Macrophages are tissue-specific immune cells that are widely present in various tissues; their functions differ depending on the site. Testicular macrophages are the major immune cells in the testes, accounting for approximately 20% of murine testicular stromal cells (17), which regulate the development of rat Leydig cells and steroidogenesis (18, 19). Specifically, testicular macrophages play a key role in fetal testicular organogenesis (20), testosterone synthesis (21), spermatogenesis (22), and testicular immunosuppression (23). Notably, the inflammatory response of testicular macrophages is lower than that of blood macrophages (24–26). Therefore, testicular macrophages are regarded as testicular guardians (27). In other words, testicular macrophages are characterized by a higher degree of immunosuppressive activity than macrophages from other tissues. Testicular macrophages are mainly polarized into pro-inflammatory (M1) and inhibitory-inflammatory (M2), the latter of which is characterized by high levels of anti-inflammatory cytokines, such as interleukin (IL)-10 and transforming growth factor-beta (TGF-β) (28). Furthermore, testicular macrophages in rats are less responsive to pathogen stimulation; and constitutively produce anti-inflammatory cytokines (29, 30), such as IL-10, which are essential for the prevention of organ-specific autoimmune inflammation (8). In contrast, under the influence of some negative regulatory factors, macrophages polarize to M1 and secrete high levels of pro-inflammatory cytokines, such as IL-1β and TNF-α (31).

Lymphocytes are cells with immune recognition functions. They can be classified as T cells, B cells, and natural killer (NK) cells according to their migration, surface molecules, and function. T cells are vital regulators of cellular and humoral immunity and can be distinguished as CD4⁺ T cells and CD8⁺ T cells based on their surface receptors. T cell receptors recognize antigenic proteins released by pathogens. Under normal physiological conditions, CD8⁺ T cells are the predominant lymphocytes in the testicular interstitial space (32). Regulatory T cells (T-regs) promote peripheral immune tolerance and exhibit immunosuppressive properties in the vasectomy model (33). Murine models of allogeneic islet cell transplantation have also exhibited the immunosuppressive effects of testicular T-regs characterized by CD4⁺ CD25⁺ (34, 35). T-regs inhibit the activation of effector T cells under normal physiological conditions (35) and increase T cells counts under inflammatory conditions (36). These observations suggest that T-regs contribute to testicular immunity. Induction of T cell apoptosis is one of the methods by which the testes suppress the immune response, and the Fas/Fas ligand (Fas/FasL) system is highly expressed in the testes (37, 38). Programmed deathligand-1(PDL-1) is significantly expressed in male germ cells. The Fas/FasL and the programmed death-1 (PD-1)/PDL-1 system can induce T cell apoptosis, thereby and protecting against islet allogeneic immune damage (39, 40). Testicular NK cells also have immunoregulatory properties, but there have been only a few studies on these cells. Previous studies have used methods such as flow cytometry to confirm that there are no B cells in the testes under physiologically normal conditions (41).

DCs are the most functional, professional antigen-presenting cells in the body and are highly efficient at ingesting, processing, and presenting antigens. DCs occur in the interstitial spaces of the testes and are immature under normal physiological conditions. The number of DCs increases significantly and they express maturation markers in the inflammatory state (42, 43), indicating that DCs play an important role in testicular autoimmunity (44). DCs minimize autoimmune responses by tolerating T cell auto-antigens under normal physiological conditions (45). However, the mechanism underlying their effects in the testes requires further study. According to Banchereau’s study (46), the maturation of testicular immunosuppressive T cells requires the assistance of mast cells.

Mast cells secrete a variety of cytokines and are involved in immune regulation and immediate hypersensitivity (type I allergy). The number of mast cells in the testes is small under normal physiological conditions. However, mast cells are heavily differentiated during inflammatory activation (47). Abnormal spermatogenesis is associated with an increased number of mast cells (48). A randomized controlled trial has demonstrated that Zaditen, a mast cell blocker, improves semen parameters, chromatin integrity, and pregnancy rates after VC surgery (49). Research on mast cells is limited, and the function of mast cells in testicular immunity is not completely understood. However, this review demonstrates, their important role in testicular immune disorders and their effects on sperm quality parameters.

In addition to regulating male sex differentiation and fertility by producing testosterone, Leydig cells can also indirectly contribute to the testicular immune microenvironment. Representing the majority of mesenchymal cells, Leydig cells have congenital antiviral infection function in rats (50, 51). Furthermore, testosterone inhibits systemic immune responses to autoantigens (52, 53). Androgen receptor-deficient murine Sertoli cells (SCs) impair immune exemption in the seminiferous tubules, suggesting that testosterone also plays a local role in maintaining testicular immune privilege (54). Leydig cells also play an important role in the innate immune defense of the testes. In microbial invasion, Leydig cells synthesize and release antiviral factors, such as TNF-α and interferon (IFN)- α, or anti-inflammatory factors, such as IL-6 and IL-1β (55, 56).

