9.4.11 Immunological infertility

Immunological infertility is the presence, in one or both partners, of an antisperm immune reaction capable of interfering with fertility variables. In about 8–10% of these couples the immunological phenomenon is on the male side, causing ‘male immunological infertility’ (1).

Since the first demonstration that a significant number of infertile men show an autoimmunity to sperm, experiments have suggested that antisperm antibodies (ASA) can interfere with the fertilizing ability of spermatozoa (2). ASA can act negatively on the motility of spermatozoa in semen, on their ability to pass through female genital secretions, or on the penetration of the oocyte. In particular, owing to in vitro fertilization techniques, it has been possible to demonstrate the effects of antibody-bound sperm directly, at the level of in vitro gamete interaction (3).

ASA can reduce the motility and concentration of spermatozoa, and can induce sperm agglutination. However, normozoospermia can be accompanied by a high percentage of antibodies bound to the sperm surface, or a high ASA titre in serum or seminal plasma. In addition, ASA can affect sperm penetration of cervical mucus. When ASA are present in cervical mucus or bound to the sperm surface, impaired sperm penetration of cervical mucus, and abnormal swimming behaviour within cervical mucus—ranging from complete immobilization of sperm, to vibratory motion with limited progression (‘shaking reaction’), to restricted tail beat frequency and loss of rotatory motion—may be observed during the post-coital test (PCT). The shaking reaction in these cases is presumably due to cross-linking of motile, antibody-coated spermatozoa to the cervical mucus gel via the Fc part of the antibody (4). ASA may also inhibit fertilization by binding specifically to membrane antigens involved in sperm–oocyte interaction. They can additionally impair the fertilization process at the levels of the acrosome reaction, of zona pellucida recognition and penetration, and of sperm–vitellus interaction (5).

The immune system can impact negatively on reproductive function at various levels (6). In particular, the following points should be recalled. First, spermatozoa are cells that are antigenically protected by a specific mechanism of immunological tolerance. Therefore, mature sperm can be considered haploid cells, distinct from the organism that produced them. Second, the same antigens come into contact with the female immune system repeatedly and on a massive scale during sexual intercourse without provoking an immune response (in the vast majority of cases). Third, the female organism allows an extraneous cell to enter and migrate along the genital tract to fertilize the oocyte, fusing with its histocompatibility antigens. Finally, the cell thus fertilized develops and is able to attach itself to the female organism, giving life to a new individual, without triggering a reciprocal rejection. Rather, a chain of events is created that is repeatable in successive pregnancies.

The first step to approach the problem of autoimmune reaction against the spermatozoa is to study sperm antigens. The antigenic nature of spermatozoa was first established experimentally in animals through active heterologous and homologous immunization in the early 20th century in the classic studies conducted by Landsteiner, Metchnikoff, and Metalnikoff. These early research results demonstrated that mature sperm carry a series of specific antigens not present in fetal life at the moment of immunological imprinting. During fetal life, the activity of lymphocytic clones, which are capable of acting immunologically against ‘self’ antigens, is suppressed, and ‘suppressor’ lymphocytes present during embryogenesis play a decisive role in the induction of tolerance towards ‘self’ structures. This series of events can prevent the development of autoimmunity in most individuals. Sperm antigens are not present in this phase of self-recognition, since spermatogenesis is not active until puberty. Therefore, when spermatogenesis takes place from puberty onwards, it must be in an environment that limits exposure of the sperm antigens to the immune system of the male host. Other sperm antigens such as blood group antigens, acrosine, HLA system antigens, hyaluronidase, and LDH-X have been studied. Surface antigens revealed by heterologous antigens, indicated by the symbols RSA-1, MA-29, and FA-1, have also been described, as have antigens identified using monoclonal antibodies for human antispermatozoa, known as S03, S37, S61, and S20. However, to date it has not been possible to fully identify an antigen that would explain the triggering of the autoimmune reaction.

The aetiopathogenetic problem of male immunological infertility has been researched widely with as yet inconclusive results; however, various modes of defence and immunoprivilege in the male genital tract have been identified. The basis for the blocking of an immune response against sperm lies in the testes. During the phases of sperm maturation, cell surface antigens undergo substantial modification, such that antigens expressed on mature sperm can differ by more than 50% from those expressed on spermatogonia (7).

Spermatogenesis is completely separate from the immunocompetent system. This is due to the presence of cellular and acellular layers that surround the tubules and separate them from the interstitium, and to the tight junctions between Sertoli cells. The latter, besides their function in nourishing male gametes, serve to completely isolate the intratubular environment by forming the blood–testis barrier. The blood–testis barrier permits the passage of soluble sperm antigens capable of activating T-suppressor lymphocytes, thus reducing the autoimmune response (immunological tolerance). Another proposed mechanism is the absence or reduction of T-helper lymphocytes in the interstitium, which would reduce the stimulation of the immunocompetent system (8).

Once the sperm have been produced, they pass through the rete testis and the efferent ducts to the epididymis for functional maturation. Because these three structures are not completely impermeable, it has been hypothesized that the dilution of the sperm, limited vascularization, and production of immunosuppressant coats on the sperm membranes might be responsible for the immune protection of the sperm. In fact, these coat substances have been found to have several types of immunosuppressant activity, including: an inhibitory effect on blastogenesis induced by mitogens on T and B lymphocytes, an alteration of the capacity of polymorphonuclear leukocytes to recognize antigens, an inhibition of the activity of cytolytic NK and T cells towards neoplastic and virus-infected cells, and antimacrophage and anticomplement activity (9).

