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.     

Detection of interleukin-6 in human follicular fluid.

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

Interrelationships among semen characteristics, antisperm antibodies, and cervical mucus penetration assays in infertile human couples

Semen characteristics, antisperm antibodies, and cervical mucus penetration studies were analyzed in 754 couples and 95 men undergoing infertility evaluation. The means for the different semen/sperm variables were within ranges published for fertile men. Ages of the men ranged from 22 to 55 years and accounted for a small amount of variation. Sperm counts were lowest in September, December, and January, and highest in April, May, October, and November. Of the sperm characteristics, morphology appeared to be associated with the most other variables. Specimens with more than 50% abnormal sperm forms were overall of significantly poorer quality in terms of sperm counts, motility, forward progression, and ability to penetrate cervical mucus. Antisperm antibodies (agglutinating and immobilizing) were detected in the serum samples of 19.0% of the men, 20.4% of the women, and 32.8% of the couples where one or both partners were positive. Agglutinating antibody titers were significantly correlated between partners. Serum titers of antisperm antibodies were associated with decreased sperm counts, motility, forward progression, and normal forms (immobilizing antibodies). Multiple correlation analysis indicated significant independent effects of sperm concentration, motility, forward progression, and antibodies on sperm-cervical mucus penetration scores of the men. In women, cervical mucus penetration was adversely affected by the presence in the serum of sperm agglutinating antibodies and of immobilizing activity in the cervical mucus.     

Evaluation of the antisperm antibodies in infertile couples afflicted with a cervical factor

OBJECTIVES: The purpose of this study was to evaluate methods used for antisperm antibodies detection in infertility. METHODS: The studied cohort comprised 38 infertile couples with a distinct cervical factor. Presence of antisperm antibodies and their levels in circulation were evaluated in sera samples of both partners and also in the cervical mucus and semen with the Latex Agglutination test. Western Blotting was applied as an additional method in antibody detection. We also assessed: the number of sexual partners, potentially allergizing sexual behaviour and other potentially sensitising factors. RESULTS: The positive antisperm antibodies were detected merely twice and only in one case there was evidence of insemination-impeding antisperm humoral response. The Western Blotting method enabled us to obtain a reaction to a range of sperm proteins which reacted with antibodies both in serum and in seminal plasma. CONCLUSIONS: Determination of infertility on immunological grounds on the basis of a single determinant on sperm presents little diagnostic value. In our view, the combination of patient's clinical status with immune-system response to a spectrum of sperm antigens provides means of infertility evaluation. We propose Western Blotting as an useful technique for detection of antisperm antibodies.     

High CA-125 concentrations in peritoneal fluid of normal cyclic women with various infertility-related factors as demonstrated with two-step immunoradiometric assay

OBJECTIVE: To determine CA-125 concentrations and total amounts in peritoneal fluid (PF) of women with various infertility-related factors throughout the menstrual cycle. DESIGN: Peritoneal fluid was obtained at laparoscopy. CA-125 was determined using the assessed two-step immunoradiometric assay (IRMA) which, in contrast to the one-step IRMA, gives valid results. SETTING: University Hospital Nijmegen, Nijmegen, The Netherlands. PATIENTS: One hundred six infertile women with a regular and ovulatory cycle were included. INTERVENTIONS: None. MAIN OUTCOME MEASURE(S): The mean PF CA-125 concentration and total amount were significantly lower during the luteal phase as compared with other phases of the menstrual cycle. No correlation was found with the presence or absence of endometriosis, adhesions, a male and/or cervical mucus infertility factor, and with patent or closed fallopian tubes. RESULTS: Peritoneal fluid CA-125 concentrations varied from 630 to 12,000 arbitrary units/mL (mean +/- SD = 3,437 +/- 2,286). Total PF CA-125 amounts (concentration x PF volume) varied from 1,760 to 13,300 arbitrary units (mean +/- SD = 30,219 +/- 26,841). CONCLUSIONS: CA-125 secretion into the abdominal cavity varies during the menstrual cycle. Retrograde menstruation is not the main source of CA-125 in PF.     