ASAs are present in 1-2% of fertile men and 5-15% of men with infertility (66). Sperm immunoglobulin (Ig) levels in the seminal plasma of men with infertility and VC were higher than those in men with infertility but without VC (67, 68). The positive rate of mixed antiglobulin reaction (MAR)-IgG in the seminal plasma of patients with VMI was more than three times that of normal seminal plasma (66). In patients with improved semen quality after surgery, the level of seminal ASAs decreased accordingly (69, 70). Animal experiments also showed similar results; i.e. the ASA of the VC rats model increased (71). The incidence of anti-sperm immune responses is associated with increased chromosomal abnormalities in the gametes. There was a significant positive correlation between the MAR-IgG percentage and sperm DNA fragmentation rate. ASAs can agglutinate sperm and bind to apoptosis-related proteins (e.g., caspase 3 and hsp70), leading to apoptosis in sperm cells (72).

ASAs affect male fertility through different mechanisms, and largely correlates with sperm autoimmunity and ASA antigen specificity ( Figure 1A ). The main role of the testicular autoimmune response is to reduce sperm movement and agglutination (73). An anti-spermatic immune response is associated with decreased sperm function and sperm quality parameters, such as DNA breaks. One study found that sperm quality in patients with ASA (+) VC is significantly reduced and correlates with the VC grade (66). Simultaneously, sperm damage is more severe in patients with ASA positive VC. The spermatozoa count in grade 2 ASA-positive VCs patients was less than half of that in ASA negative patients, whereas the reactive oxygen species (ROS) level in ASA (+)patients with infertility was 2.8 and 3.5 times higher than that of ASA (-) patients, respectively (5). However, the results of ASA testing in patients with infertility were similar regardless of whether the patient had been diagnosed with VC (2). In other words, VC may be an important cofactor for ASA production. VC increases the likelihood of immune infertility; after testicular trauma, the probability of immune infertility in patients with VC increases by a factor of 2 (66). Therefore, VC may not be the direct cause of ASA production, but VC is a synergistic factor that leads to immune sterility. However, Bozhemov et al. (66) suggested that VC is not a direct cause of the sperm autoimmune response; but a cofactor that increases the risk of ASA production. In VC patients, the odds ratio for immune infertility tripled after testicular trauma.

Multiple studies have demonstrated the central role of IL-1α and IL-1β in a number of autoinflammatory diseases (82–85). In the testes, IL-1 regulates spermatogenesis through autocrine and paracrine expression, and overexpression of IL-1 is critical for improving VMI. Studies have shown that IL-1 expression in the testes has increased in VC animal models (86, 87). Kim’s study (88) and Sahin et al. (89) found that the expression of both IL-1α and IL-β increased in the testes of the VC model, which could be reversed by treatment with herbs. However, case-control studies found that, compared with that in the control group, the level of IL-1β in the seminal plasma of patients with VC did not change significantly (9, 55, 90). ( Table 1 ) This contrasts with the increased IL-1 levels typically observed in the testicular tissues of patients with VMI. A potential explanation for this is that the liquid components of the semen originate from multiple sites, including the seminal vesicle gland and prostate, and may dilute the levels of the original substances in the testicles. Therefore, IL-1 is most likely a factor that causes infertility in patients with VC.

IL-6 is produced rapidly and briefly in response to infection and tissue damage, but the persistent dysregulation of IL-6 synthesis plays a pathological role in chronic inflammation and autoimmunity (99). Most studies (8, 55, 88, 91–94) support the view that the level of IL-6 is increased in VC animal models or patients with VMI ( Table 1 ). The concentration of IL-6 in the seminal plasma of patients with VC is much higher than that in fertile men (55, 93, 94), but lower in Finelli’s study (9). However, the study conducted by Finelli et al. included only patients with VC, excluding those with VMI the focus of our review. This may be one of the main reasons why the levels of IL-1β, IL-6, IL-8, TNF-α, and IFN-γ found in that study are inconsistent with those found in other studies. In patients with VMI, it has been reported that levels of IL-6 and ROS are elevated and that total oxidative binding energy is decreased. In vitro, IL-6 reduces sperm motility, possibly attributed to excessive production of nitric oxide (NO) (100). IL-1 and IL-6 have inhibitory effects on the acrosomal reaction similar to those of TNFα (93). In short, IL-6, like IL-1, may be an important immunoregulatory factor affecting fertility in patients with VC.

