammatory cytokines (IL-1beta, IL-6, IFN-gamma and TNF) in peritoneal fluid were evaluated by specific ELISAs. Percentage of peritoneal leukocyte subpopulations (CD3+, CD4+, CD8+ and CD14+) was analyzed by FACS using specific monoclonal antibodies. RESULTS: Leptin concentrations in peritoneal fluid correlated negatively with concentrations of IL-1beta and IFN-gamma (r(s)=-0.38, p=0.01 and r(s)=-0.31, p=0.03, respectively) and correlated positively with the percentage of CD3+ pan-T cells (r(s)=0.69, p=0.009) and CD4+ T helper cells (r(s)=0.74, p=0.036). CONCLUSIONS: Increased leptin levels in peritoneal fluid from endometriosis patients may affect local inflammatory/immune reactions, especially infiltration of CD4+ T helper cells. Thus, leptin may play an important role in the immunopathogenesis of endometriosis.