Antisperm antibodies: a critical evaluation and clinical guidelines

Journal of Reproductive Immunology 45 (1999) 159 – 183 www.elsevier.com/locate/jreprimm

Review

Antisperm antibodies: a critical evaluation and clinical guidelines Richard A. Bronson * Department of Obstetrics and Gynecology, Health Science Center, State Uni6ersity of New York, Stony Brook, New York, NY 11794 -8091, USA Received 14 March 1999; received in revised form 13 May 1999; accepted 21 May 1999

Keywords: Antisperm antibodies; Clinical guidelines; Critical evaluation

1. Historical perspective Landsteiner and Metchnikoff first demonstrated that spermatozoa were immunogenic nearly a century ago. Later, experiments by Edwards (1964), McLaren (1964), Menge (1971) and others, showed that immunity to sperm induced by intraperitoneal inoculation of females with spermatozoa of the same or heterologous species impaired their reproductive performance. In vitro exposure of spermatozoa to antisera also reduced their egg-penetrating ability. At the same time that the experimental induction of immunity to spermatozoa was shown to cause induced infertility in animals, Franklin and Dukes (1964) described the presence of sperm agglutinins in the sera of women whose failure to conceive was otherwise unexplained, and Isojima et al. (1968) demonstrated the presence of sperm-immobilizing antibodies in these women. Rumke and Hellinga (1959) found that some infertile men exhibited antisperm antibodies (ASA) in their sera and that their fertility correlated with the titre of these antibodies (Rumke et al., 1973). * Tel.: + 1-516-444-2745; fax: + 1-516-444-7740. E-mail address: [email protected] (R.A. Bronson) 0165-0378/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 5 - 0 3 7 8 ( 9 9 ) 0 0 0 4 4 - 3

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2. Contradictions in epidemiological studies Unfortunately, further attempts to show that infertility could have an immune basis in humans relied on epidemiological methods that produced conflicting data (Beer and Neaves, 1978). On hindsight, the reasons for failure of the epidemiological approach are now apparent, in that immunity to sperm is a continuum and not an all-or-nothing phenomenon. The extent to which antisperm antibodies are present in reproductive tract secretions would be expected to influence the degree of fertility impairment (Table 1). There are wide variations in the extent of reproductive failure in individual animals experimentally sensitized to sperm. Serum concentrations of sperm-directed antibodies can also vary between individual men and women. These antibodies present within the reproductive tract may be both transudates from the blood or secreted locally by submucosal plasma cells (see below). Given these two potential sources of immunoglobulins, spermreactive antibodies can be present in serum, yet not detectable in semen or within female reproductive tract (oviductal, uterine, or cervical–vaginal) secretions (Hellstrom et al., 1982; Landers et al., 1991). Conversely, local immunity to sperm has also been demonstrated in the absence of detectable humoral antibodies in both men and women. A dichotomy of immune states, then, may exist between the reproductive tract and the systemic circulation, emphasizing the difficulty encountered in simple epidemiological investigations, which have previously relied solely on serological detection of humoral antibodies as an index of immunity to sperm. Table 1 Incidence of sperm-reactive antibodies as detected by immunobead binding in sera provided by the World Health Organization Reference Banka Clinical category (no. sera)

Negative sera Positive sera (%) (%) High

Heterosexual males of infertile couples (180) Females of infertile couples (180) Fertile females (21) Fertile males (26) Vasectomized males (15) Pregnant females (32) a

Adapted from Bronson et al. (1984).

Intermediate

Low

45.6

7.2

16.1

31.1

30.0 57.1 73.7 13.3 81.2

11.7 0.0 3.5 46.7 0.0

23.3 9.5 3.5 13.3 3.2

35.0 33.4 19.2 26.7 15.6

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It is also critical to distinguish between immunity to sperm surface antigens that could theoretically play a role in gamete interactions leading to fertilization and internal antigens of spermatozoa that would not. Naturally occurring antibodies to intracellular components of sperm are relatively ubiquitous (Tung et al., 1976). Because some of these sperm-directed antibodies can be absorbed with bacteria, they appear to be cross-reactive, rather than being directed primarily against sperm-specific antigens. The antigens to which spontaneously occurring antisperm antibodies are directed are important in determining whether they will alter reproduction, yet these antigens have not been well characterized (see below). Finally, the term antisperm antibody implies not a single protein but several distinct classes of immunoglobulins (IgA, IgG, IgM, etc.), which circulate within serum and are known to play different roles in the immune defense (Goodman, 1991). These molecules are of different molecular weights and possess different numbers of antigen-reactive sites. Some interact with the complement system, while others do not. They would thus be expected to have different effects on spermatozoa. In summary, the types of immunoglobulins present, their reactivity with different sperm antigens, and their concentrations within the reproductive tract will all determine whether the immunity of a particular individual to sperm will alter reproduction.

3. The establishment of laboratory criteria that suggest immunity to sperm can cause infertility Given the complexity of the immune response and the failure of epidemiological studies to convincingly document that immunity to sperm can cause infertility, we have followed the underlying premise that humoral antibodies directed against sperm do not necessarily impair fertility unless these circulating antibodies can be shown to be present within the reproductive tract and could be documented to be present on the living sperm surface. Our development of the immunobead binding test (Bronson et al., 1981a) allowed us to identify cell-bound immunoglobulins on the spermatozoon surface, and determine the region of the sperm to which antibodies are directed, for each immunoglobulin class (IgG, IgA, IgM). As spermatozoa swim through an immunobead suspension, beads adhere to those sperm coated with antibodies following contact to and rosette around them. The pattern of bead binding then allows determination of those regions of the sperm surface to which antisperm antibodies are directed, the proportion of sperm in the ejaculate which are antibody coated, and the isotypes of these antibodies.

