Antisperm antibodies

Detection assays for antisperm antibodies: what do they test?

F.M. Helmerhorst1,3, M.J.J. Finken1 and J.J. Erwich2

1 Department of Obstetrics, Gynaecology & Reproductive Medicine, Leiden University Medical Center, PO Box 9600, NL 2300 RC Leiden, and 2 Department of Obstetrics & Gynaecology, University of Groningen, The Netherlands

The lack of a standardized and universally accepted assay for the detection of antisperm antibodies (ASA) which are specifically directed against known antigens, lack of consensus on clinical consequences of ASA (Krapez et al., 1998Go) and absence of evidence for a mechanistic explanation on how ASA impair conception, may be the reasons why physicians are not inclined to test for ASA in subfertile couples.

Balancing pros and cons of ASA testing for our patients has been discussed in a number of reviews (Marshburn and Kutteh, 1994Go; Naz and Menge, 1994Go). The ESHRE Capri Workshop Group (1998) sees `the main reason for testing for antibodies to identify couples who may warrant earlier referral for assisted reproductive technology'. In their excellent paper on unexplained infertility, Taylor and Collins concluded their chapter entitled `antisperm antibody presence and fertility', by commenting that `large prospective studies based on newer validated assay methods are needed among untreated couples to evaluate whether sperm antibody is a cause of infertility' (Taylor and Collins, 1992Go). Indeed, since the first description of a method to detect ASA (Metchnikoff, 1900Go; Metalnikoff, 1900Go), multiple assays and variations thereof have been introduced.

Not only the lack of a gold standard suggests shortcomings of these assays. Here, we will review the different assays in chronological sequence, emphasizing what the physician may expect from technical abilities of a test.

Qualitative detection

In agglutination tests (Kibrick et al., 1952Go; Franklin and Dukes, 1964Go; Friberg, 1974Go), agglutinates of spermatozoa are observed after patient's serum (and dilutions thereof) and donor spermatozoa are brought together in gelatin medium or on microslides. Agglutination can also be caused by bacteria and amorphous material within semen, as well as non-immunoglobulin proteins in serum, leading to false-positive reactions (Bronson et al., 1984Go).

In the mixed agglutination reaction (MAR) (Jager et al., 1978Go), a washed suspension of Rhesus (Rh)-positive human red blood cells (RBCs) coated with Rh-directed human immunoglobulin (Ig)G (or sometimes with IgA) antibodies are mixed with drops of semen. IgG containing ASA are then bridged with the IgG-coated RBCs after the addition of anti-human IgG antibody. When spermatozoa in the ejaculate are antibody-bound, they may form mixed agglutinates with the RBCs. The relatively large size of agglutinates limits the ability of the MAR to determine the proportion of spermatozoa which are antibody-bound or the regional specificity of antibody binding (Bronson et al., 1984Go). The regional specificity of antibody binding to spermatozoa has relevance to impairment of sperm function with studies suggesting that head-bound ASA have greater impact on fertility (Tsukui et al., 1988Go). Also in this test, non-specific Ig adhering to the sperm surface may be detected.

Complement-mediated sperm immobilization or cytotoxicity assays (Hamerlynck and Rümke, 1968Go; Husted and Hjort, 1975Go) count immobilized spermatozoa or show a dye-uptake of dead spermatozoa, when a complex of antibodies are linked with antigens on spermatozoa in the presence of complement. These type of assays are limited in their usefulness for the detection of Ig isotype IgA containing ASA, since IgA does not fix complement. Moreover many head-directed antibodies do not lead to a loss of motility, which might give false-negative results (Bronson et al., 1982Go; Mestecky and McGhee, 1987).

Antihuman antibody-coated immunobeads can detect antibodies bound to washed spermatozoa by forming agglutinates: the immunobead test (Bronson et al., 1981Go, 1982Go). The region of the sperm surface to which ASA have bound, the proportion of spermatozoa in the ejaculate that is antibody-bound and the isotypes of these antibodies can be determined (Bronson et al., 1984Go). However, the relatively large size of the immunobeads limits resolution and the number of antibody molecules bound to the sperm surface necessary for bead binding has not been determined (Bronson et al., 1984Go).

Immunofluorescence assays (IFA) (Hjort and Hansen, 1971Go; Tung et al., 1976Go) use fluorescein-tagged anti-human antibodies which bind to antibodies on the sperm surface. Internal sperm antigens exposed after plasma membrane damage or the obligatory use of methanol fixation may lead to false-positive results. Naturally occurring antibodies to these antigens occur commonly in men and women and probably play no role in subfertility (Bronson et al., 1984Go). The determination of the regional specificity of sperm-directed antibodies is limited by the low resolution of this method (Young and Smithwick, 1991Go).

