Antigenic differences between the endometrium of women with and without endometriosis

Serum and peritoneal fluid from 12 women with endometriosis, 4 women with uterine leiomyomata and 6 fertile women without endometriosis (controls) and serum from 4 women with adenomyosis were tested with a passive hemagglutination assay for antibodies against endometrium from all the controls, 8 patients with endometriosis and all patients with uterine leiomyomata and from implants from 8 patients with endometriosis. Serum antibody titers in patients with endometriosis or leiomyomata were significantly higher against endometrial or implant antigens from patients with endometriosis and 2 patients with leiomyomata than those against the controls' endometrium. Peritoneal fluid endometrial antibody titers failed to reflect these antigenic differences. Controls and patients with adenomyosis had low titers of endometrial antibodies in their serum or peritoneal fluid. Antigenic differences appear to exist between the endometrium of patients with endometriosis and that of controls.     

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.     

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.     

Effect of human seminal fluid on the growth of endometrial cells of women with endometriosis

Objectives

We investigated the effect of human seminal fluid on the growth of endometrial cells derived from women with and without endometriosis.

Study design

Seminal plasma (SP) was collected from 18 healthy fertile men. Serum, peritoneal fluid (PF) and tissue specimens of eutopic and ectopic endometrium were collected from 45 women with endometriosis and 20 women without endometriosis during laparoscopic surgery. Prostaglandin (PG) E2, hepatocyte growth factor (HGF), and estradiol (E2) levels in each sample of SP, serum and PF were measured by enzyme-linked immunosorbent assay. The growth pattern of cells derived from eutopic and ectopic endometria in response to SP was examined by 5-bromo-2-deoxyuridine (BrdU) incorporation assay.

Results

Seminal plasma was able to significantly stimulate the growth of epithelial cells and stromal cells derived from the eutopic and ectopic endometria of women with endometriosis (2–3-fold) when compared with control media. The SP-promoted proliferation of both gland cells and stromal cells derived from eutopic endometria was also remarkably higher in women with endometriosis than that of women without endometriosis. Although levels of PGE2, HGF and E2 in SP were variable when compared with other body fluids, the levels of PGE2 and HGF in SP were significantly higher than those in either peritoneal fluid or serum of women with or without endometriosis. Pretreatment of cells with individual anti-PGE2 antibody, anti-HGF antibody and two selective estrogen receptor modulators, tamoxifen and raloxifene was unable to suppress SP-mediated growth of endometrial cells. However, pretreatment of cells with combined anti-PGE2 antibody plus anti-HGF antibody or combined anti-PGE2 antibody plus anti-HGF antibody plus tamoxifen or raloxifene was able to significantly suppress SP-promoted growth of eutopic and ectopic endometrial cells.

Conclusion

Human seminal fluid enriched with different macromolecules may promote the growth of endometrial cells derived from women with endometriosis. Our findings may suggest some detrimental effect of unprotected sexual intercourse in women with endometriosis.     

Immunological aspects of surgically induced experimental endometriosis: variation in response to therapy

Elevated endometrial antibody titers were detected in serum and peritoneal fluid of rabbits with experimentally induced endometriosis. Surgical extirpation of implants or suppression with gonadotropin-releasing hormone agonist resulted in decreased endometrial antibody titers, whereas the antibody titers in untreated rabbits with endometriosis increased significantly. Endometrial implants and normal endometrial tissue had similar proteins by polyacrylamide gel electrophoresis. However, the serum and peritoneal fluid from rabbits with experimentally induced endometriosis had gamma G immunoglobulin antibodies to an endometrial protein with molecular weight of approximately 40 kD. These antibodies were absent in rabbits without endometriosis. Isolation of endometrial antigens eliciting the humoral immune response in endometriosis may aid in the development of a specific antibody marker for endometriosis.     

Histocompatibility leukocyte antigen-G is not expressed by endometriosis or endometrial tissue

OBJECTIVE: The immunological mechanisms that support persistence and proliferation of ectopic endometrial implants within the peritoneal cavity of women with endometriosis are unknown. Inhibition of natural killer (NK) and cytotoxic T-cell function has been proposed as a mechanism. We tested the hypothesis that expression of a nonclassical major histocompatibility antigen, HLA-G, might explain the local immunosuppression associated with ectopic endometrium. DESIGN: Nested case-control study of women with and without laparoscopic evidence of endometriosis. SETTING: Reproductive endocrinology clinic at a university hospital. PATIENT(S): Peritoneal fluid specimens from 10 women with revised AFS stage I-IV endometriosis and from 10 age-matched normal controls without laparoscopic evidence of endometriosis were tested for the presence of HLA-G protein. Endometriosis and normal endometrial biopsies from four patients were used to prepare stromal cell cultures directly evaluated for HLA-G protein. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The expression of HLA-G in peritoneal fluid, tissue, and cell cultures was determined by immunoblotting with a specific monoclonal antibody. RESULT(S): HLA-G protein was not detectable in peritoneal fluid specimens of endometriosis patients or controls. Moreover, ectopic and normal endometrial tissues and stromal cells did not express HLA-G. CONCLUSION(S): Immune cell inhibition in endometriosis must be mediated by factors other than HLA-G.     