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3.1. The rationale for direct immunobead binding In men, the amount of immunoglobulin bound to the sperm surface at the time of ejaculation depends on several factors: the concentration of antisperm antibodies within the prostate and seminal vesicle secretions as determined by their local production within the reproductive tract and their transudation from blood; the binding of antibodies to sperm as they transit the epididymis before ejaculation or, conversely, when they mix with seminal fluid; the elapsed time since the last ejaculation; and the affinity of different antibody molecules for various antigens on the sperm surface. Hence, the amount of immunoglobulin on the sperm surface reflects the final common pathway of several mechanisms of immunoglobulin secretion. Evidence supporting the importance of studying sperm directly in the ejaculate, in making the diagnosis of auto-immunity to spermatozoa, comes from a comparison of sperm antibodies detected in matched semen and serum specimens. In approximately 15% of cases, antibodies have been detected in serum but not on the sperm surface (Bronson, 1988c). The majority of these circulating antisperm antibodies that failed to enter seminal fluid were of low titer and directed against the sperm tail tip. In addition, antisperm IgM does not enter the male genital tract secretions, even when present in high concentration in blood. This immunoglobulin class of antisperm antibodies is only rarely encountered in sera of heterosexual men, though it is more common both in homosexual men and sera of women (Bronson et al., 1983). As regards which test to utilize to detect ASA, many would still consider immunobead binding the best available, both in terms of the low incidence of false-positive tests, and its ability to localize antibodies of all three immunoglobulin classes on the living sperm surface (Marshburn and Kutteh, 1994). This first became apparent at the WHO Workshop on Clinically Defined Sera, during which participating laboratories analyzed over 200 serum samples from known fertile and infertile men and women in a blinded manner by ELISA, agglutination, complement-dependent cytotoxicity, and immunobead binding tests and compared their results (Bronson et al., 1984, 1985). Evidence has also been presented that immunobead binding is more sensitive than the MAR test in its ability to detect immunoglobulins of the IgA class present on the living sperm surface (Meinertz and Bronson, 1988). Tests for antisperm antibodies utilizing complement-dependent sperm immobilization are dependent upon the isotype of ASA and its location on the spermatozoan surface (Bronson et al., 1982a,b). When motile sperm of fertile men were sensitized by incubation in ASA-containing sera, complement-mediated immobilization occurred only when the majority of the sperm tail was coated with immunoglobulin, but not with lesser degrees of

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Table 2 Relationship between antisperm antibody IgG binding and sperm motility in the presence of complementa Extent of antisperm antibody (IgG binding along sperm tail)

Percent motilityb (no. sera tested)

No antibody binding

86.49 1.0 (40)

Sperm tail tip 1/5 tail principal piece 2/5 tail principal piece 3/5 tail principal piece

81.19 3.9 (10) 62.09 6.8 (7) 8.09 3.4 (10) 1.091.0 (10)

a

Adapted from Bronson et al. (1982b). Expressed as mean 9standard error of the mean, following a 4-h incubation with guinea pig serum as a complement source, at 37°C. b

tail binding. There was no loss of sperm motility when ASA of the IgA class, a non-complement fixing immunoglobulin, bound to the sperm tail despite the presence of complement (Table 2; see below). Complement-fixing antibodies of the IgG class that were directed solely against the spermatozoon head did not promote significant sperm loss of motility as well. In contrast, the detection of immunoglobulins on the spermatozoon surface by immunobead binding is independent of the action of complement. These observations do not mean that there can be no improvement in current methods of detecting antisperm antibodies. The relatively large size of the immunobeads limits their resolution, and the number of antibody molecules bound to the sperm surface necessary for bead binding is not known (Bronson, 1988a). The binding of immunobeads to sperm indicates that immunoglobulins are present on the spermatozoon surface, but neither tells us the amount of antibody bound nor the antigens to which those antibodies are directed. However, new tests are likely to develop in the near future, that will be able to detect ASA directed against defined fertilizationrelated antigens. With the establishment of an index of human sperm surface proteins using a 2-D proteome (Naaby-Hansen et al., 1997), patients’ sera may now be employed on 2-D Western blots and the immunoreactivity of antisperm antibodies with groups of surface proteins defined. This analysis has proved to be a useful method that may lead to the identification of the antigens against which ASA are directed in the near future (Shetty et al., 1998). Once sequenced and cloned, their availability through recombinant DNA technology should lead to the development of ELISA-based tests utilizing purified fertilization-related antigens that may allow one to identify the specific locus of fertilization blockade for individual couples.

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To demonstrate that spontaneously occurring immunities to spermatozoa can lead to impaired fertility, our approach has been to study the function of antibody-coated sperm directly, at the level of in vitro gamete interaction. Spermatozoa from proven fertile men participating in an artificial insemination by donor program were washed free of seminal fluid and exposed to sera containing sperm-reactive antibodies from infertile men and women. Utilizing zona-free hamster eggs, the ability of such antibodybound sperm to penetrate the egg in vitro was compared with that of populations of antibody-free sperm from the same fertile individuals (Bronson et al., 1981b, 1990). In addition, because human sperm are narrowly selective in their ability to attach to the zona pellucida of other species, nonviable human eggs were utilized to study whether such antibody-bound sperm could attach to the zona pellucida, a pre-requisite to subsequent penetration (Bronson et al., 1982a; Mahony et al., 1991).

