Antifertility effects of antisperm cell-mediated immunity in mice.

C57BL/6 female mice were immunized with allogeneic (DBA/2) sperm in Freund's adjuvant either subcutaneously (s.c.), transcervically into the uterine lumen (i.u.), or with a combination of s.c. and i.u. immunization approaches. Control mice received DBA/2 lymphocytes, human erythrocytes or saline in adjuvant using the same immunization protocols. Immunization with sperm or control cells in adjuvant exclusively by s.c. or i.u. approaches did not affect subsequent fertility, although sperm-injected mice from both protocols had high titers of circulating antisperm antibodies. In contrast, mice that were immunized with sperm in adjuvant by a combination of s.c. and i.u. injections demonstrated significant reductions in fertilization rate and number of viable fetuses and an increased rate of fetal resorption when compared with non-immunized and control-immunized mice. Mice receiving sperm by the s.c./i.u. protocol had high titers of antisperm antibodies and a marked infiltration of T lymphocytes and macrophages into the uterine endometrium. To determine whether cellular immune mechanisms contributed to the infertility effect, T lymphocytes from spleens and pelvic lymph nodes of s.c./i.u. sperm-immunized mice and non-immunized mice were passively transferred to naive syngeneic female recipients which were subsequently mated. The total number of fetuses on day 15 of pregnancy was significantly reduced in mice receiving T-lymphocytes from sperm-immunized mice and a significant increase in fetal resorption sites was also observed. These mice did not have detectable titers of circulating antisperm antibodies, but had a significant infiltration of CD4+ T lymphocytes and macrophages in the uterine epithelium and endometrium. These data indicate that intrauterine antisperm cell-mediated immunity can be induced in mice by a combination of systemic and intrauterine immunizations and provide evidence for the existence of reproductive tract mucosal antisperm cellular immune responses that adversely affect fertility and pregnancy.     

Immunobiological consequence of immunization of female mice with homologous spermatozoa: induction of infertility.

Female Swiss Webster mice were immunized intraperitoneally with mouse epididymal spermatozoa or with phosphate buffered saline (PBS) and their fertility was compared by (1) incidence and size of litters, (2) number of uterine implantation sites, and (3) incidence and number of fertilized eggs in the oviducts. Statistically significant reduction in fertility was noted following two courses of injections of spermatozoa; 12% of mice injected with spermatozoa had litters compared with 80% of mice injected with PBS. The infertility did not seem to be related to a failure in fertilization since the two groups of mice had a similar incidence and number of fertilized eggs in the oviducts. All female mice were found to have a "natural' anti-acrosomal antibody. Following immunization with spermatozoa, antibodies to "postacrosomal' region, the main piece and the midpiece of the tail, as well as cytotoxic antisperm antibodies, appeared. Anti-LDH-X antibody was not detected. However, correlation was not found between infertility and antisperm antibodies or sperm granulomata that developed in the peritoneal cavities. It is concluded that female mice receiving repeated i.p, injections of mouse spermatozoa become infertile and that the infertility is related to interference with events after fertilization.     

Vasectomy and vasovasostomy in rhesus monkeys: the effect of circulating antisperm antibodies on fertility.

Rhesus monkeys develop agglutinating and complement-dependent antisperm antibodies after vasectomy. In order to study whether these antibodies affect fertility after vasovasostomy, 15 animals were given vasectomies and 6 months later vasovasostomies. Subsequently, each was mated with females of proven fertility. Five controls were given sham operations and similarly treated. During this period, each aimal was bled for serum to monitor the humoral immune response, ejaculated for semen analyses, and palpated for granuloma or fistula development. All control animals had a transient decrease in sperm density after sham vasectomy and vasovasostomy operations. The surgical procedures of vasectomy and subsequent vasovasostomy resulted in more animals having sperm of poor motility and quality. All of vasovasostomies were surgically successful in that sperm were again present in the ejaculate of each animal. The amount of sperm in the ejaculate could not be correlated with the ease of surgical procedure, presence or absence of macrophages in the ejaculum, motility, or forward progression. Only animals that had been vasectomized developed circulating antisperm antibodies. Sustained, elevated levels of antisperm antibodies most commonly occurred in monkeys that had high initial total sperm counts. Six of the experimental animals retained high levels of sperm-immobilizing antibodies after vasovasostomy. Of these, two were found to be infertile and two were classed as subfertile. Of the nine experimental animals without sustained antisperm antibody production, only one was classed as subfertile. This suggests that antisperm antibodies may in some cases impair the restoration of fertility after vasovasostomy.     