Endometrial autoantigens eliciting immunoglobulin (Ig)G, IgA, and IgM responses in endometriosis

Endometrial antigens from patients with endometriosis and fertile controls were tested against immunoglobulin (Ig)G, IgA, or IgM in endometrium, serum, and peritoneal fluid (PF) of the patients and controls by Western blot analysis. Endogenous IgG was detected in 78% of the endometria or endometriosis implants from the patients and 22% of the endometria from the controls. Endometrial IgA and IgM were detected in few controls and patients. Immunoglobulin G in the serum and/or PF of patients with endometriosis was specifically directed against antigens with molecular weights of 34, 42, 82, 94, 110, 120, and 140 kd found in the patients' endometrium or endometriosis implants. Immunoglobulin A and IgM in the serum or PF of the patients and controls were nonspecific in their reactivity. Endometrial antigens found in endometrium or implants of patients with endometriosis, and eliciting IgG responses, may be relevant to autoimmunity in endometriosis.     

Serum and peritoneal fluid proteins in women with and without endometriosis

We examined the proteins in serum and peritoneal fluid of women with endometriosis (and of healthy controls) for evidence of an autoimmune response that might account for their impaired fertility. No antibodies against endometrial glycoproteins or against "progestin dependent endometrial protein" (PEP) were found in any serum or peritoneal fluid sample. Levels of PEP were not different in serum from women with moderate to severe endometriosis (n = 6), with mild endometriosis (n = 21), or from disease-free cycling controls (n = 19). PEP levels in peritoneal fluid from mild endometriosis and from controls did not differ but were elevated ten times in fluid obtained in the secretory phase from women with moderate to severe disease. This suggests that PEP levels in peritoneal fluid reflect the extent of ectopic endometrial growth. The salient finding was a heretofore undescribed protein (mol wt 70,000) in secretory phase peritoneal fluid samples (18/20) and its absence during the proliferative phase (0/35).     

Endometrial antibodies in serum and peritoneal fluid of infertile patients with and without endometriosis

Passive hemagglutination assay was used to evaluate endometrial antibodies in serum and peritoneal fluid of 37 patients with endometriosis and 54 patients without endometriosis. The results showed that the concentration of antibody titers in serum and peritoneal fluid was significantly higher for endometriosis than control patients. The severity of endometriosis has no effect on antibody concentration. Furthermore, the concentration of endometrial antibody titers was significantly higher in serum than peritoneal fluid of patients with endometriosis. These results suggest that serum endometrial antibody assay is specific and valuable for the diagnosis and progress of endometriosis.     

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.     

An endometrial antibody assay in the clinical diagnosis and management of endometriosis

Coded serum samples from 11 normal fertile men and 17 fertile women without endometriosis (control groups) and 41 women with endometriosis were tested blindly for the presence of endometrial antibodies by use of a passive hemagglutination assay. Endometrial antibodies were either absent or present in low baseline titers in the serum samples from the control group. In contrast, 17 of the 23 (74%) patients with untreated endometriosis had elevated titers of endometrial antibodies in their serum. Of the 18 patients treated with danazol, endometrial antibodies were absent in 7 women who showed a good response at repeat laparoscopy, whereas 4 of 5 women with a poor response had significantly positive titers of antibodies. Six patients treated with danazol did not have repeat laparoscopy, but were found to have endometrial antibody titers in the baseline control range. Endometrial antibody titers in women with a good response to danazol were significantly lower than those in women with untreated endometriosis or with a poor response to danazol (P = 0.003). No correlation was observed between the antibody titers and the stage of endometriosis. The results suggest that the assay for serum endometrial antibodies may prove to be a clinically useful, noninvasive aid in the diagnosis of endometriosis. Sequential determination of endometrial antibody titers may be helpful in assessing the efficacy of pharmacologic therapy for endometriosis and evaluating the cases of patients with possible recurrence of the disease.     