3.2. Effects of antisperm antibodies on gamete interaction Although they have not been well characterized, antisperm antibodies have been shown to be directed against several different antigens (Mathur et al., 1988; Primakoff et al., 1990; Shetty et al., 1998), and each would be expected to have different effects on sperm functions. Indeed, several studies have shown various effects of antisperm antibodies on sperm function, at the level of the zona pellucida and the oolemma. Bronson et al. (1982a,b, 1989, 1990) as well as Aitken et al. (1988) have shown that antisperm antibodies obtained from different clinical sera might either inhibit, promote, or be neutral in their effects on the ability of human sperm to penetrate zona-free hamster eggs. In addition, different sperm head-directed antibodies exhibit various effects on the ability of human sperm to penetrate the human zona pellucida (Bronson et al., 1982a). Mahony et al. (1991) assessed the effects of labeling sperm from known fertile men with antisperm antibodies, using a hemi-zona assay (Table 3). The power of this approach was to eliminate the variation in sperm binding between men and zonae. Immunobead binding was used to confirm that nearly all sperm were labeled with immunoglobulin over the heads, at the serum dilution chosen. Salt-stored hemi-zonas from the same egg were inseminated with antibodyfree or antibody-labeled sperm from the same donor. A wide range in effect was observed, several sera markedly lowering the number of sperm tightly bound to the hemi-zona observed after serial zona washing, whereas other sera did not. These results further demonstrate that the functional effects of antisperm antibodies detected in different individuals may vary despite their same regional localization of binding on the spermatozoan surface. They emphasize the need for tests that allow one to determine the antigenic

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moieties against which antisperm antibodies are directed. They also indicate that for some men with auto-immunity to sperm, their spermatozoa are functionally teratospermic, despite their normal appearance in semen. As previously noted, the location of antibody binding on the spermatozoon surface, as well as immunoglobulin class, is important as regards the ability of complement to promote sperm immobilization (Bronson et al., 1982a). This observation is important not only in testing for antisperm antibodies but also places sperm at risk for loss of viability within the female reproductive tract, as a complete complement cascade is present within cervical mucus (Price and Boettcher, 1979), oviductal secretions and follicular fluid (D’Cruz et al., 1990a,b).

3.3. Effects of immunity to the spermatozoa on their transport through the female reproducti6e tract The proportion of ejaculated sperm that are coated with immunoglobulin varies markedly among men. In 154 men found to have auto-immunity to sperm as judged by direct immunobead binding, half had greater than 90% of their sperm bound, one-quarter had 50–90% bound, and the remaining quarter had less than half of their sperm coated with antibodies (Landers et al., 1991). Sperm that are antibody-bound over most of their surfaces are unable to enter cervical mucus, antibody binding to the sperm tail tip being an exception (Wang et al., 1985), yet they remain completely motile in semen. These clinical observations are consistent with experimental evidence that coating human spermatozoa with either human antisperm antibodies (Alexander, 1984; Bronson et al., 1986; Haas, 1986) or those raised in rabbits (Fjallbrant, 1969) inhibits their ability to penetrate cervical mucus in Table 3 Varying effects of ten sera containing sperm head-directed antibodies on the ability of human sperm to bind to salt-stored human hemi-zona pellucidasa Serum status

No. of sera tested

Antibody negative 3 Antibody positive 3 2 5 a

% Inhibiton of binding 1–11 B20 B50 \50

Hemi-zona Indexb 94.6 (89–99) 87.9 (85.4–91.6) 55.3 (54.4–56.1) 30.0 (18.1–46.2)

Adapted from Mahony et al. (1991). Hemi-zona index =(no. sperm bound in presence of positive serum/no. sperm bound in presence of negative serum)×100. Using non-parametric analysis of numbers of sperm adherent to the hemi-zonas, an index of less than 30–35 is considered highly inhibitory and 35–60 as moderately inhibitory. b

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Table 4 Correlation between extent of auto-immunity to sperm as determined by immunobead binding and number of motile spermatozoa in cervical mucus at post coital testinga % Antibodycoated sperm

No. of total motile sperm in ejaculate (mean9 S.D.)

No of motile sperm/hpfb (mean 9 S.D.)

100 \50 but B 100 B50

2839197 (11) 45918 (8)

2.89 2.8 7.09 5.4

134927 (5)

25.8 9 13.5

a

Adapted from Ayvaliotis et al. (1985). Cervical mucus was examined within 48 h preceding the thermal basal body temperature shift 8–12 h following coitus. Wives were free of antisperm antibodies and there was no clinical evidence of cervicitis. b

