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.     

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.     

Occurrence of serum antisperm antibodies in patients with cystic fibrosis

OBJECTIVE: To determine if acquired obstruction of the vas deferens in men with cystic fibrosis (CF) induced the development of antisperm antibodies with genital tract obstruction similar to other men. DESIGN: Serum antisperm antibodies were assayed by an indirect immunobead test and an indirect immunofluorescence assay. Both homologous (human sperm/human zona) and heterologous (human sperm/zona-free hamster ova) sperm/egg interactions were evaluated in the presence of serum antisperm antibodies from patients with CF. SETTING: Cystic Fibrosis Clinic at the University of Oklahoma Health Sciences Center, a tertiary care referral center. PATIENTS: Fifteen CF patients (10 male and 5 female), 3 non-CF antisperm antibody-positive infertile patients (2 male and 1 female), 20 fertile controls (7 males and 13 females), and 9 fertile sperm donors were used. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Serum antisperm antibody levels in patients with CF. In those patients with antisperm antibodies, determine effect of these sperm antibodies on sperm/egg interactions and complement-mediated events. RESULTS: Sera from 3 (30%) of 10 men with CF demonstrated immunoglobulin (Ig)G, IgA, and/or IgM antisperm antibodies, whereas sera from all 5 CF women and the 20 control sera were negative for antisperm antibodies. The maximal titers for IgG, IgA, and IgM antisperm antibody were 1:8, 192, 1:256, and 1:64, respectively. The immunobead binding, which was restricted to the sperm head and tail-tip or the midpiece and tail-tip, correlated with the indirect immunofluorescence pattern. Antisperm antibody-positive sera from men with CF impaired both the binding and penetration of human zonae and the penetration of hamster ova by human sperm. CONCLUSIONS: Similar to other men with congenital or acquired obstruction of their genital tract, antisperm antibodies may occur in some men with CF. Antisperm antibodies may contribute to immune sperm dysfunction in some men with CF by activated complement-mediated events and interfering with sperm/egg interactions.     

Does intraperitoneal insemination in the absence of prior sensitization carry with it a risk of subsequent immunity to sperm?

To evaluate the occurrence of antisperm antibodies in women, with no prior sensitization, 112 couples undergoing intraperitoneal insemination were tested for serum antisperm antibodies with the sperm immobilization test (SIT) and the immunobead test (IBT). A serum sample was taken from each of the 112 patients immediately before the first intraperitoneal insemination. Another sample was taken from 58 patients who underwent a second insemination procedure. In 16 of the 58 patients the IBT results were positive for one or more immunoglobulin classes. Five patients showed positive SITs. In 7 out of these 16 subjects (12%) the antibodies were bound to the head and to the shaft of the sperm tail. Five of the six patients submitted to a third intraperitoneal insemination procedure showed unchanged SIT values and IBT binding percentages. In one subject, SIT (6 months after the third insemination) became negative. Antibody production may be either a transient response to massive antigen stimulation or the first step toward systemic immunity.     

Assessment by fluorescence-activated cell sorting of whether sperm-associated immunoglobulin (Ig)G and IgA occur on the same sperm population.

Two-color fluorescence-activated cell sorting of antisperm antibody-positive sperm was used to detect simultaneously the presence of immunoglobulin (Ig)A and IgG antisperm antibodies associated in vivo on a man's sperm. Sperm positive for sperm-associated Ig were analyzed using phycoerythrin-conjugated antihuman IgA and fluorescein isothiocyanate-conjugated antihuman IgG; up to 87% of the same spermatozoa were stained with both labels. Sperm positive for only one of the antisperm antibody isotypes stained up to 90% of a man's sperm with only one fluorochrome. Immunocytochemistry studies revealed similar patterns of sperm binding for sperm-associated IgG and IgA. These results suggest that the sperm antigenic determinants reacting with antisperm IgA and IgG are present on the same sperm population at similar locations on the sperm surface.     

Relationship between circulating antisperm antibodies in women and autoantibodies on the ejaculated sperm of their partners

The relationship between antibodies on the surface of ejaculated sperm and circulating antibodies in female partners was evaluated. Of 616 couples examined by the immunobead binding test, there was a 12.4% incidence of sperm-surface antibodies in men whose wives had antisperm antibodies in their sera, but only a 6.5% incidence in partners of women who lacked these antibodies (p less than 0.025). Sperm-bound immunoglobulin G and immunoglobulin A both occurred at a significantly higher frequency (p less than 0.05) in partners of women with serum antisperm antibodies. Increased incidence of both immunoglobulin G (p less than 0.01) and immunoglobulin M (p less than 0.005) circulating antisperm antibodies in females were observed when the male partners had antibody-bound sperm. Antibody-coated sperm may activate lymphocytes in the female partners after coitus, thus leading to the production of antisperm antibodies. This may be an additional mechanism that leads to female isoimmunity to sperm and infertility.     

