Introduction

Spermatogenesis and steroidogenesis are the two major functions of the testis [1]. Testicles are mainly composed by seminiferous tubules that have germ cells (stem cells, spermatogonia, spermatocytes, and sperm) and Sertoli cells that produce inhibin B [2]. They also have interstitial region which is composed by Leydig cells that produce testosterone [3]. Sperm production in the testicles usually begins at puberty, between 12 and 13 years old, and thereafter, spermatozoa are constantly produced [1, 2].

Testicular dysfunction and infertility can be multifactorial, particularly in patients with chronic illness [2, 4–8]. Impaired health status such as malnutrition [6], obesity, alcohol, tobacco, or illicit drug use [9] may reduce fertility [10, 11]. Furthermore, hypothalamic–pituitary–gonad axis dysfunction [12–15], inflammatory disease activity or chronic renal failure [1, 2, 16], immunosuppressive drugs [17–20], genetic abnormalities (Klinefelter's syndrome and Y-chromosomal microdelections) [21], and the presence of anti-sperm antibodies (ASA) can induce impairment in male reproductive function [1, 2, 4–6, 8, 22–24].

Orchitis is defined as an acute symptomatic disease following a local or systemic infection [25]. The subacute or chronic asymptomatic inflammation of the testis, including those of non-infectious disease etiologies, is hardly ever diagnosed and for this reason, often unnoticed. Additionally, testis inflammatory diseases may also encompass the epididymis and vice versa (epididymo-orchitis) [23].

Epididymo-orchitis may be classified according to its cause such as infections (bacterial/viral) or traumatic events (testicular torsion). The most common trigger of viral orchitis is mumps [26]. Orchitis may also be associated with accessory gland infection, such as prostatitis or sexually transmitted diseases, particularly Neisseria gonorrhoeae and Chlamydia trachomatis infections, and rarely enterobacteriae [27].

Definition and Classification of Autoimmune Orchitis

Autoimmune orchitis is defined as an autoimmune aggression to testis [25] with the presence of specific testis autoantibodies and is a cause of decreased fertility in males [28] and females [29]. According to its main cause, this entity may be classified in:

  1. 1.

    Primary autoimmune orchitis-isolated infertility in presence of autoantibodies (ASA or antibodies to the basement membrane or the seminiferous tubules) [25] and without evidence of systemic disease.

  2. 2.

    Secondary autoimmune orchitis–orchitis and/or testicular vasculitis associated with a systemic autoimmune disease [25].

Epidemiology

The presence of ASA in primary autoimmune orchitis has been observed in the serum and/or in the seminal plasma or attached to the surface of spermatozoa in approximately 5–12% of infertile male partners [26, 30, 31], whereas autoimmune response against the developing germ cells of human testis has not been studied extensively [25]. These autoantibodies may also be detected in follicular fluid and cervical mucus in female partner. However, only the antibodies bound to spermatozoa are relevant for fertility [26].

These ASA were also described in men with unilateral and bilateral obstruction of vas deferens [28] and after vasectomy [30], torsion or testicular injury [32], especially in infertile patients with oligozoospermia (reduction of sperm concentration) [31, 33]. The presence of these antibodies have also been documented in other conditions including sexually transmitted diseases, varicocele, mumps orchitis, and spinal cord injury [28].

On the other hand, in secondary orchitis due to systemic autoimmune diseases, a prevalence of ASA in 7% of males with chronic rheumatic diseases was demonstrated in a recent study, even in the absence of other contributing factors to develop these autoantibodies (such as vasectomy, testicular trauma, etc.) [34].

Interestingly, ASA antibodies were reported in approximately 13–50% of systemic lupus erythematosus (SLE) patients, but a clear association with infertility was not identified [22, 34, 35]. Reichlin et al. reported IgG anti-sperm antibodies in almost half of 24 SLE patients, which were related to anti-DNA titers and disease activity, suggesting the possibility of an antibody-mediated lesion. Recently, our group confirmed an overall ASA frequency of 40% in 35 male adolescents and adults with SLE [22]. However, in this global evaluation of gonadal function, these antibodies were not associated with sperm abnormalities severity, reduction of testicular volume, hormone alterations, and cyclophosphamide therapy [22]. Additionally, epididymo-orchitis was a rare manifestation of lupus [16, 36, 37] and one case of lupus/scleroderma overlap syndrome [38].