vitro. Several studies have shown a relationship between the presence of antisperm antibodies and impaired results of post-coital tests. Ayvaliotis et al. (1985) have found an inverse correlation between the proportion of sperm that are coated with immunoglobulin and the number of sperm present within the cervical mucus after sexual relations. When all sperm were coated with immunoglobulin, it is rare to find one to two sperm per high-power field within well-estrogenized, clear cervical mucus, despite the presence of hundreds of millions of motile spermatozoa in the ejaculate. However, as the proportion of antibody-coated sperm declined below 50%, as judged by immunobead binding, the numbers of motile sperm observed in cervical mucus increased (Table 4). Hence, it appears that men who have high levels of auto-immunity to sperm, as reflected in the proportion of immunoglobulin-coated sperm in their ejaculates, appear to be functionally oligospermic. Their sperm cannot enter the reproductive tract, and the chance that they will reach the environs of the egg is diminished. This impairment of cervical mucus penetrating ability appears to be mediated through the Fc portion of the immunoglobulin molecule (Jager et al., 1981a,b). Immunoglobulins possess both an antigen-recognition region (Fab) and an effector region (Fc) that binds to various leukocytes through specific surface receptors (FcR) (Goodman, 1991). Sperm exposed to Fab preparations of antisperm IgG are able to swim through cervical mucus, whereas those labeled with intact antibody do not. Similarly, immunoglobulins of the IgA class bound to the sperm surface can be degraded by an IgA protease derived from Neisseria gonorrhoeae that cleaves the heavy chain at amino acid bond 235–236 of the hinge region (Plaut, 1983). In this manner, the Fc portion of IgA is liberated from the sperm surface. These proteasetreated sperm, although still coated with IgA Fab, exhibited an improved

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ability to penetrate into and sustain motility within cervical mucus. On this basis, we have postulated that a solid-phase component of cervical mucus possesses an unidentified receptor for the Fc portion of the immunoglobulin molecule (Bronson et al., 1987). The presence of antisperm antibodies in women may also be associated with altered sperm motion within cervical mucus. In these cases, spermatozoa initially gain entrance into the cervical mucus, in contrast to men with auto-immunity to sperm, but then subsequently become immobilized, either by shaking in place, exhibiting no forward progression, or being completely immobilized. The behavior of sperm within cervical mucus depends on the type of antibodies present within the mucus and their specificity for the sperm surface. Hence, the presence of non-complement-fixing antibodies within cervical mucus may result in sperm entrapment and shaking in place, whereas complement-fixing antibodies (when directed against the majority of the sperm tail) could lead to their complete immobilization. Because seminal plasma contains complement inhibitors (Petersen et al., 1980; Brooks et al., 1981), spermatozoa of sensitized men retain their viability within the ejaculate despite the presence of sperm-directed immunoglobulins in seminal fluid (D’Cruz et al., 1990a,b). Such sperm would only become liable to complement-mediated damage on entry of the female reproductive tract. During their residence within cervical mucus, spermatozoa are exposed to complement activity (Price and Boettcher, 1979). Present in circulation as functionally inactive precursor molecules, each complement component is activated sequentially, both by antibody-dependent and antibody-independent means, in a dynamic process which exerts its effect on cell membranes, resulting in their lysis. Studies with antibodies experimentally induced against erythrocytes indicate that the extent of plasma membrane damage depends on the immunoglobulin class of the antibody bound to the cell. Only a single molecule of IgM is necessary to lyse a red blood cell in the presence of complement, whereas approximately 1000 molecules of IgG are required (Humphrey and Dourmashkin, 1969). Antibodies of certain IgA subclasses, which do not interact with the early components of the complement cascade, are ineffective in promoting immune hemolysis. Levels of complement within cervical mucus are lower than those present in serum, and it may take as long as 6–7 h for sperm immobilization to occur. Hence, overnight post-coital testing provides a clearer indication of antibody-mediated sperm damage than does observation after a shorter interval (2 h) after sex. The carefully performed post-coital test (PCT) can be used as a means to select a group for further study of antisperm antibodies. Infertile couples in whom impaired sperm penetration of cervical mucus or abnormal motion of sperm within cervical mucus was observed at post-coital testing were tested

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for antisperm antibodies. When local cervicitis was excluded, as determined by the absence of microscopic cellularity with cervical mucus, 24% of sera from infertile men and 35% from infertile women with abnormal PCTs were found to possess sperm-reactive antibodies. Conversely, in couples with normal post-coital tests, the chance of finding high levels of antisperm antibodies proved to be small (5% of men and women tested). It should be noted that low levels of antisperm antibodies directed primarily against the tip of the sperm tail (16–35%) were found to be present in sera from both infertile and fertile couples (Bronson et al., 1984). These antibodies are rather ubiquitous and do not impair the ability of sperm to enter and penetrate cervical mucus (Wang et al., 1985). They appear to play no role in infertility. These studies suggest that a major locus of action of ASA on fertility is on sperm entrance of the female reproductive tract, at the level of the cervix, where they also become liable to complement-mediated damage.

3.4. Etiology of antisperm antibodies in men Studies using monoclonal antibodies in subhuman primates have shown that new antigens are expressed on developing spermatocytes and spermatids after the initiation of spermatogenesis (Isahakia, 1988). These antigens, to which the immune system is not tolerant, could play a role in the genesis of auto-immunity to sperm. It has been postulated that the development of auto-immunity to spermatozoa may be prevented by sequestration of auto-antigens on germ cells by the presence of the blood–testis barrier (Dym and Caviacchia, 1977; Setchell et al., 1988). That such a barrier exists has been documented by the presence of tight junctions between Sertoli cells and the inability of macromolecules to enter the luminal compartment of the seminiferous tubule after their intravenous administration. Studies in the dark mink, a seasonal breeder, have documented the presence of antisperm antibodies and orchitis following testicular regression and breakdown of the blood–testis barrier (Tung et al., 1984). There is also evidence, in mice, indicating that auto-antigenic germ cells exist outside of the blood–testis barrier which are accessible to antigen-processing cells (Tung et al., 1987; Yule et al., 1988; Saari et al., 1996), suggesting active local immuno-regulatory mechanisms may be operative within the testis. Obstruction of sperm egress has been associated with development of auto-immunity to spermatozoa. Approximately half of men who have undergone vasectomy have sperm antibodies detected in their sera (Ansbacher, 1973; Alexander and Anderson, 1979). Because unique auto-antigens are expressed on cells of the basal compartment of the seminiferous tubule, most investigators have speculated that auto-immunity to sperm

