Analytical Techniques to Measure the Presence of ASA
Methods to Measure ASA in Spermatozoa and Fluids
ASA are mainly of Ig classes IgA and IgG, whereas IgM class antibodies are rarely found in semen. These antibodies can be detected on the sperm surface and/or they can be found free in the seminal fluid, in the male or female serum, in the cervical mucus, and/or in the follicular fluid.
Among assays developed to test the presence of ASA, two are currently the most used: the mixed antiglobulin reaction (MAR) test66-67 and the immunobead binding (IB) test,68-69 or their commercially available options. Both tests detect ASA on the surface of live spermatozoa by incubating motile spermatozoa with Ig-coated particles. In the presence of ASA, these particles adhere to the sperm surface; the percentage of motile spermatozoa with bound particles and their cell surface localization are recorded by observation under the microscope. Whereas the MAR test is performed on a fresh semen sample, the IB test requires semen centrifugation to remove the seminal plasma.
Using either method, the presence of ASA can be evaluated directly on the sperm cells (direct method) or in biological fluids (indirect method) after incubating them with donor ASA-free spermatozoa. As the first step, it is recommended to determine ASA on the sperm surface, and afterward, their presence in body fluids.70 If there are insufficient motile spermatozoa to perform the direct test, indirect tests must be used because samples with poor motility may yield false-negative results. It is considered that high ASA titers in fluids are related to ASA bound to the sperm membrane, which may impair sperm performance. The World Health Organization (WHO) semen analysis manual (fifth edition) includes ASA determination as part of the basic semen evaluation, and the MAR and IB tests are detailed.71
The direct IgG and IgA MAR tests are performed by mixing fresh, untreated semen with latex particles (beads) or treated red blood cells coated with human IgG or IgA. A “bridging” antibody (anti-IgG or anti-IgA monospecific) is used to bring the antibody-coated beads into contact with spermatozoa carrying IgG or IgA. The formation of mixed agglutinates between beads and motile spermatozoa is indicative of IgG or IgA antibodies on the sperm surface (Figure 12.2). If spermatozoa do not present ASA on their surface, they will move freely, not covered with beads. Agglutinated beads will prove the reactivity of the particle antibodies and antiserum. In some cases, massive particle attachment might even cause sperm immobilization. The percentage of motile spermatozoa with bound beads/red blood cells is recorded.
In the direct IB test, spermatozoa must be devoid of seminal plasma by centrifugation. “Washed” spermatozoa are incubated with polyacrylamide beads (2-10 pm) coated with covalently bound rabbit antihuman Igs against IgG, IgA, and/or IgM, and the binding of beads to motile spermatozoa indicates the presence of Igs on the sperm surface (Figure 12.3).
In addition to the direct ASA tests, indirect tests are performed to evaluate the presence of sperm antibodies in body fluids, among them seminal plasma, blood serum, follicular fluid, and bromelain-solubilized
FIGURE 12.2 Schematic representation of the principle of the direct mixed antiglobulin reaction (MAR) test. (a) Fresh, untreated semen sample. (b) Latex beads or treated red blood cells coated with human immunoglobulin (Ig)G or IgA. (c) Anti-human IgG or IgA. (d) Mixed agglutinates composed of beads/red blood cells and motile spermatozoa (indicating the presence of IgG or IgA on the sperm surface). The percentage of motile spermatozoa with bound beads/red blood cells is recorded.
FIGURE 12.3 Schematic representation of the principle of the direct immunobead binding (IB) test. (a) Washed spermatozoa. (b) Polyacrylamide beads coated with rabbit antihuman immunoglobulin (Ig)G, IgA, and/or IgM. (c) Beads bound to motile spermatozoa (indicating the presence of IgG, IgA, and/or IgM on the sperm surface). The percentage of motile spermatozoa with bound beads is recorded.