The number of white blood cells in seminal plasma is significantly correlated with IL-8, also known as C-X-C motif chemokine ligand (CXCL) -8 (55, 73, 101). Compared with those in the control group, the levels of IL-8 in the seminal plasma of patients with VC was found to be higher (55, 95). The mechanism related to IL-6 and IL-8 has not yet been reported. Similarly, IL-17A and IL-18, as well as anti-inflammatory factors IL-10 and IL-37, have also been reported in a series of studies (9, 96–98) ( Table 1 ). In general, the current research on IL-8, IL-10, IL-17A, IL-18 and IL-37 is limited, and it is difficult to draw definite conclusions on the role of ILs in VC. Further investigation on this topic is required, is needed in the future, particularly through high-quality human studies.

Multiple studies have reported an increase in TNF-α levels in both patients with VC and in animal models ( Table 1 ). For other two studies (9, 98) did not find significant changes in TNF-α levels because they included patients different from those in the other studies, as described above. TNF-α alters mitochondrial function, increases NO production, and is inversely related to sperm motility. It also increases the production of malondialdehyde and inhibits spontaneous and induced acrosomal reactions (73). The role of pro-apoptotic TNF-α in the pathogenesis of VC-mediated dysfunction is currently under intense study (93). Animal studies demonstrated that IFN-α levels increased in the testes or serum, but human studies did not support this finding, possible reasons have been explained in IL-1. ( Table 1 ) Habibi et al. (91) found that IFN-γ levels in serum and testicular tissue were increased in rat VC models, suggesting that VC has a detrimental, time-dependent effect on cytokine levels and decreases the number of SCs, and spermatogonia, as well as the seminiferous tubules diameter and sperm indices. TGF-β balances pro-inflammatory and anti-inflammatory effects by reducing cell growth of immune precursors and plays a key role in immune tolerance (102). Similar to those of most cytokines, TGF-β levels were also reported to be increased in the testes of VC model ( Table 1 ) (71). Together, these results suggest that TNF-α and IFN-γ are potential markers of testicular damage in patients with VC.

Overall, most of the available data from patients with VC or animal VC models support the observation of elevated levels of pro-inflammatory factors in the testicular tissues and seminal plasma, to varying degrees. In particular, IL-1, IL-6, TNF-α, and IFN-γ may be key immunoregulatory factors for testicular injury in patients with VC. Simultaneously, some anti-inflammatory factors, such as IL-10, IL-37, and TGF-β, tend to be upregulated; this may be the result of the normal feedback mechanism of the body. These anti-inflammatory factors may also be of consequence to future treatment strategies, but their authenticity must first be confirmed by further studies.

PRRs can be divided into five categories, including TLRs, located on the cell membrane, and NLRs, located in the cytoplasm (103). They occur mainly on immunocytes such as macrophages, DCs, and rarely non-immunized cells. Thus, once PRRs are activated by pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs), the signal will eventually be transmitted to the downstream target gene, which regulates immunity and inflammation (104). PRRs and their specific ligands are listed in detail in a review by Wang et al. (103).

TLR2, one of the TLRs, can also recognize PAMPs and/or DAMPs (105), the latter including heat shock proteins (HSP60, HSP70, Gp96), advanced glycation end-products, high mobility group box 1, and serum amyloid A (103, 106). The average relative expression of TLR2 in the semen of men with infertility and VC was twice as high as that in fertile men, but the difference was not statistically significant (107).

NLRP3 is a subtype of the NLR family, that can be directly triggered by cytoplasmic DAMPS, such as peptides, DNA, and RNA (103). NLRP3 inflammasomes are composed of cytoplasmic sensor molecules, such as pyrin domain-containing protein 3, adaptor proteins (e.g., caspase-recruiting domain, ASC, and apoptosis-associated speck-like proteins), and effector proteins (e.g., pro-caspase-1). NLRP3 and ASC promote the cleavage of pro-caspase-1 and form an active complex, which triggers the cleavage of pro-IL-1β into mature IL-1β (65). The VC model showed a significant increase in NLRP3 gene expression after partial ligation of the left renal vein (108). Interestingly, this trend was reversed by the administration of Resveratrol (3,5,4′-trihydoxy-trans-stilbene), which is an anti-inflammatory, anti-apoptotic compound in some plants (109). We previously found a significant up-regulation of prokineticin 2 (PK2) in the VC animal model (110), whereas in the orchitis model PK2 promoted IL-1β secretion via the NLRP3 pathway (111). We speculate that this process is also involved in the onset of VMI, although this hypothesis has not been validated with rigorous experiments.