Abstract
The primary aim of this chapter is to provide practical, evidence-based information pertinent to the specific detection of anti-sperm antibodies (ASA) in males and females and the assessment of ASA results and their place in fertility evaluation and management.
7.1 Introduction
The primary aim of this chapter is to provide practical, evidence-based information pertinent to the specific detection of anti-sperm antibodies (ASA) in males and females and the assessment of ASA results and their place in fertility evaluation and management. A secondary but equally important aim is to review selected literature which is relevant to understanding why some individuals develop sperm immunity. Third, to focus briefly on whether new research and novel techniques for detecting ASAs might help to refine our understanding of their role in infertility.
7.2 Historical Synopsis
7.2.1 Early Evidence for Immuno-Infertility
Nearly a century ago “clinical trials” involving immunization of women with their partner’s semen with the aim of inducing immuno-contraception were conducted [1]. Baskin [2] reported on a study of 20 fertile women immunized 3 times intramuscularly at weekly intervals, with their partner’s whole ejaculate. All but one of the women showed sperm immobilizing activity in their serum by one week after the last injection which persisted for up to one year. Only one woman became pregnant after 12 months when the sperm immobilizing activity was no longer detectable in her serum. These trials demonstrated that women could be immunized to develop sperm immobilizing activity, and that this was associated with reduced fecundity.
Obviously, this “trial” would not get past an institutional ethics committee nowadays; however, it was notable from an historical perspective in that it stimulated significant interest in the idea that immunological responses to sperm could be involved in the development of otherwise unexplained infertility, and in the concept of an anti-sperm contraceptive vaccine. The work of Rumke in the 1970s was particularly important in establishing the veracity of immuno-infertility [3]. His work provided a rigorous demonstration of the relationship between delay to conception and the strength or titre of the ASAs in the patient’s serum.
7.2.2 Evolution of Sperm Antibody Detection Methodology
Historically, the study of human immuno-infertility was stimulated by reports of the sperm-agglutinating properties of serum from patients presenting with infertility [4–6]. It subsequently became apparent that many of the early studies had significantly overestimated the incidence of sperm antibodies in the infertile population because of their failure to distinguish specific (i.e. antibody-dependent) from non-specific agglutination. Consequently, sperm agglutination assays are no longer acceptable for detection of sperm immunity in the clinical setting.
An important step occurred in 1968, when Isojima and colleagues published a defined protocol for an immunologically specific test for sperm immobilizing antibodies in serum [7]. The sperm immobilization test (SIT) requires the dual presence of sperm-bound antibodies of immunoglobulin class G (IgG) and/or immunoglobulin class M (IgM) and active components of the classical complement cascade. If sufficient antibody and complement components are present then the majority of spermatozoa will be immobilized during a 1 hour incubation at 37ºC. The availability of the SIT provided further impetus to the study of immuno-infertility from a research perspective and also significantly improved diagnostic capability in this area. However, a more detailed understanding of the effector mechanisms leading to immuno-infertility necessitated the refinement of specific procedures for detecting and localizing antibodies bound to a patient’s spermatozoa. In this regard the development of the mixed anti-globulin reaction (MAR) by Kremer’s group provided a relatively simple means of detecting membrane-bound antibodies of IgG class on the surface of motile spermatozoa in semen and was used by this group to make important contributions relating to the role of sperm antibodies in infertility [8]. However, it could not detect antibodies of IgA and IgM classes with equal facility. Thus, there remained a need for a similar type of assay which could be set up in many laboratories for research and routine screening of the patient’s semen for sperm-bound antibodies of any of the major immunoglobulin classes.
7.2.3 Development, Validation and Application of the Immunobead Test (IBT)
As described above, sperm immunity was traditionally assessed by observing the sperm agglutinating or immobilizing properties of the patient’s serum. However, with the growth of immunological knowledge in the 1970s, it became apparent that circulating antibodies were not necessarily indicative of local antibodies in either titre or immunoglobulin class. Work on the direct IBT was initiated in this laboratory in 1979 and was first presented at the Annual Scientific meeting of the Australian Society for Immunology in 1981 and first published in 1982 [9]. The indirect version of the IBT has since proven to be an excellent test for sperm antibody screening of serum and reproductive tract secretions. A slightly different version of the IBT was developed independently by Bronson’s laboratory in New York [10]. The IBT was widely used for both routine diagnostic work and as a research tool. The research group led by Gilbert Haas published evidence that the IBT was more sensitive than radioimmunoassay for sperm antibody detection [11]. Having developed a sensitive and immunologically specific procedure, the next step was to apply it assiduously with the aim of achieving greater understanding of the mechanism by which an immunological reaction to spermatozoa, either in men (autoimmunity) or women (isoimmunity), might cause sub-fertility or infertility. Early studies with the IBT in my laboratory in Melbourne indicated that sperm autoimmunity occurred in approximately 8.5% of the male partners in couples presenting for infertility investigations [12]. The sperm antibodies were predominantly of IgG or IgA classes. IgM class antibodies were rarely detected in semen. In a proportion of men, one immunoglobulin class was obviously dominant, whilst in approximately 50% of men, both classes were equally represented. Approximately 4% of men presenting with infertility had more than 80% of their motile spermatozoa coated with antibodies of IgG and IgA classes. The aim of subsequent work from this laboratory on sperm autoimmunity was to determine whether antibodies of both classes were involved in the detrimental effects on fertility, or whether perhaps IgA, which is present mainly in the form of secretory IgA, might be the main effector molecule.