The effect of female antisperm antibodies on in vitro fertilization, early embryonic development, and pregnancy outcome

STUDY OBJECTIVE: To evaluate the extent to which human in vitro fertilization-embryo transfer (IVF-ET) alleviates immunological infertility. DESIGN: Retrospective. SETTING: In vitro fertilization program. PATIENTS: Thirty-three patients with positive antisperm antibodies undergoing 50 cycles of IVF-ET in which maternal serum was replaced by 5 mg/mL of bovine serum albumin (BSA) comprised the study group. Seventy-one patients with tubal infertility served as controls. In 50 of these, medium was supplemented with 7.5% maternal serum, and 21 were assigned to BSA substitution. RESULTS: Percentage of fertilization in the study group was significantly lower (41 +/- 31; mean +/- SD) than that of controls with maternal serum (77 +/- 15) and BSA (76 +/- 22). Early embryonic quality, as assessed by percentage of cleavage and morphological grading, was found to be inferior in patients with antisperm antibodies. The percentage of advanced embryos (greater than or equal to 4 blastomeres) at the time of transfer was 42 +/- 39 in the study group, compared with 65 +/- 23 and 75 +/- 35 for maternal serum and BSA controls, respectively. Percentage of morphologically favorable embryos (grades 1 and 2 in a 1 to 5 grading system) was 49 +/- 31 in the study group, compared with 78 +/- 35 and 74 +/- 23 for the controls. Percentage of clinical pregnancy was somewhat lower in the study group (12.5%) than in controls with either maternal serum (18%) or BSA (19%). CONCLUSIONS: Antisperm antibodies may have an adverse effect on fertilization and early embryonic development. Female immunological infertility may not be completely alleviated by IVF-ET.     

Factors that influence fertility after vasovasostomy in rats

OBJECTIVE: To determine if fertility after vasovasostomy of immunologically responsive Lewis rats differs from that of the less responsive Sprague-Dawley strain and to relate fertility to antisperm antibodies, fluid flow in the vas deferens, and testicular structure. DESIGN: Male rats received: (1) bilateral vasectomies; (2) vasectomies followed 3 months later by vasovasostomy; or (3) sham operations. SETTING: Research laboratory. MAIN OUTCOME MEASURES: Fertility was assessed by caging males with three females for 2 weeks and subsequently counting implantation sites. Antisperm antibodies were measured with an enzyme-linked immunosorbent assay, fluid flow through vas deferens segments was tested in vitro, and testicular structure was studied microscopically. RESULTS: Nearly all vasovasostomized Lewis rats were infertile (33 of 34), whereas 62% (18 of 29) Sprague-Dawley rats were fertile after vasovasostomy (P less than 0.001). In fertile Sprague-Dawley males, significant correlations existed between: (1) implantation sites or females impregnated; and (2) antisperm antibodies early after vasectomy, vas flow, and testicular morphology. CONCLUSIONS: Genetic differences affect fertility after vasovasostomy. Fertility after vasovasostomy is also influenced in a multifactorial manner by the immune response, mechanical elements, and structural changes in the reproductive tract.     

The clinical relevance of classes of immunoglobulins on spermatozoa from infertile and vasovasostomized males

Following reversal of vasectomy, conceptions occur even when antisperm antibodies are present in the seminal plasma, but this is most unusual in men with similar titers of such antibodies who are spontaneously infertile. To clarify the differences between antisperm antibodies occurring in infertile men and those associated with vasectomy reversal, we have studied 23 spontaneously infertile men and 22 men who underwent vasectomy reversal, all of whom had antisperm antibodies detected in seminal plasma by the same tray agglutination test. The class of antibody on spermatozoa was defined by a double-antibody technique using diluted rabbit anti-human IgG, IgM, or IgA or secretory component, followed, after washing, by 125I-labeled donkey anti-rabbit Ig. The results have shown that similar amounts of IgG and IgM were present on the spermatozoa, but infertile men had significantly more IgA and especially more secretory component than men who underwent vasectomy reversal. This was associated with significantly greater impairment of penetration of cervical mucus in the former group. It appears that the type of antibody on the spermatozoa may vary according to the stimulus for its production.     