Results of in vitro fertilization in women with antisperm antibodies in serum,cervical mucus,and follicular fluid

This study was undertaken to investigate the presence of antisperm antibodies (ASA) in serum, cervical mucus, and follicular fluid (FF) of women undergoing in vitro fertilization and embryo transfer (IVF-ET). IgG and IgA ASA directed mostly against sperm head were found at similar concentrations in serum, cervical mucus, and FF of 2 of 34 patients. Ninety-one percent fertilization and 100% cleavage rates, respectively, were observed in one of the two patients. No fertilization occurred in the second patient. In both women, in vitro sperm penetration tests revealed hostile mucus and repeated postcoital tests were poor. It is concluded that the sperm-cervical mucus penetration test and mucus ASA measurements are useful in establishing the diagnosis of immunological infertility.     

Comparative concentrations of steroid hormones and proteins in human peri-ovulatory peritoneal and follicular fluids

Despite the fact that both peritoneal (PF) and follicular (FF) fluids have a common ovarian origin, FF is a natural inducer of sperm acrosome reaction (AR) while PF is not. To better understand these effects, concentrations of oestradiol, progesterone and proteins in peri-ovulatory PF and FF were determined and compared. PF was aspirated by laparoscopy at the peri-ovulatory stage from women with unexplained infertility. FF was collected from patients undergoing IVF and pooled. PF and FF were tested for the presence of antisperm antibodies. Oestradiol and progesterone were measured by enzyme immunoassay, and total protein concentration was determined and analysed. The AR was determined in spermatozoa that were exposed to PF alone, progesterone-supplemented PF, progesterone, control medium, or ethanol. No antisperm antibodies were found in any fluid tested. Oestradiol and progesterone and concentrations in PF were significantly lower than in FF. Protein concentration was also significantly lower in PF than in FF, but no differences were observed between the electrophoretic patterns. When capacitated spermatozoa were exposed to progesterone-supplemented PF there was a significant increase in the percentage of AR with respect to those in PF, control medium or ethanol. These results suggest that the lack of AR-stimulating activity of PF was related to its lower progesterone concentration compared with FF.     

宫颈粘液精子制动活性与抗精子抗体的相关分析

本文用改良间接免疫胶珠法测定宫颈粘液中的抗精子抗体,同时测定宫颈粘液的体外精子制动活性。在100名不孕症妇女排卵期宫颈粘液中,检出抗精子抗体阳性14例,制动活性。在100名不孕症妇女排卵期宫颈粘液中,检出抗精子抗体阳性14例,制动活性阳性29例(包括两法同时阳性者5例)在抗精子抗体与制动活性间无明显的相关(P=0.18)。宫颈粘液精子制动试验和精子宫颈粘液接触试验用于宫颈粘液抗精子抗体的检测,缺乏相应的特异性。     

The relationship between female sexual practices and the development of antisperm antibodies

OBJECTIVE: To determine if female sexual practices are associated with the development of antisperm antibodies. DESIGN: Anonymous questionnaire. SETTING: Tertiary care; private practice. PATIENTS: Thirty-nine consecutive patients undergoing antisperm antibody testing; 32 responded. INTERVENTIONS: Cervical mucus and serum obtained at midcycle. MAIN OUTCOME: Female sexual practices were found not to be related to the development of antisperm antibodies. RESULTS: The percent (44.4) of women with antisperm antibodies who practice anal intercourse was not different from the percent (35.7) without these antibodies who engage in the same practice, and the percent (94.4) of women with antisperm antibodies who practice oral intercourse was not different from the percent (92.8) without these antibodies who engage in this practice. CONCLUSION: Female sexual practices do not appear to be related to the development of antisperm antibodies.     