The immunogold assay uses gold, instead of fluorescein-tagged antibodies and avoids the multiple washing/centrifugation/resuspensions (as with IFA) that can cause plasma membrane damage or loss (Young and Smithwick, 1991Go). All the above-mentioned methods need microscopic observation and are, therefore, susceptible to subjective interpretation.

Quantitative detection

Enzyme-linked immunosorbent assays (ELISA) (Zanchetta et al., 1982Go; Alexander and Bearwood, 1983Go; Paul et al., 1983Go; Witkin and Bongiovanni, 1983Go) use enzyme-linked anti-human antibodies, which bind to the antibodies on the sperm surface. A substrate for the enzyme is then added, and its product is measured colorometrically. ELISA requires fixation of whole spermatozoa or use of membrane extracts. Fixation of spermatozoa may lead to denaturation of sperm antigens or membrane damage, resulting in both false-negative and false-positive results; membrane extracts may not contain the relevant antigens associated with the process of fertilization (Bronson et al., 1984Go).

Radiolabelled antiglobulin assays (Haas et al., 1980Go) differ from ELISA in that the antibody-linked enzyme has been replaced by a radioisotope. In contrast to ELISA, living spermatozoa can be used for analysis and fixation is not necessary.

Radioisotopes and enzymes are quantitative probes; however, and they provide no information about the proportion of antibody-bound spermatozoa or the regional specificity of ASA. Care must also be taken to ensure that a positive assay is secondary to sperm-associated antibodies and not antibodies on seminal leukocytes (Haas, 1987Go).

As in IFA, in flow cytometry (FCM) (Haas and Cunningham, 1984Go) fluorescein-tagged anti-human antibodies are used. In contrast to IFA, living spermatozoa can be used for analysis. After incubation, samples are introduced into a cell sorter. FCM is an objective method to determine the proportion of antibody-positive spermatozoa or the quantity of antibodies bound to the sperm surface.

Detection of a specific antigen-antibody bond

The methods described so far are hindered by co-detection of undesired non-specifically bound antibodies. Immunoblotting and affinity chromatography are appropriate methods to identify specific bound ASA and have proven to be useful in identification of sperm antigens (Naaby-Hansen and Bjerrum, 1985Go), but have so far not been used in the routine laboratory investigation of the subfertile couple.

With immunoblotting (Hayes et al., 1989Go; Snow and Ball, 1992Go), antigens are first separated in a gel. The resolved molecules are transferred electrophoretically to a nitrocellulose membrane in a blotting tank. The blot is then treated with antibody to the specific antigen, washed, and a radiolabelled conjugate to detect antibodies is bound to the blot. After washing again, the blot is placed in contact with X-ray film in a cassette; the autoradiograph is developed and the antigen bands to which the antibody has bound, are visible. It should be noted that in some cases an antigen becomes so denaturated by the gel separations and blotting procedures that it can no longer be recognized by particular antibodies.

By using affinity chromatography (Roitt, 1997Go), a pure population of antibodies may be isolated. A solid-phase immunoabsorbent is prepared; this is an antigen covalently coupled to an inert support. The immunoabsorbent is placed in a column and the sample is run in under physiological conditions. Antibody to the antigen binds to the column while unbound antibody washes through. In the second step, the column is eluted to obtain the bound antibody using elution buffer, which dissociates the antibody-antigen bond. A third step towards detection of a specific bond between an antibody and antigen is to redemonstrate the antibody binding with a well-defined sperm specific antigen (Eddy and O'Brien, 1994Go). Recently, an elegant method for the identification of these antigens was demonstrated Moretti-Rojas et al. (1998). After DNA sequencing, six cDNA clones were found that express sperm proteins that elicit a specific immune response.

Conclusions

Although there is reason to accept antibody-mediated antisperm auto- or allo-immunity as a cause for subfertility, the routinely used methods are not reliable for the detection of specific ASA. On the basis of an inferior test, a positive test result may lead to an advice for a therapeutic intervention, such as condom use, antibiotics, testosterone, immunosuppressives, intra-uterine insemination or in-vitro fertilization. Their effectiveness is equivocal. The methods of immunoblotting and affinity chromatography may be promising in this field of immunology and should be placed under active scrutiny for extension to clinical use.

We entirely agree that `any link between sperm antibody presence and impaired conception must be considered hypothetical' (Taylor and Collins, 1992Go), until such time that a relationship has been found on the basis of scientifically sound trials in which reliable ASA tests have been used. At the moment, it is difficult to consider the routinely used ASA tests as an essential procedure in the fertility work-up. It is even more difficult to justify a treatment on the basis of such tests.

Notes

3 To whom correspondence should be addressed Back

This debate was previously published on Webtrack 62, 21 April, 1999

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