The IBT has been described as a relatively simple, inexpensive procedure, which takes less than 30 min to perform. Similar to the MAR test, it is very convenient, utilizing only a bench centrifuge, light microscope, and latex beads coated with anti-human IgG, IgA, and IgM [14]. IBT allows the determination of the antibody class attached to spermatozoa, the localization on the spermatozoa, and the proportions of spermatozoa coated with antibody [14]. The immunoglobulin class detected can be of clinical importance (Fig. 13.2).

Prior to conducting the assay, spermatozoa must be washed to discard any free immunoglobulins, which may be in the seminal plasma and which, if present, would alter the assay results. Thereafter, sperm concentration is adjusted to 10–25 × 10⁶ motile sperm/mL to optimize the microscopic assessment of sperm. These adjustments are occasionally necessary if the sample is oligozoospermic or asthenozoospermic. The IBT can also be conducted indirectly on reproductive fluids such as seminal plasma, follicular fluid, cervical mucus, as well as serum.

The intra-assay reproducibility of the indirect IBT was evaluated by testing aliquots of ASA-positive sera from two patients against the same donor sperm sample. The interassay reproducibility was also evaluated by testing a positive serum sample first with different sperm samples from the same donor and second with sperm samples from different donors. The results of those experiments showed that the indirect IBT has very low intra-assay variation and a high interassay variability [13]. Therefore, both the direct and indirect IBT can be considered as reliable, specific tests for the detection of sperm-bound antibodies and sperm antibodies in reproductive fluids and serum [24]. IBT was routinely applied in many andrology laboratories for the detection of ASA. However, a current lack in commercially available coated latex beads has led to its discontinuation.

The comparison between the MAR test and the IBT is of particular importance since both assays appear to be the most commonly used [37]. The two testing protocols are designed to detect immunoglobulins on the sperm surface; however, the MAR test is specific for the secretory IgA which is present in semen, while the IBT may cross-react with nonsecretory IgA present in serum [29]. To initiate a comparison, indirect tests for ASA using the commercially available MAR and IBT kits were applied to a panel of sera whose reactions in the TSAT, GAT, and SIT were well characterized. The results from assessments of 30 sera confirmed a significant correlation between the GAT, MAR, and IBT. When sera were titrated, the IBT proved to be slightly more sensitive than the GAT, while the MAR test was slightly more sensitive than the IBT [24].

A different comparative study between the IBT and the MAR test has also shown a high degree of agreement between both assays; however, the former was less accurate than the latter [29]. A similar study reported that the standard MAR protocol (direct test of unwashed semen) was found to be more sensitive than IBT on washed sperm. Other advantages for the MAR include not requiring washing the spermatozoa free of the seminal plasma, which makes it easier and faster than the IBT. It also uses less semen volume and could be applied to samples with a lower sperm concentration compared to the IBT [2, 34]. In contrast, when the MAR test was performed on washed sperm or with an indirect antibody transfer from serum or seminal plasma, the results gave mostly lower values for bead binding in comparison with the IBT. Therefore, the MAR test can be considered mostly suitable in direct assays employing unwashed ejaculates making it easier to incorporate in routine semen analysis. However, it is prudent to confirm its positive results by IBT [20].

Cross-inhibition studies have revealed high specificity between positive IBT and the presence of membrane-bound immunoglobulin. Good correlations have been reported also between the IBT and other immunoassays such as PCT and sperm-cervical mucus compatibility assays [33]. Poor correlations have been reported between the IBT and sperm agglutination test, which implies that sperm agglutination may occur due to other nonimmunological factors [14]. Therefore, false-positive results may occur with agglutination tests in the absence of ASA. Finally, it is of importance to note that there are several pitfalls associated with comparisons between the different methods of ASA detection. Many methods rely on subjective determinations and variable specimen preparations. In addition, the sensitivity and specificity for each of the testing modalities vary widely [19].

There is sufficient evidence supporting the hypothesis that ASA play a role in selected cases of infertility. The prevalence and magnitude of this role remains not well defined. In the clinical context of male infertility, the MAR test and the IBT are currently recommended for the detection of ASA. The current consensus indicates that a semen sample is to be considered immuno-compromised if more than 50 % of spermatozoa show binding in the MAR test or the IBT [1, 37].

The identification of ASA in a given sample does not necessarily indicate other inherent defects. An attempt to correlate results of MAR test with other defects in the seminal fluid revealed a significant correlation between a positive MAR test and spontaneous sperm auto-agglutination [7]. No correlations were observed, however, between test positivity and sperm concentration, motility, morphology, macroscopic features, or leukocytes concentration. Contradictory findings were identified in a different trial that correlated the results of the IgG MAR test with the semen analysis parameters of 1176 infertile males [27]. The test was only positive in 3.1 % of the cases. The positive IgG MAR test proved to correlate significantly both with the number and motility of spermatozoa. Whether the detection of ASA is associated with other deficiencies in the semen analysis or not should not infringe on the importance of the assay, which appear to be of significant value in identifying the etiology of infertility in some cases.

In a study that evaluated ASA levels in men presenting with a history of infertility, MAR test results were found to be positive in 10 % of 484 men with normal sperm counts, 23 % of 78 with low sperm motility, and 15 % of 128 with low counts. Therefore, the MAR test may be considered as a part of the routine semen analysis, since the presence of IgG antisperm antibodies can be established in about 10 % of men who might otherwise be passed as normal. In support, the evaluation of patients whose infertility remains unexplained (by routine physical and laboratory investigations) exhibits significantly elevated ASA levels in 18 % of males compared to fertile individuals. Thus, the identification of autoimmune imbalance may help to resolve some cases of unexplained infertility [12].

The assessment of ASA in the context of infertility has not been devoid of controversies. It is duly noted that “neither a specific antigen (s) nor a superior antibody detection assay exists, although both are requisite to an understanding of the significance of ASA production and for the purpose of infertility reduction” [10]. The only agreed upon current indication for performing an ASA detection assay appears to be unexplained infertility. Published data support the use of either IBT or MAR as the only reliable tests capable of assessing the presence of ASA in a clinical setting. The MAR test is easier to perform and may be more sensitive but is not suitable for testing of serum or plasma using an indirect approach.