The BTB physically divides the seminiferous epithelium into basal and adluminal compartments. Besides its function as an immunologic barrier to segregate post-meiotic TGC from the systemic circulation, it creates a microenvironment for TGC development and confers cell polarity (Fig. 3.1). At puberty, when immune competence is already established, TGC commence a new program which leads to the formation of mature spermatozoa. During this process, an array of new surface molecules is expressed on the differentiating spermatids and spermatozoa (Ike et al. 2007). Such autoantigens, therefore, do not belong to the “family” of those considered as “self” by the immune system. Therefore, under some condition in which the testicular immune privilege is broken, immune responses against the testicular autoantigens should be evoked.

Clinically, in human male infertility of immunologic origin, the presence of anti-sperm antibodies is a well-known cause of male infertility (Jodot-van de Casseye et al. 1980), and measurement and analysis of anti-sperm antibodies have been extensive. The six most common serological tests used for antibody detection include sperm agglutination, complement-dependent sperm immobilization, indirect immunofluorescence, the enzyme-linked immunosorbent assay, mixed antiglobulin reaction test, and immunobead test (Shibahara et al. 2005). Actually, male subfertile patients with significant titers of anti-sperm antibodies could be treated with steroid regimens (Hendry et al. 1979), and treatment of male autoimmune infertility with cyclosporine A has been clinically attempted (Bouloux et al. 1986). Moreover, autoantibodies against Sertoli cells, Leydig cells, and basement membrane of the seminiferous tubules were also detected in male infertility (Wall et al. 1974; Zanchetta et al. 1984; Silva et al. 2012). However, detection of the autoantibodies on sera and/or semen is not sufficient to diagnose testicular autoimmunity. The characteristic features of testicular autoimmunity include the detection of (1) inflammatory cell infiltration into the testis (=orchitis), (2) spermatogenic disturbance, (3) T cell response against target testicular autoantigens, (4) serum autoantibodies against the target antigens, and (5) binding of the autoantibodies to the target antigen-bearing cells inside the testis. Clinically, it is quite difficult to detect all these features. Therefore, immunoreactions at the level of testicular tissue have been less well studied in humans. However, there is increasing evidence that autoimmune orchitis may be an etiological factor in human reproductive failure, perhaps even more often than expected.

Testicular autoimmunity can be classified into primary and secondary ones. Primary testicular autoimmunity is defined by asymptomatic orchitis associated with anti-sperm antibodies without any evidence of systemic or local causes in infertile men. Secondary testicular autoimmunity is characterized by symptomatic orchitis and/or testicular vasculitis associated with local or systemic inflammatory disease (Silva et al. 2012, 2014). The patients suffering from secondary testicular autoimmunity typically demonstrate testicular pain, erythema, and/or swelling. Systemic autoimmunity, vasectomy, infection, tumor, or trauma may induce T cell response with pro-inflammatory cytokine production with a consequent BTB permeability alteration, anti-sperm antibody production, and apoptosis of TGC. Although the testicular autoimmunity can be classified into the two categories, the etiologies of both primary and secondary autoimmune orchitis are likely to be multifactorial and overlapped.

Of all cases of infertility, approximately 50% of cases occur in men. Although the literatures reported many causes of male infertility, approximately 65% of the cases are categorized as idiopathic spermatogenic disturbance (Fig. 3.2). The histopathology of idiopathic spermatogenic disturbance is characterized by seminiferous tubules showing Sertoli cell-only syndrome or TGC maturation arrest. In a study of patients with idiopathic male infertility, testicular biopsies showed that approximately 20% of patients had Sertoli cell-only syndrome, and approximately 50% of patients had maturation arrest (Hatakeyama 1984; Terada and Hatakeyama 1991). These features of spermatogenic disturbance were often accompanied by thickening and hyalinization of the seminiferous tubular wall (De Kretser et al. 1975; Terada and Hatakeyama 1991). Furthermore, fibrosis, sclerosis, and thickening and hyalinization of testicular interstitium are the features frequently found in the testis of infertile patients (Agarwal et al. 1987; Apa et al. 2002).