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.     

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.     

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.     

Effects of incubation time in serum and capacitation on spermatozoal reactivity with antisperm antibodies

Sperm reside within the female reproductive tract before the occurrence of fertilization. During this time they undergo surface modifications associated with changes in their functional state. To study their antigenic expression, capacitated and acrosome-reacted sperm were incubated with sera that had previously been tested for antisperm antibodies against fresh washed sperm, as detected by indirect immunobead binding. Forty-eight percent of previously positive and 20% of previously negative sera reacted differently with sperm after an extended time (18 hours) of incubation in serum or after sperm capacitation. These results suggest that current techniques of antisperm antibodies detection be modified to include testing sera after prolonged incubation times with both capacitated as well as fresh sperm.     

Intrauterine inseminations with washed human spermatozoa does not induce formation of antisperm antibodies

In this study we investigated the possible development of serum antisperm antibodies in women receiving repeated IUI. Patients acted as its own control and were evaluated before and after various (1 to 15) IUI cycles using three different assays for antisperm antibodies. It was found that only 2 out of 41 women developed antisperm antibodies. We concluded that exposure of the upper reproductive tract to washed spermatozoa during repeated IUI with partners' sperm does not significantly stimulate the appearance of serum antisperm antibodies.     

A simple method of screening for antisperm antibodies in the human male. Detection of spermatozoal surface IgG with the direct mixed antiglobulin reaction carried out on untreated fresh human semen.

A simple and rapid test for the detection of antisperm antibodies of the IgG class on freely swimming spermatozoa in fresh human semen is described. The test is based on the formation of motile mixed agglutinates between erythrocytes sensitized with incomplete anti-Rh-antibodies and freely swimming spermatozoa with surface antisperm antibodies, after mixing both cell types together with anti-IgG antiserum. Agglutination of the red blood cells serves as an internal control. The test can be applied on ejaculates with spermatozoa concentrations down to one million per ml, provided the motility is sufficient. The percentage of motile spermatozoa found to be coated with antisperm antibodies of the IgG class, and the extent of the coating, proved to be correlated with the agglutination titer of circulating antisperm antibodies and with the inhibition of sperm penetration into cervical mucus. The test can be used as a screening for the presence of antisperm autoantibodies in serum and semen.     

Heterogeneity of antigenic determinants on human spermatozoa: relevance to antisperm antibody testing in infertile couples

Sera from 1074 male and 947 female partners of infertile marriages were tested by enzyme-linked immunosorbent assay for antibodies to motile sperm purified from ejaculates of the male partners or a donor. In men, 9.2% of the sera were positive for immunoglobulin A, 7.9% for immunoglobulin G, and 5.1% for immunoglobulin M antibodies to their own sperm. In women, immunoglobulin M antibodies to the husband's sperm predominated (10.1%), with immunoglobulins G (8.3%) and A (5.9%) following. Differences between men and women in the incidence of immunoglobulin A (p less than 0.01) and M (p less than 0.005) antibodies were significant. In both sexes only about two thirds of the antibody-positive sera remained positive when donor sperm was substituted for partners' sperm in the assay. The decreased occurrences of antisperm immunoglobulins A (p less than 0.025) and G (p less than 0.01) in men and of immunoglobulins G (p less than 0.025) and M (p less than 0.01) in women were significant. Incubation of donor sperm in the husband's cell-free seminal fluid before analysis led to the acquisition of sperm reactivity with husband-specific antisperm antibodies in only one of eight women. Women with husband-specific antisperm antibodies also exhibited differences in their cell-mediated immune responses to sperm from various men. Thus sperm from different individuals vary in their ability to react with the immune system of sperm-sensitized men or women.     

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.     

