Cytokines in the male reproductive tract and their role in infertility disorders.

Cell integration between the immune and reproductive systems is the basis for normal male reproductive physiology. Cytokines are a part of the autocrine/paracrine network operating in the male reproductive tract. At the same time, immunological reactions occurring via cytokines appear to be both beneficial and/or risk factors for male fertility. As the cytokines are produced by a whole spectrum of cells in all compartments of the male genital tract, they can also be involved in a variety of andrological disorders. The monitoring of cytokines and other immune factors in seminal plasma may offer a chance to better understand the mechanisms leading to sub-/infertility. In this review, we present insights into cytokine interplay in some of the pathological conditions associated with male reproduction.

Malondialdehyde and CA II autoantibody levels are elevated in children with undescended testes.

PURPOSE: In the pathogenesis of sub-fertility/infertility and testicular cancer related to undescended testes, oxidative stress, inflammation and autoimmunity are important factors. Therefore, the present study was designed to determine serum oxidative stress markers and carbonic anhydrase (CA) II autoantibodies in boys with undescended testes (UDT), and to investigate the relationship between these parameters. METHODS: Serum CA II autoantibody titers, malondialdehyde (MDA), ischemia modified albumin (IMA), protein carbonyl content and soluble CD40 ligand (sCD40L) levels were measured in 59 boys with UDT and 30 healthy subjects. RESULTS: MDA levels were significantly higher in the UDT group compared with the control group (p = 0.003). There was no significant difference between serum IMA, sCD40L or protein carbonyl levels. CA II autoantibody titers in the UDT group were significantly higher compared with those of the control group (p = 0.048). A weak positive correlation was determined between anti-CA II antibody titers and MDA and IMA levels (p = 0.041, p = 0.005, respectively). CONCLUSIONS: MDA is the most reliable and decisive biochemical marker displaying oxidative damage in undescended testes, and an autoimmune response may be triggered by oxidative stress against CA II during the UDT process.

Cryptorchidism is not a risk factor for antisperm antibody production in post-orchidopexy males with infertility.

INTRODUCTION: Infertility in adulthood is a well-recognized consequence of cryptorchidism, even after successful orchidopexy. Autoimmune reactions against spermatozoa are associated with infertility and often found in cryptorchids. The purposes of this study were to evaluate the linkage between antisperm antibody (ASA) and cryptorchidism, and furthermore, to clarify whether ASA is involved in cryptorchidism-associated infertility. MATERIALS AND METHODS: We investigated a total of 48 infertile males with a history of unilateral (n = 30) or bilateral (n = 18) cryptorchidism who had undergone successful orchidopexy in prepuberty, and 20 age-matched fertile and healthy males were collected as controls. ASA in sperm samples was detected by the direct immunobead test, and semen analysis was performed concomitantly. RESULTS: No infertile case satisfied the diagnostic criteria of ASA-mediated infertility set forth by the World Health Organization. Decreases in both sperm concentration and motility accompanied by increases in abnormal morphology were seen in infertile cryptorchids when compared with the healthy controls. CONCLUSIONS: Testicular heat exposure in prepuberty is not a risk factor for ASA production. It is evident that the mechanisms that underlie cryptorchidism-associated infertility do not involve ASA. Poor sperm characteristics in cryptorchids resulting from thermal damage to the testes seem to be responsible for their infertility, even after successful orchidopexy.

Mannan-binding lectin-associated serine protease (MASP)-1 is crucial for lectin pathway activation in human serum, whereas neither MASP-1 nor MASP-3 is required for alternative pathway function.

The lectin pathway of complement is an important component of innate immunity. Its activation has been thought to occur via recognition of pathogens by mannan-binding lectin (MBL) or ficolins in complex with MBL-associated serine protease (MASP)-2, followed by MASP-2 autoactivation and cleavage of C4 and C2 generating the C3 convertase. MASP-1 and MASP-3 are related proteases found in similar complexes. MASP-1 has been shown to aid MASP-2 convertase generation by auxiliary C2 cleavage. In mice, MASP-1 and MASP-3 have been reported to be central also to alternative pathway function through activation of profactor D and factor B. In this study, we present functional studies based on a patient harboring a nonsense mutation in the common part of the MASP1 gene and hence deficient in both MASP-1 and MASP-3. Surprisingly, we find that the alternative pathway in this patient functions normally, and is unaffected by reconstitution with MASP-1 and MASP-3. Conversely, we find that the patient has a nonfunctional lectin pathway, which can be restored by MASP-1, implying that this component is crucial for complement activation. We show that, although MASP-2 is able to autoactivate under artificial conditions, MASP-1 dramatically increases lectin pathway activity at physiological conditions through direct activation of MASP-2. We further demonstrate that MASP-1 and MASP-2 can associate in the same MBL complex, and that such cocomplexes are found in serum, providing a scenario for transactivation of MASP-2. Hence, in functional terms, it appears that MASP-1 and MASP-2 act in a manner analogous to that of C1r and C1s of the classical pathway.