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

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

Antisperm antibody binding to human acrosin: a study of patients with unexplained infertility

OBJECTIVE: Antisperm antibody binding to acrosin was investigated by Western Blotting. The clinical significance of this binding specificity was assessed in a 2-year clinical follow-up. DESIGN: Consecutive serum samples positive for antisperm antibodies by both enzyme-linked immunosorbent assay and immunobead testing were evaluated for acrosin-binding specificity. SETTING: The patients were followed in an outpatient setting by private infertility specialists. PATIENTS: Sixty-five consecutive infertile referral patients with positive antisperm antibody were evaluated. Clinical follow-up was obtained on 8 of 9 females with evidence of antibody binding to acrosin and 19 of 26 females with no specific binding to acrosin. INTERVENTIONS: Prednisone therapy was given during six courses of intrauterine insemination with husband's sperm. All treatment decisions were made by private physicians independent of the acrosin-binding result. MAIN OUTCOME MEASURES: Pregnancy status was obtained as part of a 2-year follow-up. RESULTS: Acrosin-binding specificity was demonstrated in 10 (15%) of the 65 patients. Two of the 8 women (25%) with antibody binding to acrosin and 6 of the 19 women (32%) with antisperm antibodies but no specific binding to acrosin delivered normal children. CONCLUSIONS: Although antibody-binding specificity to acrosin could be demonstrated, a 2-year clinical follow-up showed no difference in pregnancy rates when compared with women with antisperm antibodies showing no binding specificity to acrosin.     

Failure of intrauterine insemination in male immunological infertility in cases in which all spermatozoa are antibody-coated.

OBJECTIVE: To determine if the overcoming of the cervical mucus barrier removes the interference of sperm-bound antibodies with fertility. DESIGN: Prospective case series. SETTINGS: University-based intrauterine insemination (IUI) homologous program. PATIENTS: Nineteen patients with all spermatozoa in the ejaculate coated by antisperm antibodies. As control group, 86 consecutive patients without antisperm antibodies, treated for oligoasthenozoospermia or mucus hostility. INTERVENTIONS: Intrauterine inseminations (at least 3 attempts per couple). MAIN OUTCOME MEASURES: The outcome of IUIs, demographic, and seminal parameters were compared between the two groups. RESULTS: No pregnancy occurred in the couples with male immunological infertility, treated by 110 IUIs. Twenty-three pregnancies occurred in 22 (25.6%) of the control group couples who were treated by 411 IUIs. In the group of patients without antisperm antibodies, we demonstrated that the pregnancy rate (PR)/couple in oligoasthenozoospermia without teratozoospermia was similar to that achieved in normozoospermia (35% versus 38.9%), whereas it was significantly affected by teratozoospermia (3.6%). Only three patients with antisperm antibodies had teratozoospermia. Comparing the PR per couple and per cycle between the two groups of patients (with and without antisperm antibodies), excluding the patients with teratozoospermia, significant differences resulted (P less than 0.005 and P less than 0.005, respectively). The motile sperm count was not significantly different between the two groups, which also resulted to be homogeneous for demographic data. Moreover, the motile sperm count was not different between the patients with and without antisperm antibodies, who had successful IUI. CONCLUSIONS: The analysis of this trial suggests that the failure of IUI in the treatment of male immunological infertility is imputable to antisperm antibodies when they involve all spermatozoa, regardless of semen quality.     

A radiolabeled antiglobulin assay to identify human cervical mucus immunoglobulin (Ig) A and IgG antisperm antibodies