There have been some reports which showed lymphocytic infiltration and immune deposits in specimens of testicular biopsies from infertile men (Taylor et al. 1978; Suominen and Soderstrom 1982; Salomon et al. 1982; Salomon and Hedinger 1982; Hatakeyama 1984; Lehmann et al. 1987; Aitchison et al. 1990; Agarwal et al. 1990; Hassanin and Ayad 2016). Suominen (1995) reported a case, where a surgical operation for an inguinal hernia caused an orchitis first in the ipsilateral, and few weeks later also in the contralateral testis and which finally led to an atrophy of both testes, resulting from autoimmune sympathetic orchitis. The testicular biopsy revealed that the interstitial tissue contained numerous lymphocytes and macrophages. A typical feature was characterized by disturbed spermatogenesis in the seminiferous tubules that were surrounded by lymphocytic infiltration. Occasionally, intra-tubular lymphocytes were also found in both testes (Hussein et al. 2005). Nistal et al. (2002) evaluated the focal orchitis present in cryptorchid testes of men not previously treated for cryptorchidism or antecedents of infectious diseases and detected focal lymphocytic infiltration in the interstitium and/or seminiferous tubules associated with spermatogenic disturbance in 44% of examined patients. A picture resembling autoimmune orchitis can be found in unilateral testicular obstruction (Hendry et al. 1985, 1990). Mononuclear cell infiltration around the seminiferous tubules and the rete testis with anti-sperm antibodies was noted in men with unilateral testicular obstruction. Approximately 5% of testicular biopsies from men with azoospermia or oligozoospermia had focal or general leukocyte infiltration (Suominen and Soderstrom 1982). Jahnukainen et al. (1995) evaluated the incidence of testicular mononuclear cell infiltrates in patients with carcinoma in situ and germ cell neoplasia. The results suggest that the incidence of mononuclear cell infiltration increases with increasing severity of testicular malignant changes and also that increased mononuclear cell infiltration is evident in the contralateral testis where no malignant cells can be observed. Testicular lymphocyte numbers are increased in infertile patients with sperm autoimmunity, and the predominance of CD8⁺ T cells was immunohistochemically demonstrated in the interstitium between the seminiferous tubules in them (El-Demiry et al. 1985, 1987). In infertile men with maturation arrest, Sertoli cell only, or mixed atrophy syndromes, and with cases of idiopathic infertility showing normal spermatogenesis, diagnostic testicular biopsy revealed the significant presence of CD68⁺ macrophages in the testes of all patients (Frungieri et al. 2002). These macrophages expressed the genes for IL-1 and TNF-alpha and were located in the testicular interstitium and in/around the seminiferous tubules. In Sertoli cell only and germ cell arrest syndrome, the overall macrophage number was increased over twofold. A study of phenotypical characterization of testicular leukocytes demonstrated that cell counts of all examined populations (T cells, B cells, CD68⁺ macrophages, and mast cells) in Sertoli cell-only syndrome and germ cell arrest were increased when compared with those in normal spermatogenesis (Hussein et al. 2005).

Aitchison et al. (1990) also reported several cases of granulomatous orchitis with chronic inflammation in humans. In idiopathic granulomatous orchitis, there is extensive destruction of the seminiferous tubules with tubular or interstitial pattern of granulomatous inflammation and prominent collagen fibrosis (Roy et al. 2011). Finally, the seminiferous epithelium was replaced by an admixture of large epithelioid cells, lymphocytes, plasma cells, and occasionally giant cells (Perimenis et al. 1991). Granulomatous orchitis is sometimes accompanied by inguinal or retroperitoneal lymphadenitis (Matsumura et al. 2016). Trauma and the following autoimmune responses against TGC have been postulated to be the underlying mechanism of the disease (Sporer and Seebode 1982; Wegner et al. 1994). Ischemia or preceded infection in the testis may be other origins of granulomatous orchitis (Kahn and Mcaninch 1980; Tarabuta-Cordun 1983; Rosi et al. 1984; Klein et al. 1985).