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after vasectomy broaches both the sequestration of antigens as well as these local immuno-regulatory mechanisms. The presence of these antibodies within reproductive tract secretions, noted at the time of vasovasostomy, occurs infrequently and has been correlated with an impaired chance of subsequent fertility (Meinertz et al., 1990). Men with bilateral congenital absence of the vas deferens, epididymis, or seminal vesicles (Patrizio et al., 1989), as seen in cystic fibrosis (D’Cruz et al., 1991), also are found to be at risk for immunity to sperm. Our finding that auto-immunity to sperm does not develop in these men until after puberty (Bronson et al., 1992) suggests that the immune system may become exposed to developmental antigens expressed on spermatocytes and spermatids to which it is not tolerant after activation of the pituitary–testicular axis and the initiation of spermatogenesis (Table 5). Developmental abnormalities of the formation of the blood– testis barrier, its traumatic disruption, or unilateral focal cryptic intra-testicular obstruction at the level of the seminiferous tubules could therefore lead to antisperm antibody formation. Gastrointestinal exposure to sperm has also been associated with the development of antisperm antibodies. This has been seen both experimentally in animals, where intra-rectal inoculation with sperm leads to auto-immunity (Richards et al., 1984), as well as in surveys of homosexual men. Humoral sperm-reactive antibodies have also been found in homosexual men who engaged in frequent oral–genital sex (Bronson et al., 1983; Witkin and Sonnabend, 1983; Wolff and Schill, 1985). A correlation has also been noted between sexually transmitted diseases and the presence of immunities to sperm. Recently, Witkin and his associates (Witkin et al., 1995; Munoz et al. 1996) have found an association between the presence of antisperm antibodies in men and women and immunity to the Chlamydia trachomatis 60-kDa heat-shock protein (hsp60), in asymptomatic men and women with no history of C. trachomatis infection. A soluble form of hsp60 has been Table 5 Detection of antisperm auto-antibodies in 15 males with cystic fibrosis and their pubertal statusa Patient category

Age (years, mean)

Testicular volume (ml, mean+ S.E.M.)

Serum testosterone (nmol/l, mean+S.E.M.)

Serum FSH (mIU/ml, mean+ S.E.M.)

Antibody positive Antibody negative

26.4 (range 18–33) 12.4 (range 9–19)

20.0+0.0

12.3+1.4

12.5+3.4

7.0+2

4.0+1.7

4.6+0.99

a

Adapted from Bronson et al. (1992).

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detected in semen, and this finding correlated with the presence of antichlamydia antibodies in these individuals. These circulating antibodies appear to be reactive with a specific region of hsp60, which represents a conserved epitope of the heat-shock protein (Witkin et al., 1998)

3.5. Mucosal immunity and antisperm antibodies Evidence has accumulated suggesting the production of locally secreted antisperm antibodies, within the genital tract, despite their absence in blood. These immunoglobulins are primarily of the IgA class. Secretory IgA is the major immunoglobulin present in tears, saliva, and colostrum, as well as in respiratory, gastrointestinal, and reproductive tract secretions. It is the product of two distinct cell types (Mestecky and McGhee, 1983). Secretory IgA is synthesized by plasma cells, and epithelial cells produce secretory component (SC), which acts as a regulatory transport protein for IgA. A membrane SC–IgA complex forms and is then internalized and transported to the apical region of epithelial cells. The SC–IgA complex is then released into the external secretions. Although little is known about the mechanism of IgA secretion within the male genital tract, the local production of Escherichia coli-specific IgA has been detected in men with chronic prostatitis (Fowler, 1996). A local secretory system exists in the human female reproductive tract, as suggested by the prominence of IgA-producing plasma cells in the fallopian tubes, cervix, and vagina (Kutteh and Mestecky, 1996). Kutteh et al. (1988, 1990) demonstrated by indirect immunofluorescence that tubal segments obtained at sterilization contained IgA-secreting plasma cells in the subepithelial lamina propria. Their observations suggest that if one relies solely on serological tests to diagnose immunities to sperm, results would be misleading in a significant proportion of cases. They reinforce the notion that the presence of humoral antibodies directed against sperm is not relevant to fertility unless the circulating antibodies are present within the reproductive tract. Tests capable of detecting immunoglobulins on living sperm recovered from the ejaculate are the most direct way to detect whether auto-immunity to sperm exists and, if so, to determine its extent and type (Table 6).