Antisperm immunoglobulin (Ig) A and IgG antibodies in human cervical mucus (CM) were identified by a radiolabeled antiglobulin assay. Cervical mucus samples from fertile and infertile women were exposed to a 1:3,200 dilution of 2-mercaptoethanol (2-ME), and 5 micrograms of the solubilized CM protein were assayed for the presence of IgA and IgG antisperm and anti-Candida activity by the radiolabeled antiglobulin assay. Purified human secretory IgA and IgG exposed to 2-ME retained the molecular integrity and functional activity of the untreated antibody molecules. CM aliquots collected after high-performance liquid chromatography (HPLC) fractionation were assessed for antisperm antibody activity; antisperm antibody activity was retained in the appropriate IgA or IgG CM fractions. The incidence of CM antisperm antibodies was minimally affected when the radiolabeled antiglobulin assay was performed with a motile sperm population. Approximately 70% of the CM IgA antisperm antibodies were of the IgA1 subclass; CM IgG was primarily of the IgG4 subclass. When Candida antigen was substituted for sperm in the radiolabeled antiglobulin assay, the CM antisperm antibodies were found to be exclusively sperm-specific. These data indicate that the radiolabeled antiglobulin assay using 2-ME to extract CM antibodies is a specific method for the assay of antisperm antibodies in CM.     

Are zinc levels in seminal plasma associated with seminal leukocytes and other determinants of semen quality?

OBJECTIVE: To evaluate a potential association of zinc levels with seminal leukocytes, the outcome of semen cultures; and semen quality and sperm fertilizing capacity. DESIGN: Prospective study. SETTING: Outpatient infertility clinic of a university hospital. PATIENT(S): Two hundred fifty-six randomly chosen asymptomatic males from subfertile couples. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Determination of zinc in seminal plasma by flame atomic absorption spectroscopy. In aliquots of the same ejaculates the following tests were performed: immunocytochemical round cell differentiation to determine leukocyte counts and ratios, microbial screening, and comprehensive evaluation of semen quality (sperm analysis, biochemical parameters, antisperm antibody testing, and in vitro examination of sperm ability to penetrate cervical mucus). The patients underwent medical history, clinical examination, and postcoital testing. Subsequent fertility was determined (controlled for female infertility factors). RESULT(S): The concentration of zinc in seminal plasma did not correlate in a statistically significant way with leukocytes in semen, nor was it associated with bacterial colonization. There was no statistically significant relationship of zinc in seminal plasma or serum with semen quality parameters nor with local antisperm antibody testing of the IgG or IgA class. Zinc levels did not influence sperm capacity to penetrate cervical mucus in vitro or in vivo, and did not affect subsequent fertility. CONCLUSION(S): The zinc level in seminal fluid and serum is not associated with silent male genital tract infection (indicated by seminal leukocytes); nor is it related to semen cultures in asymptomatic individuals. The lack of association with other semen quality parameters indicates that the routine determination of zinc levels during infertility investigation is not recommended.     

Antisperm antibodies in infertile and homosexual men: relationship to serologic and clinical findings

The incidence and significance of antisperm antibodies in different groups of men were evaluated by a modified enzyme-linked immunosorbent assay. In serum, 4.0% of dermatologic patients (n = 223), 9.6% of andrologic patients (n = 178), and 28.6% of homosexual men (n = 42) were positive for IgG and/or IgM antibodies. In seminal fluids, 7.3% of the andrologic patients had IgA (and IgG) antibodies to spermatozoa. Only 1 of 29 positive men had antibodies both in serum and in seminal fluid. No correlation between antisperm antibodies and IgG/IgM concentrations was found in serum, whereas in seminal plasma men with antisperm antibodies showed higher IgG/IgA concentrations than men without (IgA, 3.2 versus 1.7 mg/dl; IgG, 9.8 versus 6.3 mg/dl). It is concluded that there is a high incidence of antisperm antibodies among homosexual men, probably because of contact of spermatozoa with the immune system by passive anal intercourse. There is little correlation between antisperm antibodies in serum and seminal plasma of infertile men because of a lack of relevant antibody transfer from the serum and the formation of local antibodies in seminal plasma. Antisperm antibodies in seminal fluid are associated with elevated local IgG and IgA concentrations.     