cervical mucus. The diluted, heat-inactivated fluid suspected to have ASA is incubated with ASA-negative donor spermatozoa previously devoid of seminal plasma. Any ASA in the suspect fluid will bind specifically to the donor spermatozoa, which are then assessed in a direct test, as already described. For reliable results, it is important to allow sufficient time for the sperm-antibody interaction because it may take up to 10 minutes for the mixed agglutination to become visible. However, it should be considered that sperm motility declines with time, and the test results depend on the presence of motile spermatozoa. Both indirect MAR and IB tests can be performed (Figures 12.4 and 12.5). A description of the protocol to perform
FIGURE 12.4 Schematic representation of the principle of the indirect mixed antiglobulin reaction (MAR) test. (a) Washed antibody-free donor spermatozoa. (b) Heat-inactivated fluid with antisperm antibodies (ASA). (c) Donor spermatozoa with bound ASA. (d) Latex beads or treated red blood cells coated with human immunoglobulin (Ig)G or IgA. (e) Anti-human IgG or IgA. (f) Mixed agglutinates composed by beads/red blood cells and motile spermatozoa (indicating the presence of IgG or IgA in the fluid). The percentage of motile spermatozoa with bound beads/red blood cells is recorded.
FIGURE 12.5 Schematic representation of the principle of the indirect immunobead binding (IB) test. (a) Washed antibody-free donor spermatozoa. (b) Heat-inactivated fluid with antisperm antibodies (ASA). (c) Donor spermatozoa with bound ASA. (d) Polyacrylamide beads coated with rabbit antihuman immunoglobulin (Ig)G, IgA, and/or IgM. (e) Beads bound to motile spermatozoa (indicating the presence of IgG, IgA, and/or IgM in the fluid). The percentage of motile spermatozoa with bound beads is recorded.
Advantages and Disadvantages of the MAR Test and the IB Test
• Beads form clumps
Abbreviations: MAR, mixed antiglobulin reaction; IB, immunobead binding; ASA, antisperm antibodies.
the MAR test (direct) and IB test (direct and indirect) following the guidelines from the WHO71 is presented; moreover, information on the commercially available kits based on the MAR and IB tests is also included (see the section “Laboratory Guidelines for ASA Assessment in Sperm and Biological Fluids”).
Both the MAR test and the IB test have several advantages and limitations. Table 12.1 summarizes some of these characteristics. Several studies have compared the sensitivity obtained with both techniques but they do not always agree. The differences may be attributed to the type of method (direct or indirect) compared in each report.72-75
In addition to the MAR test and IB test procedures, the presence of ASA can be objectively evaluated using flow cytometry76,77 and radiolabeled agglutinin assays.76 For both techniques, a specific anti-Ig is labeled (with a fluorescent or radioactive marker, respectively) and mixed with the sperm sample. Flow cytometry allows the quantification of Ig level in live spermatozoa and can be coupled with immunocy- tochemistry to determine the localization of ASA on the sperm regions.
The enzyme-linked immunosorbent assay (ELISA) can also be used for auto- and iso-ASA-specific detection and quantification.78 Anti-human Igs are covalently linked to an enzyme and added to fixed spermatozoa or to sperm extracts (previously incubated with the test fluid in the indirect assay). Antibody-enzyme Ig complexes are detected by a specific enzyme substrate, resulting in a color change that can be measured. The main disadvantage of this technique is that sperm fixation may disrupt plasma membrane, altering antigen detection.
The sperm agglutination tests (SAT) are able to detect the presence of multivalent ASA (mainly IgA and IgM) in serum or semen, with the ability of cross-linking several spermatozoa. Fluid samples are heat inactivated and serially diluted, motile spermatozoa from an ASA-negative donor are added, and sperm agglutination at each dilution is determined. The agglutination tests are known as the tray-slide agglutination test54 or the tray agglutination test (TAT)79 and the macroscopic gelatin agglutination test (GAT).80 False-positive results can occur because bacteria or non-Ig proteins can also cause sperm agglutination. In addition, the sperm immobilization test (SIT)81,82 is based on the principle that surface ASA can cause loss of sperm motility in the presence of complement. This assay uses patient serum mixed with motile spermatozoa and an external source of complement, and sperm immobilization is recorded. It is worth mentioning that SIT is not useful for the detection of ASA in samples with IgA because IgA does not fix complement.