The increased number of mast cells in the testis is also associated with male infertility (Maseki et al. 1981; Nistal et al. 1984; Agarwal et al. 1987). With regard to their characteristic location within testicular tissue, two groups of mast cells could be distinguished, in both control and infertile patients: interstitial mast cells (located between Leydig and other interstitial cells as well as in the vicinity of blood vessels) and peritubular mast cells (located in the close proximity of the tubular lamina propria or incorporated in the lamina propria itself) (Jezek et al. 1999). It appeared that peritubular mast cells increased at a higher rate than interstitial mast cells in infertile patients when compared with controls. The increase in testicular mast cells in close contact to the seminiferous tubules indicates a relationship between mast cell proliferation and a dysfunction of the BTB (Haidl et al. 2011). The mast cells have been known to play a key role in the pathophysiology of various allergic, inflammatory, or fibrotic disorders, perhaps because of their chemical mediators such as biogenic amines, prostaglandins, neutral proteases, and proteoglycans (Schwartz and Austen 1984). There are two distinct subpopulations of mast cells, the connective tissue and mucosal mast cell (Irani and Schwartz 1989). Nearly all the mast cells in the normal human testes contain heparin and correspond to “connective tissue mast cells.” In the infertile patients, chondroitin sulfate containing mast cells was substantial in number, and these cells correspond to “mucosal mast cells” (Nagai et al. 1992). Therefore, in male infertility, mast cells were increased in total number, and the degree of the increase in mucosal mast cells was greater than that in the connective tissue mast cells. Actually, a mast cell blocker is clinically useful for the treatment of idiopathic oligozoospermic men (Yamamoto et al. 1995; Matsuki et al. 2000).

Other studies have provided evidence of immune deposits in testis biopsies from male patients with impaired fertility (Vu Van et al. 1978; Lehmann et al. 1987). Salomon et al. (1982) and Salomon and Hedinger (1982) reported the presence of immune complex orchitis in infertile men. In the specimens, linear deposits of IgG and complement on the basement membrane of the seminiferous tubules were found. In other biopsy specimens from infertile patients, immune complexes were found to be localized not only on seminiferous tubule walls but also on Leydig cells and various staged TGC (Morgan 1976). A case of testis biopsy from a patient with oligospermia and a large varicocele in the left testis showed carcinoma in situ with atypical spermatogonia in the right testis. This lesion was immunologically characterized by the deposit of IgG restricted to the atypical cells and the presence of circulating anti-sperm antibodies (Lehmann et al. 1986, 1987; Lehmann and Muller 1987). In immune complex orchitis, at least two different immunological mechanisms, induced locally in the testis, may be involved: an immunoglobulin deposit, in a manner specifically against TGC, and the seminiferous tubular basement membrane, in a manner nonspecifically directed to them (Lehmann et al. 1987; Lehmann and Emmons 1989). Hence, the circulating autoantibodies could specifically react with various testicular cells and tissues, but the immune deposits to the seminiferous tubule walls, blood capillary walls, and other testicular tissues could be accounted for aggregation of not only the specific antibodies but also irrelevant serum immunoglobulins in an antigen-nonspecific manner.

Collectively, the detection of lymphocytic infiltration, deposits of IgG, and deposits of complements in testis biopsy specimens indicates that inflammatory or immunological factors contribute to the occurrence of some idiopathic male infertility. However, it is quite difficult to diagnose it, because testicular autoimmune inflammation may progress chronically, torpidly, and asymptomatically. Therefore, involvement of lymphocytic infiltration and immune deposits may be no longer detectable when Sertoli cell-only syndrome and maturation arrest of some immunologic origin are completely established.

Additionally, in recent study on testicular biopsy specimens from male patients with Sertoli cell-only syndrome and maturation arrest, increased apoptosis of TGC and Sertoli cells was observed. In maturation arrest, mRNA expression of Fas ligand was upregulated in Sertoli cells and Leydig cells, while intense expression of Fas was observed in primary degenerating spermatocytes, and active mRNA expression of caspase 3 was detected in cytoplasm of both Sertoli cells and TGC. In Sertoli cell-only syndrome, mRNA expression of Fas, Fas ligand, and active caspase 3 was detected both in Sertoli cells and in hyperplastic interstitial cells (Kim et al. 2007). It indicates that upregulation of the Fas system and caspase 3 activity in the testis may result in enhanced TGC apoptosis, which is involved in the spermatogenic disturbance (Eid et al. 2002).