Detection of autoimmunity to sperm: mixed antiglobulin reaction (MAR) test or sperm agglutination? A study on 537 men from infertile couples

Two different ways of testing for antisperm antibodies were compared: the mixed antiglobulin reaction (MAR) test for demonstration of antibodies of the IgG and IgA classes bound in vivo to the sperm membrane antigens and the gelatin agglutination test for detection of nonbound antisperm antibodies in serum and seminal plasma. Samples from 537 men from infertile couples were investigated. Antibodies bound to the sperm membrane were detected in 49 men (9.1%), IgG in 44 (8.2%), and IgA in 38 cases (7.1%). Sperm agglutinins were recorded in seminal plasma from 30 men (5.6%) and in serum (titer greater than or equal to 16) from 43 men (8.0%). The investigation revealed a very close correlation between the results of MAR testing and the occurrence of sperm agglutinins in serum and seminal plasma. However, if one focuses on antisperm antibodies of the IgA class, which seem to play the major role in male immune infertility, the MAR test offered the advantage that a minor group of patients with pure IgG responses could be distinguished, and rare cases with mainly or exclusively locally produced IgA antibodies could be detected.     

Interference of antisperm antibodies with the induction of the acrosome reaction by zona pellucida (ZP) and its relationship with the inhibition of ZP binding

Objective: To determine whether antisperm antibodies can interfere with the induction of the acrosome reaction (AR) by the zona pellucida (ZP) and whether this interference also can occur in the absence of an inhibitory effect on ZP binding.

Design: Prospective in vitro study.

Setting: A tertiary care center, the Andrologic Clinic, University of L'Aquila.

Patient(s): Sera from 12 infertile patients with high titers of circulating antibodies directed against the sperm head were studied.

Intervention: None

Main Outcome Measure(s): The effect of antisperm antibodies on ZP binding was evaluated by matching antibody-exposed and nonexposed donor sperm suspensions labeled with fluorescein or rhodamine, respectively, and incubated with the same salt-stored human ZPs. The effect of antibodies on ZP-induced AR was determined by challenging antibody-exposed and nonexposed donor sperm suspensions with human ZPs disaggregated with acidic NaH₂PO₄. Acrosomal status was evaluated using fluorescein-labeled Pisum sativum agglutinin and supravital stain Hoechst 33258. In some selected cases, the effect of antisperm antibodies on the acrosomal status of sperm bound to intact ZP also was evaluated using transmission electron microscopy.

Result(s): Five of 12 sera exhibited an inhibitory effect on ZP binding. An inhibition of AR induction by disaggregated ZPs (ranging from 64% to 98%) was produced by all 5 sera with an inhibitory effect on ZP binding and by 2 of 7 sera without an inhibitory effect on ZP binding. The different effects of antisperm antibodies on AR induction by disaggregated ZP were confirmed by comparing with ultrastructural evaluations on the acrosomal status of sperm bound to intact ZP.

Conclusion(s): Antisperm antibodies can interfere with the induction of AR by ZP. This inhibition can occur even in the absence of an inhibitory effect on ZP binding. Neither effect may occur.     

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.     

Qualitative differences in sperm antibody responses in mice of different inbred strains and sexes

To better understand the immunogenetic basis and potential pathological consequences of anti-sperm humoral immunity, age-matched female mice of 9 different inbred strains were immunized with syngeneic sperm and were tested for qualitative (specificity) and quantitative (titer) antibody differences by radioimmunoassay, immunofluorescence and immunoblot techniques. All mice developed antisperm antibodies, although titers varied considerably between inbred strains. Antisperm antibodies produced in this study did not cross-react with membrane antigens on thymocytes, brain or immature testicular germ cells. Immunoblot tests identified 17 major sperm antigen bands; this approach also revealed considerable inter- and intra-strain variation in antisperm antibody specificities among female mice. In a parallel study C57BL/6 male mice demonstrated significantly lower antisperm antibody titers and an absence of response to certain sperm antigens in immunoblot tests when compared to age-matched females of the same inbred strain. These findings provide evidence that genetic factors (including sex) interact with environmental (nongenetic) factors in the control of immune responses to sperm antigens.     

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.     

Antisperm antibodies to sperm surface antigens in women with genital tract infection

Antisperm antibodies to sperm surface antigens in nulligravid women with primary upper genital tract infections were measured by the sperm mixed agglutination reaction assay. As many as 56% of women with a primary episode of pelvic inflammatory disease had antisperm antibodies. In addition, 69% of those women with no history of genital tract infection but with laparoscopic evidence of past pelvic infection had significant levels of circulating antisperm antibodies. Electroimmunoblots of sperm preparations probed with the sera of women who had either known or presumed upper genital tract infection revealed a uniformly recognized 69 kd antigen. In contrast, women with circulating antisperm antibodies before primary upper genital tract infection recognized up to five distinct sperm antigen determinants of 27, 54, 131, 146, and 174 kd. It is a distinct possibility that genital tract infections may lead to immunopotentiation of antisperm antibodies that could affect fertility.