The blood-testis barrier (BTB) is one of the tightest blood-tissue barricade in mammals. It is formed by the specialised tight occluding junctions of the Sertoli cells and is responsible for physically sequestering all the spermatogenic cells beyond the pre-meiotic leptotene or zygotene stage in the seminiferous epithelium. Sertoli cells makes various types of cell junctions such as tight junctions, gap junctions, basal ectoplasmic specialization, and desmosome-like junctions at the base of seminiferous tubules. The tight junctions between adjacent Sertoli cells involve Occludin and Claudin 11, which are linked to the actin cytoskeleton through cytoplasmic plaque proteins [4]. Cx43 is the dominant protein in gap junctions present in seminiferous tubules that allows the passage of only small molecules less than 1 kDa [5]. Cadherin-catenin complexes leads to the formation of basal ectoplasmic specialization which also plays an important role in BTB [6]. The desmosome-like junctions exist between Sertoli cells and between Sertoli and germ cells. The expression of various desmosomal proteins such as desmoglein-2, plakophilin-2, plakoglobin and demoplakin has been shown in Sertoli cells. Desmosome-like junctions regulate cell adhesion and junction assembly by interacting with tight junctions, gap junctions, and basal ectoplasmic specialization [7]. Further, hemidesmosomes are also present in the basal membrane of the seminiferous tubules formed by myoid cells. Expression of various proteins leading to the formation of specialized occluding junctions playing a role in BTB are schematically shown in Figure 1. This makes the neoantigens on the meiotic and haploid germ cells inaccessible to the immune cells present in the interstitial tissue.

However, it is now amply clear that the BTB is just one of the contributing factors towards the immune privilege in the testis [2]. Recent studies have suggested that not all antigens on the developing germ cells may be sequestered from the immune system. In fact, a proportion of meiotic germ cells antigens egress from the seminiferous tubules into the interstitium and maintain regulatory T (Treg; CD4⁺/Foxp3⁺) cells dependent systemic tolerance [8]. Further, certain sperm-specific proteins are selectively released by the Sertoli cells in the testicular interstitial fluid which can be accessed by the immune system [9]. These studies have challenged the conventional belief that all antigens on the developing germ cells are uniformly sequestered from the immune system and suggest operation of additional mechanisms to maintain systemic tolerance.

Further, most of the developing spermatogenic cells do not express major histocompatibility complex (MHC) class I and II molecules [10]; although, the mature human sperm does express them [11]. Thus, the delayed expression of MHC molecules makes these cells less immunogenic and may play a role in the escape of recognition by the immune cells. Moreover, several testicular cells exhibit human leukocyte antigen-G (HLA-G) and HLA-E, which are implicated in suppression of the adaptive immune response [12].

Along with Leydig cells, several immune cells are present in the interstitial compartment [13], which are shown in Figure 1. Together these cells contribute towards maintaining the tolerogenic immune state in the testis (summarized in Table 1). Testicular macrophages represent the most abundant immune cells in the testis in most species, and exist in close physical and functional association with Leydig cells [14–16]. Majority of the macrophages in the testis are of the alternately activated M2 type (CD163) and produce interleukin-10 (IL-10) that have immunosuppressive and anti-inflammatory activities [17]. Beside the macrophages, dendritic cells are also found in the interstitium, although they are less in numbers under normal physiological conditions. Expression levels of MHC II antigens, co-stimulatory molecules such as CD80 and CD86 and chemokine receptor 7 (CCR7) suggest that the testicular dendritic cells are tolerogenic under normal physiological conditions [18]. The numbers of both macrophages and dendritic cells increase in infertile patients with chronic inflammation and their functions are also compromised [19]. In addition, suppressor T cells (CD8⁺), NK cells, and Treg cells are encountered in the interstitium [20, 21]. Although they are sparsely distributed under normal circumstances, their numbers increase in infections and autoimmune conditions [13, 22]. Granular leukocytes like mast cells are also present in the interstitium and are found to be increased in testicular inflammatory conditions [23]. However, the roles of NK cells and mast cells in maintaining testicular immune previlege is not clear.