More recently, human testicular biopsies from infertile men with focal lymphocytic infiltrates were probed with antibodies against high-mobility group box protein 1 (HMGB1), and it was found that HMGB1 was translocated from the nuclei in the testes of infertile men with spermatogenic disturbance and lymphocytic infiltrates (Aslani et al. 2014). Klune et al. (2008) reported that activated macrophages and monocytes secrete HMGB1 as a cytokine mediator of inflammation. HMGB1 is among the most important chromatin proteins, and the presence of HMGB1 in the nucleus depends on post-translational modifications. When the protein is not acetylated, it stays in the nucleus, but hyper-acetylation on lysine residues causes it to translocate into the cytosol. Furthermore, interaction of HMGB1 and TLR-4 results in upregulation of NF-kappa B, which leads to increased production and a release of cytokines in macrophages. Therefore, the binding of HMGB1 to TLR-4, which mediates HMGB1-dependent activation of macrophage cytokine release, may play an important role in the onset and progression of autoimmune diseases, involving human orchitis (Aslani et al. 2014).

Therefore, testicular biopsy has provided various information of immunological aspects on the spermatogenic disturbance; however, it must be kept in mind that testicular biopsy itself may injure BTB focally, leading to testicular autoimmunity (Hjort et al. 1974, 1982).

Many studies suggest the involvement of humoral immunity in male infertility. Anti-sperm antibodies have been the center of attention in immunology and immunopathology of reproduction. Indeed, the presence of anti-sperm antibodies has been observed in approximately 5–12% of infertile male partners, compared to 0–2% of the general male population (Silva et al. 2012). Circulating anti-sperm antibodies detected in other studies ranged from 8 to 30% in unselected men with infertile marriages (Francavilla et al. 1999, 2007). Moreover, circulating antibodies against immature TGC, Sertoli cells, Leydig cells, and basal lamina of the seminiferous tubules were detected in serum samples from infertile patients (Wall et al. 1974; Hatakeyama 1984; Zanchetta et al. 1984; Silva et al. 2012).

Patients with high titers of anti-sperm antibodies had evidence of testicular damage with decreased testicular volumes, elevated serum FSH and LH levels, and reduced sperm density and motility, indicating the induction of the spermatogenic disturbance (Handelsman et al. 1983). Chlamydia trachomatis, Ureaplasma urealyticum, and Mycoplasma hominis compared to other sexually transmitted diseases cause statistically significant increase of anti-sperm antibodies concentration in blood serum, as well as in ejaculate. Local infection by them may induce orchitis and epididymitis, which results in inflammatory and toxic damage to the seminiferous epithelium, which in turn plays significant role in development of testicular autoimmunity (Tchiokadze and Galdava 2015).

The anti-sperm antibodies in males have been shown to have an inhibitory effect on fertilization. There is a possibility that fertilization-related antigens may be the targets of these anti-sperm antibodies. Such antigens are consisted of sperm surface antigens and acrosomal antigens. The former include PH-20, PH-30, fertilization antigen-1 (FA-1), sperm agglutination antigen-1, and lactate dehydrogenase-C₄, and the latter include SP-10 (Hall et al. 1994; Shibahara et al. 2005). Acrosin, hyaluronidase, RSA-1, MA-29, FA-1, SO3, S37, S61, S20, and YLP₁₂ are also human sperm autoantigens presumably involved in anti-sperm immune response (Ishidori et al. 1988; Naz 2004). In particular, treatment with FA-1 was demonstrated to remove anti-sperm autoantibodies from spermatozoa of infertile men and results in increased rates of acrosome reaction (Menge et al. 1999). Seminal autoantibodies against laminin-1 are also important in infertile men (Ulcova-Gallova et al. 2008). In males, laminin is found on not only the surface of spermatozoa but also basal lamina of the seminiferous tubules. Sperm agglutination antigen-1 (SAGA-1) was characterized as a polymorphic, highly acidic, glycophosphatidylinositol-anchored glycoprotein on the surface of human spermatozoa (Diekman et al. 2000). It is unique in that SAGA-1 core peptide is identical to CD52, a glycoprotein on the surface of human lymphocytes. Therefore, autoimmunity to CD52 may be an important factor in the etiology of immunologic male infertility.