Macrophages and infertility: enhancement of human macrophage-mediated sperm killing by antisperm antibodies

The mechanism by which antisperm antibodies inhibit fertility is not completely understood. Macrophages may play a role in mediating infertility by interacting with sperm and destroying gametes. Experiments were conducted evaluating the effect of antisperm antibody on the phagocytosis and lysis of sperm by human peritoneal macrophages in vitro. Sperm from a fertile man treated with sera from normal men and women or medium alone had 5 to 280 molecules of IgG/sperm, as determined by a 125I-labeled anti-human IgG monoclonal antibody assay. By contrast, sperm treated with sera containing antisperm antibodies had 310 to 1240 molecules of IgG/sperm. Peritoneal macrophages harvested from infertile women with tubal/adhesive problems mediated phagocytosis and lysis of 111In-labeled sperm which was enhanced by treatment of the sperm with sera containing antisperm antibodies (39.0% +/- 1.5% versus 76.3% +/- 3.2% phagocytosis, and 3.3% +/- 0.3% versus 23.3% +/- 2.3% lysis of sperm [control versus antibody-treated]). The likelihood of fertilization in couples with antisperm antibody may be determined not only by the antibody but also by the presence of genital tract macrophages capable of destroying the antibody-coated sperm.     

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.     

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.     

Vasectomy: consequences of autoimmunity to sperm antigens.

Data from studies examining the effects of vasectomy in a large number of nonhuman primates vasectomized for periods ranging up to 14 years are summarized, and these findings and speculations are used as a framework with which to review the subject of autoimmunity and vasectomy. Attention is directed to autoimmunity to sperm antigens following vasectomy (factors affecting antisperm antibody levels, characteristics of circulating antisperm antibodies, antisperm antibodies in seminal plasma, and cellular immunity following vasectomy), and immunopathology of antisperm autoimmunity (local effects on the male reproductive tract and systemic effects on the male reproductive tract). The 6 hypotheses that have been advanced to explain individual variations in dynamics and types of antisperm antibodies produced following vasectomy are reviewed. 3 tests are commonly used to detect free antisperm antibodies after vasectomy: 1) the spermagglutination test; 2) the sperm immobilization test; and 3) the immunofluorescence test. Spermagglutinating (SA) antibodies, the most common type of antibody produced after vasectomy, occur in approximately 2/3 of vasectomized men and in a majority of vasectomized rhesus monkeys. Sperm-immobilizing (SI) antibodies are also produced in a large percentage (40%) of vasectomized men and rhesus monkeys. About 30% of vasectomized men also have antiprotamine antibodies.     

Immunological approach to male contraception

Sperm surface antigens may induce an immune response in mammals. In humans, the presence of antisperm antibodies has been noted in the blood, seminal plasma, cervical mucus, and follicular liquid. Because they provoke immobilization and/or agglutination of sperm and a diminution of the rate of fertilization in vitro, these antibodies are believed to be a factor in some unexplained infertility, as for example after surgical reversal of vasectomy. These observations have led to research oriented toward development of a nonhormonal contraceptive method based on the immunological capacities of some sperm antigens. Possible secondary effects and the modes of action remain poorly understood. Antisperm immunoglobulins in the male should be induced by epitopes that are specific to sperm excluding the proteins of the sperm membrane, so that they will be without effect on functions other than fertilization. The epitopes should be located on the sperm surface and should be of post-testicular origin because of the risk of orchitis posed by testicular antigens. The antisperm immunoglobulins should also be present in the area surrounding he sperm in the male genital tract because of the limited permeability of the tract to immunoglobulins and other large molecules. The antisperm immunoglobulins should not be inhibited in the male by the immunosuppressive activities of the seminal plasma or in the female by capacitation. In vivo and in vitro observations indicate that various functions may be altered by antisperm antibodies. The antibodies may immobilize the sperm, block interaction with the oocyte at the level of the zona pellucida, block adherence to the vitelline membrane, or cause anomalies in embryonic development. In rats, isoimmunization against epididymal cofactor of the sperm zona pellucida receptor results in reduced sperm mobility and reduced fertility. The same study was conducted in 12 rams selected for homogeneity of sperm characteristics. Isoimmunization of rams induced transient asthenospermia. Most sperm antigens involved in fertilization are poorly understood. Observations in the ram suggest that embryonic mortality following isoimmunization should be studied.     