Infertility in mice after unilateral vasectomy

The effects of unilateral vasectomy upon fertility and antisperm antibody production were studied using DBA/1J mice. Thirty-six males underwent either unilateral vasectomy, unilateral orchiectomy, or sham surgery. In vivo effects upon fertility were monitored by matings. Antisperm antibody titers were measured monthly. In vitro fertilization was performed in the presence of serum obtained 4 months postoperatively, and serum testosterone levels were also determined. After 3 months, only 1 male in the vasectomy group induced a pregnancy (1 of 12), while all but 1 of the males in the two control groups induced a pregnancy (20 of 21). The geometric mean antisperm antibody titer was 1:169 in the vasectomy group, while the orchiectomy and sham surgery groups had titers of 1:4 and 1:14, respectively (P less than 0.0001). The percentage of eggs fertilized in vitro in the presence of serum from experimental mice fell from 80% in the two control groups to 40% in the unilateral vasectomy group. Unilateral vasectomy induced infertility in DBA/1J mice and an antisperm antibody response. Sera containing these antibodies inhibited in vitro fertilization. This suggests that infertility after unilateral vasectomy may be immunologically mediated by antisperm antibodies.     

Spermatozoal antibodies in cervical mucus

The indirect immunofluorescent technique performed on spermatozoa provides a suitable method for detecting spermatozoal antibodies in cervical mucus. Blood sera and preovulatory cervical mucus samples from 13 women with infertility of unknown origin were tested extensively with this technique. Spermatozoal antibodies in cervical mucus were detected in 3 out of 13 patients. The specificity of these antibodies was shown to be IgA in two cases and IgG in the third case. One IgA antibody was also present in the blood serum of this particular patient, however in a low titer (1:4). Blood serum from the patient with an IgG antibody in cervical mucus contained the same antibody in a high titer (1:64). The results werecompared to the concentration of immunoglobulins in cervical mucus. In view of present knowledge our data are consistent with the following theories about circulating spermatozoal antibodies. IgA antibodies result from local immunization in the female genital tract and may leak to the general circulation. IgG and IgM antibodies result mainly from general immunization. IgG antibodies may diffuse to female genital tract secretions when present in a sufficiently high concentration. IgM antibodies are rarely found in the female reproductive tract.     

Beta-endorphin and met-enkephalin in peritoneal and ovarian follicular fluids of fertile and postmenopausal women

Immunoreactive (IR) beta-endorphin (beta-EP) and met-enkephalin (MET-ENK) have been found in peritoneal fluid (PF) and ovarian follicular fluid (FF). Gel chromatography also revealed the presence of coeluting IR beta-lipotropin and gamma-lipotropin. IR beta-EP and IR MET-ENK levels in healthy menstruating women were from 10 to 40 times higher than those present in circulating plasma, which indicated a possible local production. The highest concentrations of IR beta-EP in FF were found in the largest follicles, whereas in the PF they correlated with the luteal period of the menstrual cycle and with progesterone concentrations. No relevant changes in IR MET-ENK were detected in the FF or in the PF in relation to the phase of the menstrual cycle. In postmenopausal women, the concentrations of the two IR opioid peptides were undetectable in both fluids.     

Concentration of lactoferrin and interleukin-6 in cervical mucus from patients being treated for infertility

Background  The concentrations of the iron-binding protein lactoferrin (LF) and interleukin-6 (IL-6) were measured in the cervical mucus of patients being treated for infertility throughout the menstrual cycle. Methods  A total of 251 cervical mucus samples were obtained from the patients throughout the menstrual cycle. One hundred and fifty samples were from primary infertility patients with unexplained infertility and 101 samples were from secondary infertility patients as a control. The concentrations of LF and IL-6 were measured by enzyme immunoassays. The standard curve of LF concentrations ranged from 1.6 to 50 ng/mL. Results  The mean LF and IL-6 concentrations in the cervical mucus of primary infertility patients were higher than that of the control patients (P = 0.04,P = 0.032, respectively) The LF and IL-6 concentrations were highly correlated (P < 0.0001). Conclusion  Elevated levels of IL-6 and LF in the cervical mucus were obtained from primary infertility patients. We speculate that LF might also be one of the causes of infertility and might play an important role in reproductive processes in the cervix.     

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.