The incidence of epididymitis is greater than that of orchitis in humans, and susceptibility to anti-sperm antibody formation after damage to the epididymis or vas deferens increases with increasing distance of the damage from the testis. One important consideration must be the very different immunological environments of the testis, where sperms develop, and the epididymis, where sperms mature and are stored. Compared with the elaborate BTB, the tight junctions of the epididymal ducts are much less effective. Unlike the seminiferous epithelium, immune cells are commonly observed within the epididymal epithelium and can even be found within the lumen of the epididymis (Hedger 2011). Therefore, epididymal sperm autoantigens tend to be easily accessible to immune cells, resulting in anti-sperm antibody production.

It is noted that naturally occurring human anti-sperm autoantibodies, detectable by immunofluorescence, had a peak incidence of 90% in both sexes before puberty (Tung et al. 1976). Thereafter, the incidence declined to approximately 60% and persisted through life. Actually, it remains vague whether production of anti-TGC or anti-sperm autoantibodies is a cause or a result of testicular autoimmunity. In other words, it can be said that these autoantibodies become the cause, the result, or the both in each case. There is another possibility that those natural anti-sperm autoantibodies may downregulate the specific autoimmune responses through some immune network system based on “idiotypic network theory,” proposed by Jerne (1974). The BTB damage in testicular autoimmunity results in production of anti-TGC autoantibodies, and male infertility may result from the binding of anti-TGC or anti-sperm autoantibodies (Tung 1998; Lustig and Tung 2006). These autoantibodies present in local secretions of the germ cell tract rather than serum are associated with this type of male infertility. Primary testicular autoimmunity can be defined by isolated and asymptomatic orchitis with the autoantibodies but without evidence of local or systemic disease or injury. Most cases of the primary disease are torpidly and chronically advanced. On the other hand, secondary testicular autoimmunity accompanied by the autoantibodies involves orchitis associated with endogenous factors such as cryptorchidism, varicocele, torsion, tumor, or systemic autoimmune diseases and orchitis associated with exogenous factors such as trauma, biopsy, vasectomy, and infection. These are testicular autoimmunity associated with identified pro-inflammatory causes. Obstruction or injury of the male reproductive tract is associated with anti-TGC or anti-sperm antibody formation, since leakage of large quantities of TGC or sperm antigens from the tract can induce the antibody production. Anti-sperm antibodies attendant to vasectomy may be responsible for infertility in some patients following vasovastomy (Witkin et al. 1982; Lee et al. 2009). Anti-sperm antibodies are common in patients with congenital absence of the vas deferens associated with cystic fibrosis (D’Cruz et al. 1991; Lustig and Tung 2006). The role of anti-sperm antibodies in the pathogenesis of varicocele-related male infertility has been also noted. The prevalence of varicocele-related immune infertility is about 15% male subjects from infertile couples (Bozhedomov et al. 2014). Varicocele is not an immediate cause of autoimmune reactions against spermatozoa but is a cofactor increasing a risk of anti-sperm antibody formation. These disorders correlate with the level of sperm oxidative stress; reactive oxygen species production in anti-sperm antibody-positive varicocele patients is 2.8 and 3.5 times higher than in anti-sperm antibody-negative varicocele patients and control fertile men, respectively.