Blocking of human fertilization in vitro by sera with sperm-immobilizing antibodies

Serum samples with sperm-immobilizing antibody activity from six women were examined for ability to block sperm-egg interaction by a zona penetration test where human follicular ova matured in vitro were used. Exposure of spermatozoa from a fertile healthy donor to the sera impaired binding to and penetration through the zona pellucida of the spermatozoa completely in five cases and incompletely in one case. Successful fertilization in vitro was achieved by using fetal cord serum instead of autoserum of the patient included in the in vitro fertilization and embryo transfer program. These results suggest that interference with sperm-egg interaction may be an additional mechanism of infertility that is caused by antisperm antibodies.     

Effect of therapy on infertile couples with antisperm antibodies

One hundred seventy-eight couples with positive antisperm antibody titers in serum and genital secretions were offered treatment with prednisone. Of 60 couples who received prednisone only, 43% conceived. Of 25 who had no therapy, 48% conceived. Fifty-four patients treated with prednisone received additional therapy and 31% conceived. Ten of 39 patients not treated with prednisone but receiving other therapies conceived. Cytotoxic antibodies were reduced in 30% to 42% of serum samples and in 24% to 33% of genital secretion samples. In those couples with decreased cytotoxic antibodies pregnancy rates were 40% to 60% compared with 0% to 23% in those with decreased hemagglutinating antibody titers. Our data suggest that prednisone did not improve overall pregnancy rates; pregnancy rates were comparable in both groups treated with other therapies; donor insemination was the most successful of the alternative therapies; reduction of cytotoxic antibody titers after prednisone treatment was associated with increased pregnancy rates.     

Antisperm antibodies and in vitro fertilization

The purpose of this study was to investigate the influence of antisperm antibodies in the male, the female, or both partners on the outcome of in vitro fertilization treatment. The results in terms of ongoing pregnancies in the male and female antibody-positive group were the same as in the antibody-negative group. In the double antibody-positive group two of the three patients became pregnant. When high levels of antisperm antibodies were present on the spermatozoa, the fertilization rate was significantly reduced. In the female positive group no clear relationship between the antibody titer and the fertilization percentage could be detected. Abnormal semen quality was responsible for a much lower fertilization rate than the presence of antibodies. The conclusion of this study is that in vitro fertilization provides an equal chance of conception in couples with antisperm antibodies in comparison with couples with no antibodies if the other semen parameters are normal.     

Dose previous Chlamydia trachomatis infection influence the pregnancy rate of in vitro fertilization and embryo replacement?

The association between previous chlamydial infection, as reflected by the presence of chlamydial antibodies (specific serum immunoglobulin G antibodies with a titer greater than or equal to 32) and pregnancy outcome after in vitro fertilization and embryo replacement was studied in 121 infertile women with tubal damage. The antibody prevalence was 74.4%; the overall pregnancy rate was 26.4%. No difference in seropositivity was detected between those who became pregnant and those who did not (71.9% versus 75.3%). The geometric mean titers were also similar in the two groups. Even after subdivision of the cases into primary or repeated in vitro fertilization attempts, or after stratification of the material according to the number of embryos used for replacement, there was no correlation between chlamydial antibodies and pregnancy rate. Thus, past infection with Chlamydia trachomatis did not influence the outcome of in vitro fertilization and embryo replacement treatment in this study.     

Antisperm antibodies in women: variability in antibody levels in serum, mucus, and peritoneal fluid.