3.6. Etiology of immunity to sperm in women Although women are regularly inoculated intra-vaginally with spermatozoa during coitus, this activity is usually not associated with the development of immunity to sperm. Yet the female reproductive tract is not an immunologically privileged site, as demonstrated by the presence of antiCandida antibodies in women with yeast vaginitis. Experimental intra-vagi-

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Table 6 Detection of antisperm antibodies in 170 women at risk for immunity to sperma Serum

Positive Negative

Vaginal flush Positive

Negative

41 (24.1%) 5 (2.9%)

79 (46.4%) 45 (26.4%)

a

Antisperm antibodies were detected in women with impaired post coital tests by immunobead binding by in vitro antibody transfer following incubation of known antibodynegative sperm in serum or saline vaginal flushes that had been concentrated by ultra-filtration. Of vaginal flushes, 74% contained IgA, 55% IgG and 15% IgM isotypes. Adapted from Bronson (1987).

nal inoculation of women with poliovirus in women has been shown to lead to the formation of locally produced antiviral antibodies in vaginal secretions (Orga and Orga, 1973). Immuno-inhibitory substances have been detected and partially characterized in seminal plasma (James and Hargreave, 1984). They both protect sperm from immunological damage and may prevent sensitization of a woman to sperm antigens after coitus. 19-Hydroxyprostaglandin E, a potent immunosuppressive agent, has been found in high concentration seminal fluid of men and subhuman primates (Samuelsson, 1963; Templeton et al., 1978). Other possible immunosuppressive factors include polyamines, transglutaminase, and a high-molecular weight Fc receptor-binding protein (Thaler et al., 1989). Spermatozoa themselves have been shown to be immunosuppressive in rodents. However, because semen samples in vasectomized males also exhibit immunosuppressive activity, this observation suggests that active components are derived not solely from testicular, epididymal, or spermatozoan origin. Studies on fractions obtained by gel filtration of seminal fluid and chromatographic techniques support the view that the inhibitory effects of seminal plasma are due to a range of molecules of widely different molecular weights and binding affinities for specific ligands (Lord et al., 1997). Using serum-free culture conditions, evidence has been presented of interference by seminal fluid in the immune function of T cells, B cells, and NK cells and macrophages. The effects of human seminal plasma on immunologically active cells include a reduced ability to bind antigen and to differentiate or proliferate in response to mitogens, as well as a failure of phagocytosis in antibody-dependent cell lysis. Anti-complement activities have also been demonstrated (Petersen et al., 1980; Brooks et al., 1981). Lymphocytes that express a T-suppressor phenotype have been detected within seminal fluid as well and could play a role in local immunosuppression (Witkin, 1988). Nature then might provide the means

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through the common exposure at coitus to seminal fluid-derived suppressors, as well as spermatozoa, to prevent the development of immunity of sperm in women. Conversely, would the lack of immunosuppressor activity of seminal fluid lead to the development of antisperm antibodies? These questions, unfortunately, currently have no answer.

3.7. Treatment: why treat in the absence of proof? The accumulated evidence from laboratory-based studies provides circumstantial evidence that immunities to spermatozoa can potentially impair processes leading to fertilization. However, there are currently no prospective studies demonstrating a decreased fecundity in those couples in whom ASA are detected, when compared with couples in the absence of immunities to sperm. While we have maintained that such studies are needed as proof of immunological infertility (Bronson and Tung, 1992), these data are not likely to be available in the near future. Given the low incidence of significant immunities to sperm in men and women (which are found in approximately 3–5% of unselected infertile couples), many centers will need to participate in a prospective study of the effects of ASA on fertility, and patient acquisition will be slow. In addition, we have performed a retrospective analysis of pregnancies in women treated for infertility, whose husbands were found to exhibit an auto-immunity to sperm but were not themselves treated (Ayvaliotis et al., 1985). Pregnancy rates varied from 15.3 to 66.7%, depending upon the proportion of spermatozoa coated with immunoglobulin, as judged by immunobead binding. The results suggest that the number of cases needed to obtain sufficient power to detect differences in fertility between antibody-positive and antibody-negative groups will be large. Currently utilized tests, including immunobead binding, also do not identify those sperm-associated antigens to which ASA are directed. Antibodies directed against different antigens would be expected to play differing roles in impairing processes leading to successful fertilization, and this appears to be the case. As noted earlier, when transferred to sperm of known fertile men, antisperm antibodies detected in sera of infertile couples have varying effects on gamete interactions in vitro, as judged by both hemi-zona assays and sperm penetration of zona-free hamster eggs. It is then currently difficult to select homogenous groups of couples with immunities to sperm for study. For these reasons, a prospective analysis of pregnancy rates of men or women with immunities to sperm is not possible at this time. I would argue, however, that one need not wait for clinical proof through prospective analysis of couples with immunities to sperm that they cause infertility to recommend treatment. There is sufficient evidence garnered through laboratory investigation to suggest that these antibodies

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directed against sperm may alter their ability to enter the female reproductive tract and to successfully fertilize. In addition, treatments are currently available in their presence. The use of the immunobead binding test also provides useful information that allows the laboratory to optimize the preparation of spermatozoa for intrauterine insemination (see below).