In general, anti-TGC and anti-sperm antibodies of IgG class are regarded as an important factor for male infertility; however, those of IgE class might also participate in the male infertility. The increased number of mast cells in the testis was found in cases of male infertility (Maseki et al. 1981; Agarwal et al. 1987; Hussein et al. 2005; Welter et al. 2011). Activated mast cells secrete enzymes, e.g., hyaluronidase, protease, cytochrome oxidase, phosphatases, histamine, serotonin, and heparin. Considering that these substances are important in the laying down of collagen, proliferation of mast cells may result in increasing fibrosis and hyalinization of seminiferous tubular wall and the surrounding interstitium, leading to the spermatogenic disturbance. Mathur et al. (1981) suggested that there may be a local IgE antibody response in the idiopathic infertile males. In response to seminal antigens, mucosal plasma cells are triggered to produce IgE antibodies locally in the male reproductive tracts. This possibility is supported by the elevated levels of IgE in the serum and the semen samples of infertile males with high anti-sperm antibody titer. The high affinity of these antibodies for mast cells induces release of various substances from mast cells described above. Intra-testicular T cells may stimulate mast cells, with the resultant increase of their cell number and stimulation of their secretion in the testis. However, it is yet unclear whether mastocytosis in the testis is a cause, or a result, of the testicular fibrosis in infertile male patients. Altogether, the analysis of epidemiological and prognostic studies may support the opinion that anti-sperm and/or anti-testis autoantibodies are a relative, rather than absolute, cause of immunologic male infertility (Francavilla et al. 1999, 2007).

Systemic inflammatory diseases causing male infertility include primary vasculitis such as polyarteritis nodosa, Behcet’s disease, and Henoch-Schönlein purpura (Dahl et al. 1960; Lie 1988; Pannek and Haupt 1997; Silva et al. 2012, 2014). The overall frequencies of orchitis and anti-sperm antibodies in rheumatic diseases are 2–31% and 0–50%, respectively. The presence of anti-sperm antibodies was reported to be present in 13–50% of systemic lupus erythematosus patients (Silva et al. 2012). Orchitis occurs in 2–18% of patients with polyarteritis nodosa. Epididymo-orchitis was also observed in 4–31% of Behcet’s disease patients. Orchitis was evidenced in 7–21% of Henoch-Schönlein purpura patients. Testicular vasculitis may be a part of systemic vasculitis or may exist as isolated testicular vasculitis. A study of patients with testicular vasculitis demonstrated that 51% of patients had isolated testicular vasculitis and 49% had systemic testicular vasculitis (Hernandez-Rodriguez et al. 2012). Non-granulomatous inflammation affecting medium-sized testicular vessels occurred in most patients with both isolated and systemic testicular vasculitis. Among systemic testicular vasculitis, polyarteritis nodosa was the most frequently diagnosed (63%), followed by Wegener’s granulomatosis (17%) (Shurbaji and Epstein 1988). Focal non-granulomatous orchitis was also reported in a patient with Crohn’s disease (Piton et al. 2015). Pathological examination of the testis revealed a focal inflammatory infiltrate predominantly composed of lymphocytes accompanied by few plasma cells, lacking giant cells or granulomata. Granulomatosis with polyangiitis is a systemic necrotizing granulomatous vasculitis, which predominantly affects small-sized blood vessels in the upper/lower respiratory tract and kidneys. In the patients, orchitis is also found but rare, reported in <1% of cases in large cohorts (Alba et al. 2015).

Humans who have inherited the human class I MHC allele HLA-B27 have a markedly increased risk of developing the multi-organ system diseases termed HLA-B27 syndromes. Clinical features in HLA-B27 syndromes include anterior uveitis, ankylosing spondylitis, reactive arthritis, psoriatic arthritis, and inflammatory bowel disease such as ulcerative colitis and Crohn’s disease. Although autoimmune orchitis has not been reported in men with spondyloarthritis, these patients showed reduced sperm motility, higher plasma luteinizing hormone and follicle-stimulating hormone, and lower testosterone levels compared with control subjects (Villiger et al. 2010; Ramonda et al. 2014).