Testicular involvement in connective tissue diseases is typically caused by medium-vessel vasculitis as described in polyarteritis nodosa (PAN) [39]. Orchitis or testicular tenderness occurs in 2–18% of patients with this disease [39–43], although autopsy studies identified frequencies as high as 93% [39]. In contrast, isolated form of PAN with testicular involvement was rarely reported [40].

Acute clinical epididymo-orchitis was also observed in 4–31% Behçet's disease [39]. This manifestation could be recurrent in 12% of patients with this vasculitis and other associated symptoms include oral ulcers, uveitis, arthritis, and penile ulcer [44]. Orchitis was evidenced in 7–21% of Henoch–Schönlein purpura [39, 45, 46]. There were two reported cases of this urologic alteration in relapsing polychondritis [39] and two cases in rheumatoid arthritis [39, 41]. Scrotum and testicular calcinosis in juvenile dermatomyositis was described in two pre-pubertal patients of our idiopathic inflammatory myopathies population [7]. The frequencies of orchitis and ASA in autoimmune rheumatologic diseases are shown in Table 1.

Table 1 Orchitis and anti-sperm antibodies in autoimmune rheumatic diseases
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Immunopathogenesis

The immunopathogenesis of autoimmune orchitis is shown in Fig. 1. Testis has a unique immune condition because it is able to tolerate autoantigens expressed in germ cells [26, 47] after puberty when immunocompetence is established [48]. It is believed that the underlying pathogenic mechanism involved in the testicular autoimmune disease is a T-cell response to antigens or microorganisms that have permeated the testis barrier [25]. Indeed, the prevention of this autoimmune disease involves the blood–testis barrier, a structure that protects the access of germ cells antigens to the immune cells and antibodies [48].

Fig. 1
figure 1

Immunopathogenesis of autoimmune orchitis

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Studies on experimental autoimmune orchitis (EAO) have helped to partially elucidate not only organ-specific autoimmunity [49, 50], but also the systemic and local regulation that normally prevents disease in the testis [26]. The most likely mechanism for blood–testis barrier breakdown during inflammation is cytokines. An increase in tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) levels may downregulate occluding expression in Sertoli cells with a consequent disassembly of its tight junctions [51]. Macrophages, endothelial, and Leydig cells secrete chemokines (MIP-1 alfa, MIP-1) that bring about the outflow of immune cells to the testicles [50]. Dendritic cells with a mature immunogenic state determine immune response against spermatic antigens in the testis induced by EAO [52].

Moreover, immune cells secrete other pro-inflammatory cytokines such as IFN-γ, IL-1, IL-6 [48, 51], IL-12, IL-17, and IL-23, which modify the normal testicular environment [52]. These mechanisms induce inflammation with increased numbers of germ cells expressing TNFR1, IL-6R [52], and Fas/Fas-ligand system leading apoptosis of spermatocytes and spermatids [53, 54]. In addition, the mast cells release proteases in testicular intersticium and peritubular area and probably contribute to fibrosis [50]. Recent data have also demonstrated that activation of toll-like receptors identified in mouse Sertoli cells increases these pro-inflammatory cytokines [55].

The histopathology progression of orchitis can also be learned from studies in EAO [56] and in the early stages is characterized by peritubular and/or interstitial mixed cellular infiltrates [25]. Subsequently, there is mononuclear cells infiltration into the interstitium and sloughing of germinal cells begins to occur, with granuloma formation, necrosis, and complete absence of spermatogenesis [49]. EAO is a T-cell-mediated disease in which the inflammatory CD4 (Th1) T-cell mechanism is the critical pathway that targets the spermatogenic germ cells for autoimmune attack [49]. In spite of the knowledge of local, genetic, and immunological factors, the underlying mechanism of EAO is still not completely understood [25].

In human autoimmune orchitis, the typical pathological feature in testicles resembles those observed in EAO and is characterized by a severe vasculitis leading to inflammation and infarction of reproductive system. Acute and chronic inflammation [16] with lymphoplasmocytic infiltrate [42], fibrinoid necrosis of the vessels [40, 42], and seminiferous tubules [57], and periarteritis in varying amounts are present [39]. Infarction was observed in testicular lesions of PAN [42] and in rheumatoid arthritis [39].

Semen alterations are the direct consequence of this process, and there are few studies regarding the possible triggers at molecular levels. In this regard, there are some evidence suggesting a possible role of reactive oxygen species (ROS) in male infertility [58]. Excessive generation of ROS in the reproductive tract not only affects the fluidity of the sperm plasma membrane, but also the integrity of DNA in the sperm nucleus [59]. In addition, the extent of sperm DNA damage has been reported to be closely associated with impaired sperm function as well as male infertility [60]. Moreover, the degree of DNA damage seems to correlate with various fertility indices including fertilization rate, embryo cleavage rate, implantation rate, pregnancy rate, and live birth rate [61–63].

