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.     

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.     

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.     

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.     

Identification of human sperm antigens reacting with antisperm antibodies from sera and genital tract secretions.

OBJECTIVE: To identify sperm antigens reacting with antisperm antibodies relevant in human infertility. DESIGN: The reactions of separated sperm antigens with antibodies present in sera and genital tract secretions from infertile and fertile females and males were examined by immunoblotting techniques. SETTING: The patients were followed in an outpatient setting of a hospital clinic. PATIENTS: One hundred consecutive infertile males and females, referred for determinations of antisperm antibodies, comprised the study group. Fifty hospital and faculty employees with proven fertility served as a control group. RESULTS: A high proportion of sera from fertile and infertile humans contained antibodies reacting with at least one sperm antigen. However, two discrete bands of antigenic proteins with molecular weights of 44 and 72 kd reacted significantly more frequently with serum antibodies from infertile females than from fertile females. No apparent correlation could be demonstrated between any particular antigen and serum antibodies from infertile males. Nevertheless, antigenic proteins of 62 kd were identified as the major sperm antigens reacting with antibodies present in seminal plasmas from infertile males. CONCLUSIONS: The major sperm antigens reacting with systemic antibodies differ from the antigens recognized by local antisperm antibodies. Sperm antigens exhibiting relative molecular weights of 62 kd are major antigens reactive with local antisperm antibodies from infertile humans.     

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.     

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.     

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).     

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.     

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.     

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.     

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.     

子宫内膜异位症不孕患者腹腔液淋巴细胞转化活性及可溶性白细胞介素-2受体的含量

本文采集了15例子宫内膜异位症不孕患者及13例健康生育妇女的腹腔液，分别测定腹腔液淋巴细胞转化活性及淋巴细胞培养上清液中可溶性白细胞介素-2受体（SIL－2R的含量。结果表明，子宫内膜异位症不孕患者淋巴细胞转化活性增强，SIL－2R含量增加。这些变化可能与异位症不孕的发生密切相关。     

Alterations in expression of endometrial endothelial nitric oxide synthase and alpha(v)beta(3) integrin in women with endometriosis

OBJECTIVE: To determine the expression of endometrial endothelial nitric oxide synthase (eNOS) protein and alpha(v)beta(3) integrin in patients with and without endometriosis. DESIGN: Case-control cohort study. SETTING: University-based tertiary care center. PATIENT(S): Endometrial biopsy samples were obtained from 9 fertile women with regular cycles and 30 infertile women with varying severity of endometriosis. Peritoneal fluid levels of nitric oxide were determined in 13 infertile women with a normal pelvis and 12 infertile women with endometriosis. MAIN OUTCOME MEASURE(S): Expression of eNOS and alpha(v)beta(3) integrin protein in the endometrium and peritoneal fluid levels of nitric oxide. RESULTS: In patients with endometriosis, expression of eNOS was significantly increased in the glandular and luminal epithelium, with no significant changes in the stroma. Peritoneal fluid levels of nitric oxide were unchanged, and expression of alpha(v)beta(3) integrin expression in glandular and luminal epithelium was significantly decreased compared with controls. A significant negative correlation was observed between luminal expression of eNOS and alpha(v)beta(3) integrin and between glandular expression of eNOS and luminal expression of alpha(v)beta(3) integrin. CONCLUSION(S): The nitric oxide pathway may play a role in the pathogenesis of 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.     

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.     

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.     

Autoimmune reaction factors of infertility in patients with endometriosis]

The presence of immunoprotein depositions in endometrium and antiendometrial autoantibodies in serum was detected in 59 infertile patients with endometriosis, 45 infertile patients without endometriosis and 11 fertile women by the methods of immunohistochemistry determination and immunodiffusion assay. The immunoprotein depositions in endometrium of endometriosis were significantly increased. The antiendometrial auto-antibodies in serum were positive in 32.2% for infertile patients with endometriosis, 6.7% for infertile patients without endometriosis and 0% for fertile women. This suggested that an autoimmune response might be an etiologic factor of infertility in patients with endometriosis.     

子宫内膜异位症患者辅助性T细胞亚群免疫状态的研究

目的　探讨辅助性T细胞 (Th)亚群在子宫内膜异位症 (内异症 )发病中的作用。方法　采用酶联免疫吸附法检测 30例内异症患者 (内异症组 )及 2 0例非内异症患者 (对照 1组 )血清及腹腔液中白细胞介素 (IL) 2、6的水平 ;用免疫组化技术分别检测IL 2、IL 6在内异症组患者异位内膜组织和 10例子宫肌瘤患者 (对照 2组 )的正常子宫内膜组织中的表达。结果　内异症组患者血清及腹腔液中位数IL 6水平分别为 5 3、2 1ng/L ,对照 1组患者血清及腹腔液中位数IL 6水平分别为 2 5、0 9ng/L ,两组妇女血清和腹腔液中IL 6水平分别比较 ,差异均有统计学意义 (P <0 0 5 ) ;内异症组Ⅲ～Ⅳ期患者血清及腹腔液中位数IL 6水平分别为 13 6、4 1ng/L ,Ⅰ～Ⅱ期患者血清及腹腔液中位数IL 6水平分别为 3 7、1 6ng/L ,Ⅲ～Ⅳ期患者与Ⅰ～Ⅱ期患者血清及腹腔液中位数IL 6水平比较 ,差异均有统计学意义 (P <0 0 5 ) ;内异症组IL 2 /IL 6比值在血清及腹腔液中分别为 0 7、1 1,均分别低于对照组的 0 8、6 2 ,差异也有统计学意义 (P <0 0 5 )。内异症组患者腹腔液与血清IL 6水平呈正相关 (r =0 74 5 ,P <0 0 1) ,血清及腹腔液中IL 6水平与IL 2 /IL 6比值均呈负相关 (r =- 0 4 0 6 ,P <0 0 5 ;r =- 0 4 80 ,P <0 0 5 )     

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.