Sertoli cells, peritubular cells, and Leydig cells express and secrete several immunomodulatory molecules that are critical for maintaining the immune privilege environment in the testis (Table 1) [24, 25]. Sertoli cells produce galectin-1, which has an immunosuppressive property [26, 27]. In addition, Sertoli cells secretes anti-inflammatory cytokines and activin that help in suppressing the immune response [28]. Peritubular cells also help in making testicular environment immune tolerant by expressing activin A and TLR [29]. Under testicular inflammatory conditions, production of proinflammatory molecules such as tumor necrosis factor (TNF), IL-1, IL-6, monocyte chemotactic protein-1 and nitric oxide production is increased whereas IL-10 production is decreased [13, 30, 31]. Leydig cells secrete macrophage MIF [32] that prevents cytotoxic activities of T cell and NK cells, thereby maintaining a tolerogenic environment. In addition, Leydig cells also secrete testosterone that is known to have immunosuppressive activity [33].

Formation of antibodies against the spermatozoa represent the most common and widely studied immune dysfunction of the male reproductive system. It is also listed as one of the factors causing infertility in men [34]. The presence of anti-sperm antibodies (ASA) can target the sperm for immune destruction, lead to a decrease in sperm motility, inhibit sperm penetration through the cervical mucus, and can affect sperm function by blocking receptors or molecules essential for fertilization thus leading to ‘immune infertility’ [35–38]. In contrast to other autoimmune disorders where immune response is generated against a specific dominant autoantigen, ASA are found to be directed towards multiple antigens, which may vary among individuals [39, 40].

There is a significant debate on the types of methods and source of samples to be used for detection of ASA. Although several studies had investigated the presence of ASA in seminal plasma and serum, it is now increasingly clear that only antibodies physically bound to the sperm are clinically significant [36, 41, 42]. Therefore, WHO has recommended detection of ASA on the surface of the ejaculated sperm by the direct antiglobulin-based tests such as the mixed anti-globulin reaction (MAR) test and the immunobead-binding test (IBT). These tests determine the percentage of motile sperm bound to the antibody, and a threshold of 50% of antibody-bound motile sperm are considered pathological [38, 43, 44]. Although ASA are found to be of immunoglobulin G (IgG) and IgA classes, it is suggested that presence of IgA-ASA is indicative of a mucosal immune response, which can impair sperm-cervical mucus interaction. Lastly, these tests are recommended to be used in conjunction with sperm function tests such as the post-coital test (PCT), which would determine the clinical consequence of ASA-bound sperm [36].

ASA are observed in 2–21% of infertile men and several epidemiological studies have reported different incidence of ASA, mainly due to the differences in the study population, detection methods and thresholds chosen [34, 38, 45–47]. There has been intense debate on the association of ASA with infertility as ASA bound sperm are also found among a small proportion of fertile men indicating that presence of ASA may not always lead to infertility [45, 48]. This may be due to a variety of factors such as localisation of ASA to the spermatozoa (head, neck or tail region), functional relevance of the antigen(s) sequestered by ASA, immunoglobin classes involved and their proportion [37, 38]. Several studies have associated ASA with reduced sperm counts and motility [47, 49]; however, inhibition of sperm penetration through the cervical mucus presents the main mechanism by which ASA interferes with fertility [37, 50]. Furthermore, ASA-positive infertile men had higher incidence of acrosome disorders, sperm DNA fragmentation, and increased reactive oxygen species (ROS) activity in washed spermatozoa samples of ASA group, all of which are detrimental to male fertility [46]. A recent large-scale retrospective study of infertile couples found that presence of 100%-positive MAR test in normozospermic infertile men was significantly associated with negative PCT outcome, suggesting that a 100%-positive MAR test can be a sole determinant of male infertility [34].