7.3 Mechanisms by Which Sperm Antibodies Affect Fertility
7.3.1 The Effect of Sperm Antibodies on Sperm Migration through Cervical Mucus
Initial research in this area commenced with a comparison of IBT results with the outcome of the semen-cervical mucus interaction tests [13]. These studies indicated that there was a strong association between the presence of IgA class antibodies and a strong shaking reaction in the semen-cervical mucus contact test (SCMCT), lending support to the hypothesis advanced by Kremer and Jager [14] that IgA class antibodies formed a bridge between spermatozoa and the cervical mucus micellular structure. These bridges tethered the sperm to the mucus framework, preventing normal penetration of the sperm through the mucus. By implication, the operation of this mechanism in vivo would ultimately prevent sperm transport to the normal site of fertilization in the ampulla of the fallopian tube.
In females, the uterine cervix is known to be a highly competent mucosal immune site [15], which contains many IgA-positive plasma cells located in the sub-epithelial layers of the endocervix. Most of the IgA in cervical mucus is secretory IgA consisting of two IgA monomers linked by J-chain and secretory piece. The secretory IgA antibodies directed against potential pathogens and occasionally sperm can immobilize the invaders by cross-linking them to the cervical mucus strands, effectively blocking their progress to the upper reaches of the reproductive tract [14]. There are obviously protective mechanisms which normally preclude such immunological reactions to sperm in the majority of women. However, in a small percentage of couples these protective mechanisms are somehow circumvented or disrupted, resulting in local and often circulating ASA production and reduced chances of natural conception. In women with otherwise unexplained infertility, sperm antibody activity has been detected in cervical mucus in more than 10% of cases [16].
Thus, the overall conclusion of these investigations was that IgA was the most important effector molecule leading to inhibition of the normal process of sperm migration through mid-cycle cervical mucus. Although high-titre sperm antibodies will totally block sperm migration through cervical mucus, many patients have intermediate antibody levels which only partially inhibit mucus penetration. Hence, it was vital to determine whether ASAs might affect other aspects of sperm function.
7.3.2 Effects of Sperm Antibodies on In Vitro Fertilization (IVF)
Retrospective analysis of IVF results by Clarke and colleagues [17] provided some of the first evidence that ASAs from female serum could inhibit the fertilization of viable human oocytes by human spermatozoa. They observed a fertilization rate of only 15% for patients who had significant titres of IgG and IgA class ASA in their serum, which at that time was used as a supplement in the IVF culture medium, versus 69% for those patients where replacement serum was used during the fertilization culture. Their later results confirmed that very high titre ASA of IgG immunoglobulin class in female serum could effectively inhibit fertilization of fresh human oocytes [18]. Subsequent reports from other laboratories have also indicated that high level ASA can inhibit human fertilization [19–21]. Consequently, it is now generally accepted, at least with strong sperm immunity, that ASA can block sperm functions such as cervical mucus penetration and fertilization and thereby impair fertility.
7.3.3 Post-Fertilization Effects of ASA on Fertility
Definitive studies in various animal models have shown an association between ASA and pre- or post-implantation embryonic degeneration [22]. Why should ASA react with embryos? First, the sperm membrane is integrated as a mosaic into the zygote membrane during the process of fertilization, so that sperm antigens are incorporated, although at relatively low densities, into the developing embryo [23]. Second, embryonic gene expression commencing from the four to eight cell stage results in the synthesis of various developmental antigens which can cross-react with sperm antigens [24]. Consequently, during embryo development and perhaps particularly around the time of blastocyst hatching, there is a chance for the ASA to bind to cross-reacting embryonic antigens and potentially cause embryo degeneration or possibly prevent implantation.