OBJECTIVE: To look for patterns of antisperm antibody expression in women by exploring the levels of antisperm antibodies in different body fluids. This was achieved by studying sequential serum samples from individual patients and by comparing the levels of antisperm antibodies in serum from a number of patients with the levels of antisperm antibodies in cervical mucus or peritoneal fluid (PF). DESIGN: Prospective studies were performed on sequential serum samples within a menstrual cycle. Retrospective studies were done to compare antisperm antibodies in serum and mucus or PF. The immunobead assay was used to measure antisperm antibodies in these fluids. SETTING: Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire. PATIENTS: A random sample of patients undergoing evaluation for infertility. RESULTS: The levels of antisperm antibodies in sera drawn from patients at different points in a menstrual cycle stimulated by the presence of exogenous hormones did not change during the follicular phase of the menstrual cycle. Also, in many samples, the antisperm antibody level in serum did not correlate with the antisperm antibody levels in mucus or PF. CONCLUSIONS: The data suggest that measurement of antisperm antibodies at a single point in time or from a single fluid is not sufficient when evaluating a woman for immunological infertility. The data also suggest that numerous and complex factors contribute to the expression of antisperm antibodies in women.     

Immunological consequences of vasectomy and vasovasostomy in rhesus monkeys

The effects of circulating antibodies on fertility after vasovasostomy was studied in rhesus monkeys. After vasectomy there was rapid rise in antibody levels against spermatozoa; these reached a peak after 2 weeks, rather more rapidly than has been reported in man. Antibody levels declined after this period. Monkeys with high antibodies were also more likely to have granulomas and fistulas. After vasovasostomy several monkeys became fertile again with sperm counts in ejaculate reaching a high level at 3 months, although levels were lower than before vasectomy. Some males retained a high and sustained level of sperm immobilizing antibodies in plasma, sometimes for 18 months or more. These males produced fewer spermatozoa and were more likely to be subfertile or infertile after vasectomy. Almost all the monkeys without antibodies were fertile. Presence of antibodies at vasovasostomy was not a definite indication of subsequent infertility but did provide an indication of likelihood of continuing sterility. These are similar to observations of men undergoing vasectomy and vasosostomy.     

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.     

Antispermatozoal antibodies in men with urethritis

Antispermatozoal antibodies have been measured by the tray agglutination test (TAT) in three groups of patients with urethritis attending a clinic for sexually transmitted diseases and in one control group of men without urethritis. In 3 of 17 (17.6%) patients with acute nongonococcal urethritis (NGU) the serum TAT was positive at titers of 1:16 or more; and in 1 of these patients in whom Chlamydia was grown, the TAT titer rose from 1:4 before treatment, to 1:8 at 2 weeks, and 1:16 at 4 weeks, indicating probable immunization against sperm antigens at the time of infection. A rise in titer from 0 to 1:8 occurred in a second patient with NGU. Six (15.6%) of 39 patients with recurrent NGU and 2 (16.6%) of 12 patients with gonorrhea followed by postgonococcal urethritis also had positive antisperm antibody titers of 1:16 or more. None of 27 control subjects had positive antisperm antibody titers. These observations indicate that development of antisperm antibodies can be stimulated in some individuals by NGU.     

Detection and titration of class-specific antisperm antibodies in serum using an enzyme-linked immunosorbent assay

An antisperm antibody enzyme-linked immunosorbent assay (ELISA) that uses whole unfixed sperm and detects immunoglobulin G (IgG) and IgA antibodies in serum was developed. Donor sperm were washed and plated on poly-L-lysine-treated microtiter plates. The patient's sera were diluted to concentrations of 1:4 to 1:256 and incubated with sperm. Positive and negative sera had been previously tested for IgG antisperm antibody activity with a radiolabeled antiglobulin assay. Samples were considered positive when the mean absorbance of triplicate wells was greater than 2 SD above the pooled negative mean. Intra-assay variation was 7.9 and 9.6% for pooled negative and positive controls, respectively. Identical titers of control positive serum were consistently detected. A correlation of 0.83 was observed between ELISA IgG serum titers and radiolabeled antiglobulin results (N = 12). All negative samples tested negative in both assays (N = 21). Some serum samples showed IgA antisperm antibodies. Determination and titration of class-specific antibodies in serum should facilitate initial screening and follow-up of patients at risk for antisperm antibodies.