Several studies have investigated the probable causes and risk factors for ASA production. However, only chronic obstructions to the male reproductive tract is proven to be a clear risk factor. Surgical procedures like vasectomy is widely accepted to be the most common cause of ASA formation. This is evident from the fact that most men (70%) develop ASA after undergoing vasectomy due to the extravasation of sperm, and consistently report high ASA titres post-vasecctomy [51, 52]. There is also an association between ASA and obstructive azoospermia and congenital absence of vas deferens [51, 52]. Although ASA are also reported in cases of testicular trauma, torsion, varicocele and other infectious and inflammatory conditions, there are several conflicting reports and lack of a concensus about a causal relationship [53]. Studies have also shown an increased incidence of other autoantibodies such as antinuclear antibodies and thyroglobulin antibodies in infertile men with ASA, suggesting that there may be an underlying genetic component towards ASA formation [54, 55].

Previously, treatment of male infertility due to ASA used immunosuppressive drugs such as glucocorticoids. These drugs supressed the production of ASA, and led to limited success in improving prospects of pregnancy. However, treatment with glucocorticoids is frequently accompanied by side-effects, and hence are not recommended as the first line of treatment for ASA realted infertility [35, 56].

Assisted reproduction techniques (ART) are the current methods of choice in cases of male infertility attributed to sperm autoimmunity. However, success rates of ART like in vitro fertilisation (IVF) is negatively affected by the presence of ASA. Studies have shown that men with sperm-bound ASA experienced low fertilisation rates in IVF [57, 58], which could probably be due to ASA impeding the sperm-egg interactions. Intracytoplasmic sperm injection (ICSI), which involves direct injection of isolated spermatozoa into the oocyte cytoplasm, has been successfully used to overcome this problem. ICSI has yielded fertilisation and pregnancy rates comparable with ASA negative patients [59]. It has therefore become the best treatment modality in cases of male infertility due to sperm-bound ASA.

Acquired immunodeficiency syndrome (AIDS) caused by the HIV has emerged as one of major health issue of the last century affecting both developed and developing nations. Development of antiretroviral therapy has significantly extended the life expectancy of HIV infected individuals. One of the main challenges in cure of HIV is the emergence of viremia after completion of suppressive antiretroviral therapy. A leading factor contributing to this is the persistence of replication-competent HIV in cellular reservoirs like CD4⁺ memory T cells. Testis has been postulated to be one such site due to its suppressed immunoregulatory environment [73, 74].

There has been an intense debate on the status of testis as a viral compartment and sanctuary site during HIV infection [75]. It was initially thought that testis may contribute negligibly to the seminal viral load as there has been no difference in the seminal HIV-RNA after vasectomy [76]. However, resident testicular macrophages express the CD4, C-X-C motif chemokine receptor 4 (CXCR4), and CCR5 receptors for HIV-1 entry, which make them susceptible to infection, and can potentially constitute a reservoir for HIV [77, 78]. There has been considerable controversy regarding the susceptibility of testicular germ cells to HIV infection [79–81]. It has been shown that though HIV is unable to infect germ cells, it binds to these cells using alternate receptors. The cell-associated infections through contact with infected testicular macrophage or lymphocyte are possible, leading to low level of viral replication and production [82]. Studies have shown that in HIV infected men (on suppressive antiretroviral therapy) with suppressed viral load (< 50 copies of HIV RNA/mL) in blood still harbour low levels of integrated HIV DNA in at least one testis [83]. It remains to be seen whether these can give rise to replication-competent viruses [84]. A recent study has further revealed that there is significant blood-testis genetic compartmentalisation of HIV DNA mainly due to differential clonal expansion of the infected immune cells in the blood and testis [85]. These findings may pose further complications for HIV eradication.

The ZIKV has been declared as a global public health emergency by the WHO after a major outbreak in 2016. The main mode of ZIKV transmission is through the bite of Ades mosquito in endemic regions. Although most ZIKV infections are asymptomatic, systemic infections can lead to development of common symptoms like fever, rashes, arthralgia, arthritis, myalgia, headache and Guillain-Barre syndrome in severe cases [86]. Besides the vector-borne transmission, sexual transmission has also emerged as an important route, with male-to-female transmission being more frequent [86]. These findings pose a grave concern as vertical transmission of the virus from infected mother to the developing fetus can cause various teratogenic effects such as the congenital Zika syndrome, which involves serious complications like microencephaly, cerebral abnormalities, intra uterine growth restriction and eye and hearing defects [87, 88].