3.8. Treatment: whom to treat and when A judgment as to whether an individual should be treated for immunity to sperm is far easier to make for men than for women. Spermatozoa are easily accessible for study. The degree of their impairment of penetrating ability of cervical mucus is directly related to the extent of auto-immunity. Men whose spermatozoa are all or nearly all (\70%) antibody-coated by at least one immunoglobulin class and fail to penetrate cervical mucus require treatment. When coated over the sperm head, not only do these sperm encounter difficulty penetrating cervical mucus, but should they reach the ampulla of the oviduct, fertilization may be impaired. Conversely, when B50% of sperm are antibody-bound, the number of sperm seen at postcoital testing is often no different from that of men without auto-immunity to sperm, and other causes of infertility should be investigated. The diagnosis of clinically relevant immunity to sperm in women is more difficult, given our inability to adequately sample secretions of the uterus and fallopian tubes. Immunoglobulin secretion within the female reproductive tract is under hormonal control, and each of the reproductive compartments (cervix, uterus, fallopian tubes) exhibits different mechanisms in the regulation of antibody transport (Parr and Parr, 1996). As an example, estradiol lowers the content of immunoglobulins within cervical mucus while stimulating the active transport of IgA and transudation of IgG into the uterine lumen. While it would be ideal to evaluate tubal fluid or cervical mucus for the presence of sperm antibodies, this is not often possible. Extraction of antisperm antibodies from cervical mucus, a hydrogel, is difficult and may result in damage to these immunoglobulins. A high incidence of ‘immunological background noise’ is also present in women (Table 1). In our study of sera from known fertile women, supplied by the World Health Organization reference bank, 40% contained immunoglobulins that reacted with the tail end piece of spermatozoa (Bronson et al., 1984, 1985). These results suggest that there is a continuum in the extent of immunity to sperm and that those mechanisms in women that prevent immunization to paternally derived antigens are imperfect. Hence, care must be exercised in distinguishing between a positive result and a clinically significant result, whether based on immunobead binding or any antisperm antibody assay. Results of these tests should not be interpreted in the absence of clinical correlates.

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When sperm-reactive antibodies are present in serum, the post coital test is impaired, and other causes of hostile cervical mucus have been excluded (occult cervicitis, altered pH, or poor cervical mucus production due to an insensitivity of mucus-secreting cells to estrogen stimulus, as seen in prenatal diethylstilbestrol exposure), the diagnosis of immunological infertility is strongly suggested. The finding of ASA at the site of fertilization, within peritoneal fluid and in uterotubal flushings retrieved at laparoscopy, or in vaginal secretions or cervical mucus, reinforces the diagnosis of immunological infertility. In these women sensitized to sperm, large numbers of spermatozoa are often observed within cervical mucus, either immobilized or displaying restricted motion.

3.9. How to treat Observations from recent clinical experience in the treatment of immunological infertility reinforce the notion that the major locus of action of antisperm antibodies is through their impairment of sperm transport to the site of fertilization and their shortened longevity within the female reproductive tract. In those couples with ASA who proceed with IVF, in vitro fertilization rates have been found to be high in the presence of circulating antisperm auto-antibodies (Rajah et al., 1993; Acosta et al., 1994; Pagidas et al., 1994; Vazquez-Levin et al., 1997), and only when nearly all sperm ( \ 70%) are coated over their heads with immunoglobulin, as reflected in MAR or immunobead binding assays, is there be a significant fall in the likelihood of fertilization (Lahteenmaki, 1993; Yeh et al., 1995). In the latter situation, the possibility exists that fertilization-related antigens (likely to be specific gamete receptors and their ligands) may be the targets of these antibodies. As the etiology of auto-immunity to sperm in men remains in large part unknown, treatments must be empirical, being directed not against the cause, but, rather, against the abnormal response. Evidence suggests that the use of corticosteroids for immunosuppression is relatively ineffectual, benefiting only approximately 20% of treated men (Hendry et al., 1990). When carefully timed to follicular maturation in superovulated, hormonally and sonographically monitored cycles, recent evidence suggests that IUI results in an increased chance of achieving pregnancy. The rationale for IUI is to place within the uterine cavity a large population of living sperm that were excluded after coitus because of the presence of antisperm antibodies either on their surface or within cervical mucus. In theory, this would increase the likelihood that sperm might enter the fallopian tubes and reach the egg. Using careful sonographic and hormonal monitoring of follicular maturation, insemination can be timed to within a few hours of the expected ovulation. The accuracy of timing is

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important in that antibody-bound spermatozoa have a theoretically shortened survival time within the female reproductive tract, where they become opsinized by binding complement components, leading either to their phagocytosis by macrophages or their immobilization (London et al., 1985). There are no controlled, prospective studies of the efficacy of IUI in the treatment of infertility mediated by antisperm antibodies, in men or women. However, retrospective case reviews suggest that IUI is beneficial in conjunction with the use of clomiphene citrate or gonadotropin. The large study of Margalioth et al. (1988) illustrates this thesis. These authors reviewed the outcomes of intrauterine insemination in a group of women with impaired post coital tests who were also found to have antisperm antibodies in their sera. Monthly pregnancy rates in the first 3 months of treatment were 5% following IUI in the natural cycle, 9.7% following IUI in association with the use of clomiphene citrate, and 14.3% per cycle following gonadotropin stimulus and IUI. These differences were statistically significant (P B0.05). The majority of women conceived during three cycles of treatment, and 40% of women who failed to conceive in clomiphene-stimulated IUI cycles conceived with the subsequent use of gonadotropins. The likelihood of pregnancy was also lower for those women possessing sperm head-directed antibodies compared with those directed against the spermatozoon tail. A recent large prospective study of infertile couples (Guzick et al., 1999) provides supporting evidence of these trends, in that pregnancy rates were highest following IUI in gonadotropin-stimulated cycles when compared both with clomiphene-stimulated and unstimulated cycles. Although one could argue that the presence of antisperm antibodies in serum does not in itself constitute proof of an immunological basis of infertility in women, and that other factors could have introduced bias in the study of Margalioth et al. (1988), the data do suggest the use of IUI in this clinical circumstance in a limited trial. In a similar manner, IUI appears to be beneficial in men with auto-immunity to sperm, particularly when performed following the use of laboratory techniques in which ejaculation is performed directly into a buffer solution prior to sperm recovery. Sperm washing may provide some benefit, by eliminating low-affinity antisperm antibodies within seminal plasma that may have bound to spermatozoa during their residence within the vagina following coitus. Unfortunately, the affinities of immunoglobulins for antigens on the sperm surface are high, and once antibody binding to spermatozoa has taken place, simple sperm washing will not remove these antibodies from the sperm surface (Haas and D’Cruz, 1988). Techniques that lead to dissociation of antibody–immunoglobulin complexes (low pH or high ionic strength) are associated with irreversible loss of sperm motility.

