Antisperm Immunity and Infertility

Jin-Chun Lu; Yu-Feng Huang; Nian-Qing Lu

Disclosures

Expert Rev Clin Immunol. 2008;4(1):113-126. 

In This Article

Evaluation of ASA-assay Methods

In clinical andrology, an ASA assay has been considered to be a necessary step for the assessement of male infertility. This practice is generally accepted even though there is still some disagreement surrounding the true meaning of antisperm immunity, and there remains a good deal of controversy about the test, which is regarded as the most suitable for the detection of antibodies directed against sperm antigens. There are many ASA assay methods, each with advantages and disadvantages. However, until now, it was difficult to address which test is optimal.

The requisite of an ASA assay is the preparation of sperm antigens. Sperm antigens used in the assay were prepared by different methods:

  • Antigen extract was made by treatment of freeze-thawed sperm pools with 0.3 mol/l lithium 3,5-diiodosalicylate or detergents, such as Tween-100 and NP-40;

  • Sonicated sperm antigens: whole spermatozoa were disrupted with sonic bursts on ice, and the supernatants were obtained by supercentrifugation;

  • Cavitated sperm antigens: sperm pellets were placed in the chamber filled with nitrogen, and the cavitated sperm suspension was made by the sudden release of pressure;

  • Various recombinant sperm antigens prepared by gene engineering;

  • Sperm antigen epitopes prepared by phage-display techniques.

The sperm pool used in the first three methods should also be obtained by Percoll technique, Ficoll or swim-up so as to prevent leukocyte contamination. It appears to be accepted that outer sperm-membrane antigens are of relevance, for ASAs are not able to penetrate the outer cell membrane of vital sperm.[69] The exception to this rule concerns acrosomal antigens, for the inner acrosomal membranes of the spermatozoon become externalized and antigens are presented during the AR, which are otherwise not recognized.[4] Owing to sperm antigens from different individuals varying widely the method to select adequate sperm donation is yet to be fully elucidated.

Sperm agglutination tests include glass agglutination test, the tray agglutination test and the gelatin agglutination test . Unfortunately, this assay requires fresh motile spermatozoa. Moreover, individual sperm may vary in their antigenic expression.

In agglutination tests, agglutinates could be formed due to bacteria and amorphous material within semen, nonspecific immunoglobulin, as well as nonimmunoglobulin proteins in serum, leading to a false-positive reaction. The regional specificity of antibody binding limits the formation of agglutinates of a relatively large size. In addition, agglutination tests could not be used to identify the classes of immunoglobulins implicated. Therefore, these tests are not recommended for routine analysis.

Sperm immobilization test (SIT) involves counting motile sperm under the microscope, and is subjective as it relies on the judgement of the examiner with respect to sperm motility. Komori et al. analyzed motile sperm by a digital method using Sperm Quality Analyzer instead of the conventional method,[70] which may be of more value in the measurement of motile sperm in determining sperm-immobilizing antibodies. However, because complement-mediated sperm-immobilization assays are limited in their usefulness for the detection of IgA, since IgA does not fix complement; and many sperm head-binding antibodies do not lead to a loss of sperm motility, which might give false-negative results, SIT is also not recommended.

Immunobead test (IBT) and mixed antiglobulin reaction (MAR) methods are very easy, inexpensive, and rapid, such as with similar sensitivity and specificity, detection mechanism and operation and therefore, were recommended by the WHO as the 'standard' laboratory technique for the determination of sperm-bound ASAs.[71] They can be used to identify the classes of ASAs and the proportion of sperm-binding antibodies, but are not able to identify specific antigens, the number of antibody molecules bound to the sperm surface and the place of fixation because the relatively large 'labels' (immunobeads, latex particles and erythrocytes) limit resolution. Moreover, the spermatozoa from different donors were found to differ significantly in the degree and location of immunobead binding after exposure to the serum sample containing ASAs.