Importantly, the most consistent evidence for autoimmune orchitis is the presence of ASA [26]. They can be found in seminal fluid and plasma in men, and also in cervical mucus, oviductal fluid, or follicular fluid in women [25]. The presence of ASA can lead to the immobilization and/or agglutination of spermatozoa, which may significantly impair sperm motility affecting acrosome reaction, cervical mucus penetration, zona pellucida binding [26] and sperm–egg interaction [31]. Also ASA can prevent implantation and/or arrest embryo development. Interestingly, only ASA bound to sperm surface and acrosomal antigens are relevant for fertility [28], whereas the finding of iso-ASA in men and women's sera do not appear to have clinical implications [64]. These later antigens include PH-20, PH-30, FA-1, sperm agglutination antigen-1 (SAGA-1), and SP-10 [28]. In addition, males with ASA had more often HLA DR6 and DQ7 compared to controls [65].

Clinical Manifestations

The usual clinical manifestation of the primary autoimmune orchitis is isolated infertility [25] since most cases of orchitis is asymptomatic. The signs and symptoms of orchitis, when present, are often difficult to distinguish from those of other acute scrotal processes, mainly acute testicular torsion [25]. Generally, the scrotum is inflamed and swollen, and there may be localized testicular pain during palpation, which may be associate or not with fever. Mild thickness of the peritesticular membranes may be also evidenced in some patients [23].

An immunologic basis for some cases of infertility has been identified in a significant number of infertile men, suggesting that ASA may have a harmful effect in fertilization [66]. The term infertility is used to describe the situation where a couple does not succeed in achieving a spontaneous pregnancy in spite of attempting to conceive during at least 1 year of unprotected intercourse [67]. Immunologic infertility is characterized by the presence of antibodies against spermatozoa in the serum and/or in the seminal plasma or on the sperm surface. Secondary autoimmune orchitis is rare and is mainly associated with primary vasculitis such as PAN, Schönlein–Henoch's purpura and Behçet's disease [39].

ASA Detection

The ability of sperm to induce antibodies has been documented for almost 50 years [68]. Approximately 10% of men undergoing evaluation for infertility will have antisperm antibodies in serum, seminal plasma, or else, directly attached to sperm [69].

The current consensus is that antibodies bound to sperm within the semen are the most important. Therefore, assays for sperm-bound antibodies are regarded to be the most appropriate. In addition, only IgG and IgA classes are found in significant quantities within the reproductive tract and should be included into the ASA assay [70].

The diagnosis of antibody-mediated infertility lacks a pathognomonic clinical picture. However, there are some parameters that guide an ASA assay recommendation [71]:

  1. 1.

    Agglutination or clumping in the absence of clinical infection in semen analysis

  2. 2.

    Low sperm motility usually less than 30% of total motility

  3. 3.

    Poor penetration of mucus on sperm–cervical mucus contact testing

  4. 4.

    Diagnosis of unexplained infertility

The most useful method for ASA detection are those that evaluate antibodies on the surface of spermatozoa [28] such as the direct immunobead binding test that detects antibodies on the sperm cell surface to Fc portion of the ASA (sperm head, mid-piece, and/or tail) using immunobeads rabbit anti-human Ig (IgA, IgG, and IgM). At least 50% of the motile spermatozoa must be coated with immunobeads in order to validate the clinical significance of test. Negative control should have a score <10% bead attachment and positive control a score >20% bead attachment [72]. Alternatively, there is other more economical tests that may be used for screening [30] that employs sheep erythrocytes instead of immunobeads to detect and localize antibody-bound sperm. Additional methodologies to detect ASA in infertile males include postcoital test and the hemizona assay [28]. However, independent of the methodology used, the lower limit of detection of sperm antibodies is not known compromising the clinical relevance of these assays.

Imaging

Several different imaging methods can be used for the diagnosis of orchitis, such as color Doppler sonography and magnetic resonance imaging [8], and they are essential for the differential diagnosis with testicular torsion [73] and intrascrotal masses (abscesses, tumors, and hydroceles) [39].