Peritoneal fluid-mediated enhancement of eutopic and ectopic endometrial cell proliferation is dependent on tumor necrosis factor-alpha in women with endometriosis

OBJECTIVE: To determine the effect of autologous peritoneal fluid and tumor necrosis factor-alpha (TNF-alpha) on proliferation of endometrial cells from women with endometriosis. DESIGN: Endometrial cells from eutopic and ectopic endometrium were cultured in vitro with peritoneal fluids or recombinant TNF-alpha for 72 hours before DNa synthesis determination by 3H-thymidine labeling and liquid scintillation counting. SETTING: An institute for the study and treatment of endometriosis and university-based research laboratories. PATIENT(S): Thirty-five women with endometriosis and 17 controls without endometriosis. MAIN OUTCOME MEASURE(S): In vitro incorporation of 3H-thymidine in endometrial cells was examined. RESULT(S): Peritoneal fluid from women with endometriosis enhanced proliferation of autologous and heterologous endometrial cell cultures from women with endometriosis. The soluble TNF-receptor etanercept blocked the ability of peritoneal fluid from women with endometriosis to enhance proliferation of eutopic or ectopic endometrial cells. Recombinant TNF-alpha also enhanced proliferation of eutopic and ectopic endometrial cells from women with endometriosis. In contrast, autologous peritoneal fluid, heterologous peritoneal fluid from women with endometriosis, and recombinant TNF-alpha failed to enhance, and often inhibited, the proliferation of eutopic endometrial cells from controls without endometriosis. CONCLUSION(S): Endometrial cells from women with endometriosis can utilize factors in peritoneal fluids, such as TNF-alpha, to facilitate proliferation in ectopic environments. Endometrial cells from women without endometriosis do not share this ability, suggesting that this abnormality is etiologically related to development of the disease. Therapy with agents that block the effects of TNF-alpha may be warranted.     

Alterations in humoral immune responses associated with recurrent pregnancy loss

OBJECTIVE: To investigate the reactivity of maternal antibodies with endometrium-derived antigens and to correlate their association with recurrent pregnancy loss (RPL). DESIGN: Prevalence study. SETTING: Academic research center. PATIENT(S): Nulliparous women (n = 10), women with RPL (n = 15), pregnant women (n = 8), and multiparous women with a normal obstetric history (n = 20). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Reactive antibodies were analyzed by Western immunoblot techniques and quantitated by densitometry. RESULT(S): Antibodies from women with RPL and multiparous women recognized antigens ranging from 10-120 kd on normal endometrium and endometrial tumors. Antibodies from most women with RPL (10/15) and from multiparous women (15/20) recognized 65-kd and 80-kd proteins in normal endometrium. Antibodies from women with RPL recognized 21-kd and 28-kd antigens (12/15 and 13/15, respectively) in endometrial tumors at a significantly greater rate (than did antibodies from multiparous women (5/20 and 8/20, respectively). Women with RPL had significantly lower levels of asymmetric IgG compared with controls. CONCLUSION(S): Recurrent pregnancy loss may be linked with the failure to elicit asymmetric IgG and a unique immunologic recognition of endometrial antigens.     

Immunoglobulin content of the endometrium in women with endometriosis

A study of the immunoglobulin content of endometria from women with and without endometriosis has shown that, in women with endometriosis, both IgG and IgA are more commonly found in the interstitium of the endometrial stroma than is the case in endometria from women without this disease. It is thought that the increased stromal content of immunoglobulins in endometriosis is simply a passive reflection of elevated serum IgG and IgA levels. Both the incidence and extent of positive endometrial glandular epithelial staining for IgG and IgA are markedly increased in women with endometriosis: the excess of intraepithelial IgA is probably simply a consequence of the excess of stromal IgA, but the increased epithelial staining for IgG lends support to the concept that many women with endometriosis develop autoantibodies directed against an endometrial epithelial antigen. No relationship could be demonstrated, however, between IgG deposition in the endometrium of women with endometriosis and a history of infertility.     

Cytolysis of Eutopic and Ectopic Endometrial Cells by Peripheral Blood Monocytes and Peritoneal Macrophages in Women with Endometriosis

Objective: To compare the ability of peripheral blood monocytes (PBM) and peritoneal macrophages (PM) to mediate the in vitro cytolysis of endometrial cells from eutopic and ectopic endometrium in women with endometriosis.

Design: Prospective study of immune function.

Setting: Institute for the Study and Treatment of Endometriosis and university-based research laboratories.

Patient(s): Twenty-four women with endometriosis (15 in stage I/II, 9 in stage III/IV) and 4 patients treated with GnRH agonists.

Intervention(s): Peritoneal fluid and peripheral blood were sampled and eutopic and ectopic endometrium were biopsied during diagnostic laparoscopy.

Main Outcome Measure(s): Lysis of autologous endometrial cells.