In addition, these two methods have different standards judged for positive, in general, for 10%, but also 20 or 50%. Moreover, it is impossible to discriminate which antibodies are of clinical relevance and which are not.[4]

Immunofluorescence assays (IFAs) seem to be valuable tools in the diagnosis of immune infertility, in particular when impairment of the acrosome function is suggested.[49] However, internal sperm antigens exposed after plasma membrane damage or the use of methanol fixation may lead to false-positive results. Moreover, the determination of regional specificity of sperm-binding antibodies is limited by the low resolution of these methods.

IFAs can only detect the immunoglobulin class of the antibody concerned and the proportion of sperm-binding antibodies, but are not able to detect the number of antibody molecules or antigens involved in the binding. Moreover, it is impossible to discriminate which antibodies are of clinical relevance and which are not,[4] and results based on IFAs are unreliable and lacking in correlation with continuing infertility. Thus the routine use of IFAs is not recommended. The use of an immunogold assay, which is similar to IFA, has the same shortcomings.

ELISA could be used to detect ASAs by the antigen absorbed in the solid phase, and antihuman globulin conjugated to alkaline phosphatase or horseradish peroxidase as a developing reagent. The ELISA assay enables detection of IgM, IgG and IgA ASAs in serum, cervical mucus and seminal plasma samples. Moreover, ELISA is more sensitive, less subjective, does not need fresh sperm and easier to perform than other commonly used microscopical methods (SIT, IFA, IBT and MAR). However, ELISA requires fixation of whole sperm or use of membrane extracts. Fixation of sperm may lead to denaturation of sperm antigens or membrane damage, resulting in false-positive or false-negative results. Membrane extracts may not include relevant antigens associated with the process of fertilization. Recently, our group investigated the variations of the results on the detection of ASAs among four different brands of ELISA kits, with up to ten-times the difference of positive rates (Lu J-C et al., unpublished data) , indicating that the detection methods of ASAs urgently need to be standardized, especially the preparation procedures for sperm antigens.

Radiolabeled antiglobulin assay (RIA) is another means of identifying ASAs. Similar to ELISA, RIA provides no information about the proportion of antibody-bound sperm or the regional specificity of ASAs. Both RIA and ELISA were hampered by the fact that the antigens were not biochemically defined and those relevant for the process of fertilization might not be included in the antigen preparation. In addition, seminal plasma itself interferes with the antigen-antibody reaction in such an immunoassay.[72]

Hjort[73] thought that flow cytometry seemed to be a promising technique to determine the exact amount of IgA and IgG on individual sperm. However, this method does not always provide objective and reproducible results.[74] Moreover, few medical centers and university laboratories have access to a flow cytometer, and the practical use of flow cytometry has been limited by the need for a skilled operator and expensive instrumentation.[75]

Immunoblotting and affinity chromatography were reportedly the minors methods for the detection of ASA.[76] However, antigens may be denaturated during the antigen preparation. In addition, a lymphocyte proliferation assay was used to detect CMI. However, Focacci et al.[77] observed no relationship between serum ASAs, and sperm CMI, indicating that the lymphocyte proliferation assay did not appear to be useful in the diagnosis of immunological infertility.

The ASAs detected by different methods are not identical. Comparative investigations using various assays to detect levels of antibodies to human spermatozoa have produced widely varying results. A possible explanation for the observed wide variation in results from different methods to detect ASAs may be the different target antigens used in assays. Comparison of methods between laboratories could be improved only if equivalent target sperm preparations were used. It is important to validate assays, such as the ELISA, before widespread application to patient screening particularly. In general, no single test to detect the presence of ASAs has correlated precisely with subsequent fertility.

In conclusion, although there is reason to accept antibody-mediated antisperm immunity as a cause for subfertility, the routinely used methods are not reliable for the detection of specific ASAs. The methods described so far have been hindered by codetection of undesired nonspecifically bound antibodies. Moreover, the lack of a standardized and universally accepted assay for the determination of ASAs may be the reasons why physicians are not inclined to test ASAs in subfertile couples.[4] At the moment, it is difficult to consider the routinely used ASA tests as essential procedures in the fertility work-up. It is even more difficult to justify a treatment on the basis of such tests.

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