Regarding autoimmune diseases, it has been observed that serial sections of affected testicles have uniform lesions, and therefore, a 3 × 3 × 3-mm biopsy should be sufficient to detect testicular manifestations [39]. Surgical complications of testicular biopsies are rare since minimal hematomas or infections are detected in less than 2% of this procedure [25, 39]. Furthermore, the risks for an immune response of clinical significance for fertility after biopsies are also low [39].

Treatment

Symptomatic patients with orchitis should receive analgesic treatment associated with local cooling, scrotal elevation, and bed rest [25]. Local anesthesia by a nerve block using 5 ml of 1% lidocaine around spermatic vessels may be necessary for those with severe tenderness; however, this procedure will not last longer [39].

In secondary autoimmune orchitis with or without testicular vasculitis, glucocorticoids [40], immunosuppressive agents (such as azathioprine and cyclophosphamide) [16, 37, 45, 57], and intravenous immunoglobulin [7] have been described as treatment options. Dapsone has been reported to be useful in Behçet's disease [74]. The impact of untreated testicular vasculitis on gonadal function and fertility is unknown; however, it seems to be relevant [25].

On the other hand, the treatment of infertility and the real significance of ASA are controversial, and there are no standardized treatment protocols. In uncontrolled studies of ASA-mediated infertility in males, oral glucocorticoids (commonly used to suppress antibody production) and non-steroidal anti-inflammatory drugs had been reported to have beneficial effects [23, 75]. The adverse events of glucocorticoids [30], however, particularly the risk of hip osteonecrosis, makes this option unattractive to most infertile couples [31]. One study described a significant increase in the number of pregnancies amongst those receiving prednisolone (20 mg twice daily on days 1–10 of the female partner's menstrual cycle, followed by 5 mg on days 11 and 12) for more than 3 months compared to placebo group [76]. Immunoinfertility treatment also includes other immunosuppressive agents, such as cyclosporine [77].

Sperm-washing technique can be used to remove bound antibody in assisted reproductive technique. Rapid dilutional washing of the seminal fluid may remove unbound antibodies; however, antibodies that are already bound will suffer little impact due [78].

Assisted reproductive technologies such as intrauterine insemination (IUI), in vitro fertilization (IVF), and ICSI are options therapy for male infertility due to the presence of ASA [28, 30, 77]. The conception rate per cycle was 3–10% by IUI with untreated semen [28]. A significant improvement was obtained using sperm sample collected in sterile medium from males with high levels of ASA (predominantly IgG antibodies). In fact, successful pregnancies were observed in 64% of couples after three IUI cycles, with 47% conception rate following the first cycle [79]. These benefits for immune infertility needs, however, to be defined in randomized prospective studies of IUI. When IVF is used to treat immune infertility in cases of male-factor infertility, early embryonic cleavage, the fertilization, and pregnancy rates was found to be lower than those observed for other indications, with overall pregnancy rates ranging from 14% to 35% per cycle [80]. However, in cases of female-derived ASA, IVF offers fertilization and pregnancy rates comparable to those associated with other diagnoses usually indicated for IVF procedure [81, 82].

ICSI seems to be a promising alternative for patients who failed IUI or IVF [28, 31]. This technique is also considered to be the treatment of choice for patients with severe sperm autoimmunity [2], particularly in males with low semen counts or motility [83]. Using ICSI, the numerical sperm requirement for egg fertilization is as low as one viable sperm for each retrieved oocyte [84]. This technology has the potential to overcome antibody-mediated infertility by bypassing the interaction between sperm and the zona pellucida as well as oocyte membrane [85]. The pregnancy success rates for ASA-positive infertile men were reported to be within the same range as population of ICSI patients with severely abnormal seminal parameters [86–88]. However, the same studies showed that embryo quality is poorer in ASA-positive than in ASA-negative ICSI cases, suggesting that ASA may affect post-fertilization events. Nonetheless, antisperm antibody testing has low sensitivity in predicting low fertilization and does not appear to be cost-effective when selectively ordered as part of an IVF workup in clinical settings at this time [89].

Future Directions

In spite of the major advances in understanding the underlying mechanism of autoimmune orchitis, there are at least a few areas of research progress that we expect in the near future. The first is a reliable, standardized testing protocol for antisperm antibodies, which will clarify the real contribution of this immunologic phenomenon in male infertility. The focus on genomic integrity, particularly the degree of DNA damage, and its possible association with autoimmune orchitis or antisperm antibodies has not previously evaluated and may be the missed link of translating molecular alterations from bench to bedside. Finally, research directed at a better identification of underlying mechanism of autoimmune orchitis will certainly provide a more precise target therapy for these patients.