The chemical organization of ASA antibodies has become a complex matter. After pioneering work of molecular mapping of the sperm epitope reacting with female immobilizing antibody [55], we have also found common carbohydrates both linked with protein and lipid carriers on human sperm [35]. An interesting conclusion coming up from these studies was a bimodal curve received with virtually all antisperm monoclonal antibodies after sperm periodate oxidation, while more gentle N-deglycosylation carried out with cocktail of enzymes seemed to open sperm glycocalyx allowing to penetrate antibodies uncovering the previously seen cryptic sperm determinants [35]. Unlike, however, in Koyama and co-workers paper [33] where by using chemical deglycosylation (trifluoromethyl acid with sodium periodate) they abrogated all ASA reactivity; in our hands, high amounts of periodate did not remove the reactivity completely, so we could speculate that some antigenic portions were hidden beneath the cell membrane or alternatively there was retained binding mediated through the protein (lipid) carrier. While Tsuji [56] has managed to strip off (by strong oxidation) all the carbohydrates, the conclusion was that immobilizing properties strongly depend on carbohydrates while the remaining activity of antibodies recognized the portion that was digested off by proteolytic enzymes.

The intriguing observation on sperm N- and O-deglycosylations led us to explore the partial carbohydrate digestion, although the revealed pattern by Western blotting and immunoprecipitation (reaction of antibody with denatured or ‘native’ antigenic conformations) was quite confusing; see Fig. 6.2 and Table 6.1).

After immunoprecipitations obtained with various families of antisperm antibodies (from infertile individuals), three types of reactions can be seen. For example, O-deglycosylation procedure in same cases did not change the pattern of reaction when comparing to N-deglycosylation or the ‘native’ sperm extract (lanes 1 vs. 2 vs. 3). In some variants, O-deglycosylation procedure (lanes 4, vs. 5 vs. 6) produced more bands with ASA polyclonal sera than after N-deglycosylation, and in some cases (lanes 16, 17, 18), O-deglycosylation abolished the antibody reaction when comparing to the other applied sperm extracts (N-deglycosylated or ‘native’ ones).

In the next series of experiments, reactions were performed when the sperm antigens were selectively or simultaneously deglycosylated and antisperm antibodies reacted both in Western immunoblotting and immunoprecipitation techniques (Table 6.1). The relatively less number of positive reactions revealed in N-deglycosylation sperm does not allow to infer highly sensitive N-linked binding since technical difficulties in protocols for liberation N-glycans have not been yet satisfactorily solved [32].

However, it can be concluded (from the Western immunoblotting) that there is more (sensitive) O-deglycosylated sites on human sperm than N-deglycosylated ones what could indirectly confirm our earlier reports on enhancement of ASA antibodies binding due to limited N-deglycosylation [35]. Further, it can be clearly concluded that N- and O-deglycosylation procedures applied simultaneously significantly diminished the number of bands precipitated or immunoblotted in comparison to each of the applied procedures alone.

It should be further underlined upon glycemic and proteomics analyzes the high amounts of fucosylated glycoconjugates in reproductive compartment [34] of both sexes concerning, e.g. glycomics of uterine fluid or glycemic gradient complexity in different regions of epididymis. The carbohydrate sequences involved in reproductive tracts must take part in creation: (a) immune privilege in the human reproductive system; (b) immunologic interface between self (female reproductive tract epithelium) and non self (placenta, sperm cell components); and (c) operating milieu for effective response to pathogens. All these tasks an incredible to be precisely fulfilled without any errors if both arms of innate and adaptive immunities are in full action. Yet, in reproductive age relatively low number of errors occurs resulting in anti-gametic response formation. This may be at the expense of immunological deviations arising after reproduction is completed opening the space for malignancies in already redundant compartment.

We may again emphasize the diversity of antisperm antibody reactions which recognize abundantly glycosylated human sperm entities. It seems that there is sufficient data to underline once more a complex nature of ASA reactions (1) being mediated by carbohydrate epitopes (with background of ‘natural’ antibodies), (2) sex-dependent differences in sperm recognition expressed by auto- and isoimmune reactions, and (3) genuine characteristics of ASA reactions developed pre-puberty that deserve further epidemiological interest.