These functions, demonstrated in vitro, have the physiological effect of protecting the sperm in the male genital tract and also during the first stages of passage through the female genital tract. Pathological alterations of these protective functions can play an important role in susceptibility to sexually-transmitted diseases, in the development of male and female neoplasia, and in the reduction of immunological activity such as that typical of some acquired immune deficiency syndromes. Such alterations can also trigger antisperm autoimmune responses.

Another mechanism of antisperm reaction has been proposed based on the Fas/Fas ligand system following the discovery of Fas ligand production by Sertoli cells in experimental models (10). This substance is capable of acting as an immune suppressor, promoting tolerance at the level of the testes. Soluble Fas ligand has been found in varying concentrations in samples of human semen, but as yet is uncorrelated with seminal characteristics.

During the first decade of the 21st century, researchers investigating the antisperm immune reaction in the immunology of reproduction have made several other important findings. First, an andrological pathology that could predispose an autoimmune reaction has not been identified. However, there may be a genetic predisposition to such a reaction. Second, certain pathological conditions such as trauma, torsion of the spermatic cord, vasectomy, cryptorchidism, and varicocele can induce antisperm antibodies both in blood serum and seminal plasma. Third, inflammatory processes can also result in predominantly local production of secretory IgA that goes directly into seminal plasma (11, 12). Finally, because testicular cancer directly affects the gamete production site, it has been postulated as a trigger for an autoimmune response. The presence of ASA may in fact be explained by local effects caused by the cancer, such as raised scrotal temperature connected with blood flow alterations, or disruption of the blood–testis barrier, with a massive release of sperm antigens that stimulate antisperm immunization. For this reason, some investigators have considered autoimmunity to be closely correlated with testicular cancer. In contrast, in a recent study it was found that patients in the first stages of testicular cancer present with a low percentage of ASA (13). Patients with testicular cancer also showed mean semen parameters above WHO reference values (14, 15). These data support the hypothesis that testicular cancer may not be a possible cause of antisperm autoimmunization and infertility. This is a reassuring conclusion, because testicular cancer patients who bank their semen not only have a good chance of recovering spermatogenesis approximately 2 years after therapy (16), but also have a minimal ASA autoimmune response, which will enable their return to future fertility.

Because of the absence of specific symptoms, diagnosis of immunological infertility is based on laboratory analysis. Antisperm antibodies can be identified from both blood serum and semen; in the latter case they are found either on the surface of spermatozoa or in the seminal plasma.

One of the most debated topics in reproductive immunology was the establishment of a universally accepted Standard Protocol of tests for antisperm antibody (ASA) detection.

A purified molecular antigen clearly involved in the immune reaction and in immune infertility has not yet been identified; therefore, the only way to study antisperm immunity has been to use the sperm cell as the antigen. To reduce interassay variability in the antigen component it would be useful to test a biological sample with various methods in parallel and repeat each test using different donors. Among the methods utilized in the search for a soluble antibody (in serum or seminal plasma) are the gelatin agglutination test (GAT) and the tray agglutination test (TAT). GAT is a flocculation test in gelatin, which uses motile spermatozoa as the antigen (17, 18). TAT utilizes as the antigen a suspension of only motile spermatozoa obtained by swim-up from semen with normal parameters. Antibody titres of 1 to 32 or greater in serum and of 1 to 16 or greater in seminal plasma are considered clinically significant.

To date, other methods such as the immune radio binding test (IRB) and the enzyme linked immunosorbent assay (ELISA) (19) do not provide results correlated with immunologic infertility.

The mixed antiglobulin reaction test (MAR Test), SpermMAR test, and immunobead test (IBT) are used to identify antibodies bound to the sperm surface. The MAR Test is based on a modification of Coombs’ test, and detects IgG antisperm antibodies sensitized with anti-D antiserum as a marker of antibody–antigen reaction. The great simplicity and rapidity of detection makes the MAR test a valid tool for the routine screening of antibodies bound to the surface of the spermatozoa. It is limited by the fact that it allows the detection of only IgG class antibodies (20). The SpermMAR test is a modification of the MAR assay and employs latex particles coated with IgG and IgA as markers of the reaction instead of erythrocytes. The procedure is identical to that of the MAR Test (21). The IBT uses polyacrylamide beads coated with antihuman IgG, IgA, and IgM. The reaction takes place between the N-terminal groups of the beads and the carboxylic group of the Fc fragment of the antihuman immunoglobulins, creating a complex of bead, antibody, and immunoglobulins. This technique has the advantage of evaluating all the Ig classes found on the sperm surface, and it is correlated with other tests (22). The MAR test, SpermMAR and IBT can all be employed as indirect tests to evaluate antisperm antibodies in the serum and seminal plasma. The only limitation of these tests is that they cannot be used in samples with severe oligozoospermia or hypomotility.

A recommended approach is to use at least one direct method (MAR or IBT). Indirect macroscopic (GAT) and microscopic (TAT) tests (23, 24) can be reserved for laboratories specialized in reproductive immunology.

Proposed treatments of immunological forms of male infertility have been carried out using immunosuppressive drugs, such as steroids. However, to date, because of the difficulty in selecting homogeneous case histories, this cannot be considered an evidence based medicine approach. In cases showing a high ASA positivity (MAR test or IBT >90%) an ICSI programme is recommended (25), emphasizing the fact that ICSI outcomes are not influenced by ASA levels on sperm (26).