Result(s): Peripheral blood monocytes were significantly more cytolytic than peritoneal macrophages against autologous uterine endometrial cells. However, PBM and PM displayed a similar degree of cytolysis against a hepatoma cell line. Ectopic endometrial cells were significantly more resistant to cytolysis by autologous PBMC than were matched eutopic endometrial cells, and were completely resistant to cytolysis by autologous PM.

Conclusion(s): The reduced capacity of PM from women with endometriosis to mediate the destruction of endometrial cells coupled with the increased resistance of ectopic endometrial cells to macrophage-mediated cytolysis may facilitate the survival of these cells within the peritoneal cavity of women with endometriosis.     

Spontaneous Apoptosis of Endometrial Tissue is Impaired in Women with Endometriosis

Objective: To evaluate spontaneous apoptosis in single-cell suspensions of eutopic and ectopic endometrium from women with endometriosis and in eutopic endometrium from fertile controls without endometriosis.

Design: Paired specimens of eutopic and ectopic endometrial tissue from patients with endometriosis and eutopic endometrium from controls were assessed for spontaneous apoptosis.

Setting: Institute for the Study and Treatment of Endometriosis and university-based research laboratories.

Patient(s): Fertile controls (n = 10) and women with untreated endometriosis (n = 16).

Intervention(s): None.

Main Outcome Measure(s): Spontaneous apoptosis assessed with an ELISA-based cell death detection kit.

Result(s): Spontaneous apoptosis (monitored by absorbance) of eutopic endometrium from patients with endometriosis and fertile controls was 0.63 ± 0.1 and 1.43 ± 0.11, respectively. Among patients with endometriosis, spontaneous apoptosis of ectopic endometrium was 0.26 ± 0.06. Decreased apoptosis of ectopic versus eutopic endometrium was observed independent of cycle phase.

Conclusion(s): The susceptibility of endometrial tissue to spontaneous apopoptosis is significantly lower in women with endometriosis than in fertile controls. We suggest that decreased susceptibility of endometrial tissue to apoptosis contributes to the etiology or pathogenesis of endometriosis.     

Serum VEGF (vascular endothelial growth factor) concentration in patients with endometriosis

The theory of Sampson that endometrial cells and fragments desquamated during the menstrual period are transported through fallopian tubes into the peritoneal cavity where they implant, proliferate and develop into endometriotic lesions is generally accepted. Accumulating data suggest that deficient immunity against retrograde endometrium during menstruation may be involved in the pathophysiology of endometriosis. Recent studies in women with endometriosis demonstrated functional changes in several immunologic components in the peritoneal fluid as well as in sera of those patients. Among others it was shown that a wild pattern of cytokines take part in events occurring during endometrial cells implantation, proliferation and forming of endometriotic lesions. One of them VEGF seems to play a very important role in neovascularisation and implantation of ectopic endometrial lesions. In present study we evaluated the concentrations of VEGF in serum of patients with endometriosis and showed negative correlation between AFS score and VEGF concentration in peritoneal endometriosis. Above results do not confirm former observations indicating the role of VEGF in endometriosis pathogenesis.     

CA 125 in serum, peritoneal fluid, active lesions, and endometrium of patients with endometriosis

Ca 125 levels in serum and peritoneal fluid were measured in 39 patients with endometriosis and 18 patients with normal pelvic anatomy at laparoscopy, and the presence of this antigen in endometriotic tissue and endometrial mucosa was also investigated. Serum CA 125 concentrations were elevated in patients with Stage III or IV endometriosis compared with control subjects (32.9 +/- 11.2 versus 16.4 +/- 8.9 U/ml, means +/- SD; p less than 0.001). CA 125 values were greater than 35 U/ml in 36.8% of women with Stage III or IV endometriosis and in none of the control subjects. No significant differences in CA 125 levels in peritoneal fluid were found between patients with endometriosis and control subjects. The immunohistochemical studies found CA 125 in 10% of the endometriotic lesions and 37.5% of the endometrial samples of patients with endometriosis and in 33.3% of the endometrial samples of control subjects.     

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.     

Localization of anti-endometrial antibody binding in women with endometriosis using a double-labelling immunohistochemical method

Summary. Anti-endometrial antibody binding was localized using a double-labelling immunohistochemical method on frozen sections of endometrium taken from a woman without pelvic disease. Serum from 40 women with endometriosis was tested and, as controls, serum samples from 20 adult males and 20 umbilical cords. The method allowed compensation for endogenous immunoglobulins in endometrium and accurate localization of anti-endometrial antibody binding in the cytoplasm of the glandular epithelium. Significantly more women with endometriosis (14/40) were found to have anti-endometrial antibodies than controls (1/40) ( P < 0.001; χ²). There was no correlation between disease severity and the presence of anti-endometrial antibodies or the intensity of staining.