Studies have shown that high loads of ZIKV RNA can persists in semen for longer durations (more than 6 months) than other body fluids like blood and urine [86, 89, 90]. Studies have reported persistent shedding of infectious ZIKV in semen for up to 30 days from the onset of symptoms, and presence of ZIKV RNA for much longer [90], suggesting that there is risk of sexual transmission even after symptoms are resolved. Moreover, sexual transmission has also been reported between asymptomatic individuals [91], and there are reports of detection of ZIKV in semen of vasectomised men [92]. These studies strongly suggest the tropism of ZIKV for the male genital tract even beyond the testis and epididymis. Studies have shown a transient decline in semen parameters like sperm count and motility along with disturbances in gonadotropin and steroid hormones in ZIKV infected men, suggesting a possible direct effect on spermatogenesis [93]. Mice models of ZIKV infection have demonstrated progressive testicular atrophy post-infection [94]; therefore, long-term effects on fertility in men cannot be ruled out and needs investigation.

ZIKV has been shown to infect a variety of testicular cells including the macrophages, peritubular cells, Leydig cells, Sertoli cells, and germ cells; and even produce replication competent viral particles [95]. It has been postulated that the ZIKV-infected macrophages can affect the tight junctions of the BTB and invade the seminiferous epithelium to disseminate the virus to the germ cells. Additionally, ZIKV can penetrate the adluminal compartment via the Sertoli cells, which are quite susceptible to ZIKV [96]. These findings implicate the testis as a potential long-term reservoir for ZIKV, even after peripheral clearance. Additionally, ZIKV can directly bind and infect the mature spermatozoa via the tyrosine kinase 3 receptors on the mid-piece [93, 97].

The coronavirus disease-19 (COVID-19) pandemic caused by SARS-CoV-2 has spread worldwide and infected millions of men. Although controversies exist on the detection of SARS-CoV-2 RNA in semen [98], studies have detected viral shedding in seminal plasma during the active phase of infection [99]. However, unlike the ZIKV, SARS-CoV-2 does not persist in semen for longer periods and viral RNA is not detectable within 2 weeks of recovery [100]. The SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) and the S protein cleavage enzyme TMPRSS2 are detected in testis [101]. Viral RNA and protein are also detected in the testis of men with COVID-19 implying that the coronaviruses can have an adverse impact on testicular health [102]. Indeed, degenerating germ cells have been reported in the testis of men with SARS-CoV-2. Sloughing of the spermatocytes was also noted along with swelling of the Sertoli cells and reduced number of Leydig cells [102–104].

SARS-CoV-2 infection systemically is associated with immune dysfunction in the testis. Significant interstitial edema and inflammatory infiltration with T cells are detected in testis of men infected with SARS-CoV-2 [103]. In addition, transcriptome profiling has revealed that inflammatory cytokines are upregulated in COVID-19 patient testes while genes asscoaited with spermatogenesis were reported to be downregulated. These data suggest that SARS-CoV-2 infection may lead to dysfunction of the genes that regulate spermatogenesis and inflammation-related pathways, thereby causing inflammatory immune attack in the testes and defects in spermatogenesis. Indeed, immunohistochemical staining demonstrated infiltration of CD3⁺ T lymphocytes, CD20⁺ B lymphocytes, CD38⁺ activated B cells, CD68⁺ macrophages CD138⁺ plasma cells in the interstitial compartments of testis from patients with COVID-19, such infiltration were rarely detected in controls [102]. There is also IgG precipitation suggesting that SARS-CoV-2 might trigger a secondary autoimmune response and fuelling the primary pathogenesis of viral orchitis and consequent testicular damage [102].