Autoimmune polyendocrine syndrome, also called autoimmune polyendocrinopathy, is a heterogeneous group of rare diseases characterized by autoimmune activity against multiple endocrine organs, although non-endocrine organs can be also affected. The skin and nails, ovary and testis, eyes, thyroid, and digestive system are affected. In regard to the male hypogonadism, anti-sperm autoantibodies and autoantibodies against steroidogenic enzymes and other antigens expressed in the Leydig cells were detected (Tsatsoulis and Shalet 1991. Maclaren et al. 2001). In the sera of patients, anti-testis-specific protein 10 antibodies were also detected (Reimand et al. 2008; Smith et al. 2011). The testis-specific protein 10 is a highly expressed protein in the testis and plays a key role in spermatogenesis. Therefore, autoimmune responses against these antigens may affect the spermatogenesis in the patients. Actually, the immunosuppressive action of cyclical intermediate-dose steroid therapy led to a significant improvement in semen parameters (Tsatsoulis and Shalet 1991).

More recently, some cases of orchitis were also reported in patients with multi-organ IgG4-related disease involving a broad range of organs and tissues such as the pancreas, biliary tract, liver, breast, thyroid gland, lymph nodes, skin, aorta, pituitary gland, meninges, lacrimal gland, lung, and reproductive organs (De Buy Wenniger et al. 2013; Karram et al. 2014; Migita et al. 2014; Lin et al. 2015). In these cases, dense interstitial lymphoplasmacytic infiltrate composed of IgG4-positive plasma cells and epithelioid histiocytes (macrophages) was observed around the seminiferous tubules and in the epididymis, and prominent interstitial fibrosis was also found.

In regard to infectious diseases, the best-known orchitogenic viruses in humans are the mumps virus and human immunodeficiency virus (HIV) (Aiman et al. 1980; Chabon et al. 1987; De Paepe and Waxman 1989; Jala et al. 2004; Chandrashekar et al. 2015). Anti-sperm antibodies have been detected in both mumps and acquired immune deficiency syndrome (AIDS) patients. In AIDS patients, marked spermatogenic disturbance was noted with Sertoli cells predominantly lining the hyalinized seminiferous tubular wall. Furthermore, the testicular blood vessels were thickened, and mononuclear inflammatory infiltration composed of lymphocytes and macrophages was seen, and the many lymphocytes were CD4⁺ (Pudney and Anderson 1991). Five to 37% of adults with mumps infection develop orchitis. In mumps virus-affected testis, lymphocytic infiltration was followed by the spermatogenic disturbance with atrophy and fibrosis of the seminiferous tubules with the resultant transient or permanent infertility. The mumps virus does not appear to induce TGC transformation and may not be directly injure TGC. Actually, in the mouse, it was demonstrated that mumps virus induced innate immune responses in Sertoli and Leydig cells through TLR 2 and retinoic acid-inducible gene I signaling, resulting in the production of proinflammatory cytokines, including TNF-alpha, IL-6, monocyte chemotactic protein-1, IFN-alpha, and IFN-beta (Wu et al. 2016). By contrast, mumps virus did not induce the cytokine production in TGC. Clinically, serum IL-6 and IFN-gamma levels were elevated in severe mumps cases. Additionally, serum IL-10 level was also elevated in almost all patients with mumps (Wang et al. 2014). AIDS patients often suffer from orchitis, hypogonadism, oligozoospermia, or azoospermia. Lymphocytic infiltration, loss of TGC, and interstitial fibrosis were observed in AIDS patients (Rogers and Klatt 1988; Yoshikawa et al. 1989). It is speculated that the perivascular accumulation of CD4⁺/HIV⁺ cells and cytokine production could affect the integrity of the BTB and favor the development of autoimmune orchitis (Lustig and Tung 2006). Hepatitis B and C viruses and herpes simplex virus have been also found in the interstitial tissue and/or seminiferous tubules in men (Melaine et al. 2003). Granulomatous reactions in the testis have been ascribed to bacterial infections, including tuberculosis, syphilis, leprosy, and brucellosis (Kahn and Mcaninch 1980; Kumar et al. 1982; Roy et al. 2011; Bosilkovski et al. 2016). In most of these cases, the epididymis is primarily and predominantly involved and the testis is secondarily affected. Granulomatous inflammation involves other conditions such as sarcoidosis, malakoplakia, and granulomatous seminoma and lymphoma (Kleinman et al. 1983; Eyselbergs et al. 2011; Bosilkovski et al. 2016). Although these infectious diseases elicit specific immune responses against target virus or bacteria in the testis in early stage, the induced inflammation may break the BTB later and be followed by autoimmune responses in the testis, supporting a diagnosis of autoimmune orchitis.