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Ejaculation directly into a washing buffer appears beneficial in terms of both maximizing sperm recovery and minimizing the amount of antibody coating sperm (Bronson, 1988b; Jeulin et al., 1989; Elder et al., 1990). The process of antibody coating of sperm within semen is complex. Witkin (1988) has shown, in rabbits, that immunoglobulins may enter the male reproductive tract via the epididymis. If this is the case in humans, antibody coating of sperm may occur prior to ejaculation as well as thereafter. Conversely, immunoglobulins are also present in prostatic secretions (Rumke, 1974; Fowler, 1996), and sperm become exposed to them only after semen liquification. Rapid dilution of the ejaculate and mixing of semen could, in theory, be beneficial on this basis. In addition, transportation of semen collected at home to the laboratory prior to sperm processing would allow time for post-ejaculatory sperm antibody coating and agglutination, which would be avoided by processing semen immediately after ejaculation. Pregnancy rates associated with IUI, in the treatment of men with auto-immunities to sperm, have improved substantially since adopting washing techniques requiring ejaculation of semen into medium rather than into dry containers. While older reports describe 3–10% per cycle rates of conception associated with IUI (Haas, 1991; Francavilla et al., 1992), a recent study utilizing ejaculation into a buffered medium of men who demonstrated high levels of antisperm antibodies (Ombelet et al., 1997) described pregnancy rates of 64% following three cycles of IUI, with a 47% conception rate in the first cycle. Our own unpublished experience confirms these results. If IUI fails, IVF currently appears to offer an excellent chance of conception in couples with documented immunities to sperm. Antisperm antibodies within follicular fluid can be removed by washing the cumulus oocyte complex, and any residual immunoglobulins that may remain within the cumulus oophorus that surrounds the egg do not usually appear to significantly interfere with sperm penetration. While older studies, such as that of Mandelbaum et al. (1987), reported lower rates of fertilization in vitro when patient sera was used to supplement culture media, IVF in women with antisperm antibodies has been successful at rates in serum-free medium have been nearly comparable to those in their absence (VazquezLevin et al., 1991; Hershlag et al., 1994; Diatoh et al., 1995). In men with auto-immunity to sperm, as previously noted, the diminished number of antibody-coated sperm entering cervical mucus after coitus markedly lowers the chances that the gametes will meet. IVF circumvents this problem of sperm transport and ensures the meeting of spermatozoa and egg. Fertilization rates achieved by sperm obtained from men with auto-immunity to sperm have been high in several reports, suggesting that

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impaired sperm transport (functional oligospermia) has been the primary basis of their infertility (Lahteenmaki, 1993; Pagidas et al., 1994; Sukcharoen and Keith, 1995; Ombelet et al., 1997). In contrast to women with immunities to sperm, however, in whom follicular fluid containing antisperm antibodies can be washed from the egg, immunoglobulins in the ejaculates from men with auto-immunity to sperm remain bound to the sperm surface after their recovery from seminal fluid. Although antibodies present on the sperm tail do not significantly prevent fertilization in vitro, sperm head-directed antibodies have the potential to alter the spermatozoan egg-penetrating ability, as previously described in both the hemi-zona assay and the zona-free hamster egg penetration test. Fortunately, these effects may only become apparent when more than 70% of the sperm population used in IVF are coated with immunoglobulins (Clarke, 1988; Lahteenmaki et al., 1995; Yeh et al., 1995; Diatoh et al., 1995). In theory, results will also depend on the sperm antigen to which these antibodies are directed. Should they be directed against antigens that have no role in fertilization, the process of penetration by the spermatozoon would not be impaired. This has clearly been documented under experimental conditions, after the generation of antisperm monoclonal antibodies (Saling and Lakoska, 1985). Unfortunately, no clinical test can predict this outcome before an actual attempt at IVF. Hence, when head-directed antisperm antibodies are detected by direct immunobead binding on all sperm, intracytoplasmic sperm injection (ICSI) should be performed, to ensure high rates of fertilization (Lahteenmaki et al., 1995; Nagy et al., 1995; Clarke et al., 1997). While the argument has been made that the routine testing for ASA is not cost effective in the use of ART, given its low incidence and the high likelihood of fertilization, this is a value judgement. Should one wait for the occasional case of failed fertilization to perform tests for antisperm antibodies retrospectively, as has been suggested? How does one judge the emotional cost to the couple who has gone through these procedures and failed to conceive? Had an immunobead binding test been performed, for a very small individual cost relative to the total cost of an IVF cycle, these couples could have been placed in a high risk category for failed IVF and the need for ICSI discussed.

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