There is also some evidence for post-fertilization effects associated with ASA in humans. Concerning negative effects, Warren Jones reported in 1981 that around 50% of pregnancies conceived in women with ASA subsequently ended in first trimester spontaneous miscarriages [25]. Similar observations have been reported by other groups [16,26]. In the latter study, it was found that 7/16 (44%) of women who miscarried were positive for ASA in their serum, compared with only 2/17 (12%) of women who had successful ongoing pregnancies. Examination of the immunoglobulin classes of the antibodies revealed that IgA was significantly (p<0.01) more common in those women who miscarried. The IgA class antibodies in serum may be a marker for local secretory IgA in the female reproductive tract. However, despite the strong evidence in rabbits [27], it is still not known whether IgA class ASA in humans are embryotoxic. In another clinical study [28], it was found that of 173 women referred for a history of three or more consecutive spontaneous miscarriages, there was a significantly higher incidence of sperm immobilizing antibodies when compared with the infertile group. Interestingly, they also observed a higher incidence of ASA in the group of women shown to have an immunological basis for their recurrent miscarriages (e.g. couples sharing at least three HLA determinants, or couples with the female showing a relatively low response to her partner’s lymphocytes in mixed lymphocyte culture). Other groups have reported a significant association between ASA and some autoantibodies such as anti-phospholipids, which may be involved in deleterious effects on the foetus. In contrast to the studies cited above, which have reported an association between ASA and recurrent miscarriage, others have not seen a statistically significant association [29]. Further investigations in this area would be useful, particularly focusing on the possible involvement of sub-surface sperm antigens which react with IgA class ASA. It is important to note that sperm antibodies specific for sub-surface antigens are unlikely to be detected by assays such as the IBT or the MAR which are designed to measure reactivity with membrane antigens on motile sperm. It could be very informative to conduct a clinical investigation of IVF patients with repeated implantation failure or early spontaneous miscarriages, using a new generation of highly specific ELISA and immunofluorescence assays in conjunction with the MAR (unfortunately immunobeads are no longer available so the original IBT has become obsolete).
7.4 Possible Causes of Immuno-Infertility
What information is currently available regarding the development of, or predisposing factors for, sperm immunity? In men, sperm auto-immunity often develops after vasectomy or genital trauma or due to cross-reactions with infectious organisms. Observations of potential relevance to understanding the underlying causes of ASA in women include evidence that they are more likely to have detectable sperm antibodies if their male partner also has ASA in his semen [30]. Another important observation was that in about one-third of cases women apparently react only to their partner’s sperm antigens, rather than to sperm-specific antigens [31]. This observation requires further investigation because if confirmed, it would indicate that many female patients with immunity to their partner’s sperm may be going undiagnosed. Several hypotheses have been proposed in order to explain the origins of sperm immunity and the observed association between male and female sperm immunity in a proportion of couples [32].
7.5 Current Laboratory Procedures for Detection of Sperm Antibodies
There are a few methods which are more suitable for use in the routine diagnostic laboratory, including the MAR, the immunosphere binding assay (IBA) and the SIT. Although there are many ELISA kits available on the market, some of which could prove useful for research applications, I am not convinced that this procedure is suitable for routine screening of patient sera during infertility investigations. I believe that it is indicative that no laboratories report ELISA results for sperm antibody detection in the Australian External Quality Assurance Scheme for Reproductive Medicine (EQASRM). This scheme sends four sample distributions per year and laboratories enrolled for the sperm antibody module receive four serum samples in each distribution. It is vital that any laboratory performing diagnostic sperm antibody tests is enrolled in an appropriate EQA scheme.
For the commercial sperm antibody detection kits it is important to follow the manufacturer’s instructions provided with each kit and that any variations are detailed in the laboratory quality system. For “in-house” procedures such as the SIT it is necessary to document any variations from published protocols, to perform internal controls and if possible to participate in an appropriate EQA scheme. The following sections provide extra information relating to particular tests.
7.5.1 Mixed Anti-Globulin Reaction (MAR)
The MAR detects antibodies bound to the surface of motile sperm (Figure 7.1) and can be used in the direct form for detection of antibodies attached to the surface of sperm in the ejaculate, or in an indirect form designed to detect circulating antibodies present in blood serum from the male or female partner. As shown in Figure 7.1, the antibodies are detected by cross-linking of latex beads coated with human immunoglobulin (IgG) to antibodies bound to the surface of motile sperm (an analogous reaction can be used for IgA class sperm antibodies). The cross-linking is caused by an anti-human immunoglobulin antiserum (anti-globulin). As with any immunological test, it is vital to know that the detection system is effective in detecting sperm antibodies in a known positive sample, and that any nonspecific binding of the latex beads to sperm is minimal as indicated by testing a known negative sample. The positive and negative controls should be performed at regular intervals during the life of each MAR kit (e.g. weekly or fortnightly). An important internal control is to note and record the presence of significant bead-bead clumping during every test. The bead clumping is caused by the added anti-globulin binding specifically to the human immunoglobulin attached to the latex beads and is the same mechanism which causes mixed agglutination between beads and motile sperm coated with sperm antibodies. The vast majority of beads should be in clumps with or without sperm – weak bead clumping indicates that something is not right and should be investigated. If the same thing happens with a repeat test, then perform positive and negative sample controls – if the positive control is weak or negative, this suggests that the kit may have deteriorated during storage (e.g. it may have frozen due to defective refrigeration or become contaminated). If indicated, it may be necessary to open a new kit, test it against the known controls and repeat the patient’s test. Some laboratories (e.g. reference or research laboratories) may wish to undertake more rigorous kit evaluation by performing crossed-inhibition tests on new kits. For example, an MAR-IgG kit should show positive controls inhibited by pure human IgG but not by pure IgA or IgM. My laboratory recently performed a crossed-inhibition test using a serum which gave 100% of motile sperm coated with IgA class antibodies on the sperm head. Addition of pure IgA completely inhibited the binding, whereas pure IgG gave no inhibition. This demonstrated the specificity of the kit for detection of antibodies of IgA immunoglobulin class.