Shiraishi et al. (2009) examined anti-sperm antibody in the sera of 70 males with systemic autoimmune disease and 80 healthy controls by usage of the indirect immunobead test and the sperm immobilization test. Among 70 males with systemic autoimmune diseases, five were positives, with incidence of 7.1%. However, no positives existed in 80 healthy males. Therefore, systemic autoimmune diseases may be one of the risk factors for developing anti-sperm antibody in men.

Although anti-sperm antibodies have been noted in testicular autoimmunity in men for long years, the occurrence of delayed-type hypersensitivity (DTH) responses against testicular antigens can be observed in cases of human immunologic infertility (Anderson and Hill 1998), indicating that cellular immunity against the testicular antigens may be also critical for testicular autoimmunity. Leukocyte migration inhibition was positive in infertile males with cryptorchidism, varicocele, genital tract infections, idiopathic oligozospermia, and vasectomy (Nagarkatti and Shanta 1976; Polidori et al. 1980). Recently, clinical studies have shown that macrophage numbers increased in the testes of patients with the spermatogenic disturbance of different etiologies (Frungieri et al. 2002). Previously, patients suffering from prostatic cancer received intra-testicular injection of BCG bilaterally instead of orchidectomy (Frick et al. 1983). Testicular biopsy of the patients revealed that the seminiferous tubules were partially or completely atrophied with hyalinization. Sertoli cells were vacuolated, and mononuclear infiltration was evident in the testicular interstitium. However, tuberculous structures were not seen. It was also reported that immune deviation toward a Th17 immune response was associated with testicular damage in azoospermic men (Duan et al. 2011). Th17 cells are a subset of T helper cells producing IL-17 and create inflammation and tissue injury in autoimmune disease. This indicates that control of not only humoral but also cellular immunity against testicular antigens is important for studying developmental pathways of orchitis and immunotherapeutic approaches for the disease.

A crucial role of Treg-mediated tolerance on prevention of orchitis was indicated by the autoimmune polyendocrinopathy, a condition of spontaneous autoimmunity in a number of endocrine glands, which is characterized by defective CD4⁺CD25⁺ Treg function (Kriegel et al. 2004). In patients with autoimmune polyendocrinopathy, expression of mRNA and protein of Foxp3 was significantly decreased (Kekalainen et al. 2007). This suggests that loss of active suppression in the periphery could be a hallmark of this syndrome.

However, there is still little evidence regarding the role of cellular immunity in male infertility in men. In the majority of patients with testicular autoimmunity, there is a chronic and asymptomatic development of the inflammatory reaction. Therefore, this disease is very difficult to be diagnosed at the ongoing stage, and it is possible that many clinical findings of histopathology of idiopathic spermatogenic disturbance are those of the post-active inflammation stage of autoimmune orchitis. In other words, the biopsies from infertile men represent the final stage of the pathological process, and thus the reactive changes such as lymphocytic infiltration might be no longer detectable and only the spermatogenic disturbance remains (Fig. 3.3). Therefore, cellular immune responses may be more critical for male infertility than has been suspected in the past in cases of idiopathic spermatogenic disturbance.

Hatakeyama (1984) observed the human testes more thoroughly than anyone else previously and reported on the autopsy findings of approximately 400 independently examined patients with or without male infertility, out of which death resulting from some disease occurred in approximately 160 cases, and sudden deaths determined by medical examiners occurred in approximately 240 cases. He documented that autoimmune inflammation should occur in the testes and the epididymides more frequently than in any other organ and that approximately 70% of idiopathic spermatogenic failure is associated with autoimmune phenomena. Here, the immunological fragility of the testes will be discussed using the findings from Hatakeyama’s autopsy reports and those from animal experiments.