Oxidative, inflammatory and immunologic status in children with undescended testes.

BACKGROUND: In order to better understand the pathogenesis of risk of future sub-/infertility in children with undescended testes (UDT), we designed this prospective study to examine the oxidative stress, inflammatory response and autoimmunity in children with UDT. We examined the concentrations of malondialdehyde (MDA), interleukin-6 (IL-6) and antisperm antibodies (ASA) in children with UDT and healthy controls. METHODS: The UDT group consisted of 88 boys (aged 1-14 years, unilateral in 67 and bilateral in 21 cases), and 44 boys with normal descended testes served as a control group. Clinical evaluation revealed no testicular or other system abnormalities. MDA was used as lipid peroxidation index. IL-6 levels were measured using a commercial enzyme-linked immunosorbent assay kit. ASA was determined with an anti-human spermatozoa immunoglobulin G test. RESULTS: Mean age values ± SD were 4.6 ± 3.2 in the UDT group and 4.7 ± 3.4 in the control group (P= 0.872). MDA and IL-6 results for the UDT and control groups were significantly different (P= 0.003 and P= 0.019, respectively), but those for ASA were not (P= 0.473). The mean MDA and IL-6 values were significantly higher in bilateral cases than the respective values in the unilateral cases (MDA: 4.03 ± 3.68 vs 3.49 ± 5.22, P= 0.015; IL-6: 7.70 ± 6.86 vs 3.48 ± 6.50, P= 0.001) (P= 0.015). CONCLUSION: The results indicate that children with UDT are exposed to high levels of oxidative stress and inflammatory reaction. This could negatively affect the future fertility in these children.

Weak association of anti-sperm antibodies and strong association of familial cryptorchidism/infertility with HLA-DRB1 polymorphisms in prepubertal Ukrainian boys.

BACKGROUND: Cryptorchidism is a frequent syndrome occurring in 1-2% of males within the first year of age. Autoimmune reactions, particularly directed to testicular elements and/or spermatozoa have been found to be often associated with cryptorchidism. Therefore we investigated in this study the frequency of HLA class II alleles in order to recognize possible genetic predisposition for antisperm antibodies development in prepubertal boys with diagnosed cryptorchidism in Caucasoid population. METHODS: Sixty prepubertal boys with cryptorchidism and sixty healthy boys were examined for anti-sperm antibodies by indirect immunobead test as well as for their HLA-DRB1 and -DQB1 alleles using DNA obtained from peripheral blood leukocytes. The typing of HLA-DRB1 and -DQB1 was performed by using PCR-SSP low resolution method. RESULTS: Allele frequencies of HLA-DRB1 and HLA-DQB1 did not differ between boys with cryptorchidism and control boys. However, weakly significant differences in DRB1*04 (p corrected=0.0475) and DQB1*06 (p corrected=0.0385) were seen between cryptorchid patients with and without AsA, but none of these two patient groups differed significantly in HLA class II frequencies from controls except for AsA-negatives and HLA-DQB1*06 (p corrected=0.0247). On the other hand, comparison of cryptorchid boys with familial cryptorchidism and/or infertility to control boys revealed highly significant (p corrected=0.0006) difference in HLA-DRB*11 frequency, whereas boys with sporadic cryptorchidism did not differ from control. A much weaker, but still significant difference in DRB*11 frequency was also observed between boys with bilateral cryptorchidism and controls (p corrected=0.037), whereas patients with unilateral cryptorchidism were not different from control in frequency of any HLA-DRB1 or -DQB1 allele tested. CONCLUSIONS: Predisposition to produce anti-sperm antibodies seems to be only weakly associated with HLA class II genes, although this question requires further study on much larger population sample. It is plausible that familial and sporadic cryptorchidism may present distinct genetic background. The same may, to lower extent, apply to bilateral and unilateral cryptorchidism.

Cryptorchidism and long-term consequences.

Cryptorchidism has been on the rise for several decades and can be observed with frequency of 1-2% of males within the first year of age. It may appear as an isolated disorder or can be a consequence of genetic and endocrine abnormalities connected with somatic anomalies. Its genetic background still seems to be unclear although a range of genes can be responsible for the development of this syndrome. Cryptorchidism can be associated with serum testosterone level although the often co-existing hypogonadotropic hypogonadism may also indicate the involvement of pituitary hormones. Recently, environmental factors have been blamed for cryptorchidism induction. Autoimmune reactions in conjunction with steroid hormones regulating immune response can be also partly responsible for cryptorchidism etiology. The appearance of antisperm antibodies can be considered as a marker or a serious side-effect of uncorrected cryptorchidism. If so, it could be implied that early surgery (orchidopexy) should be beneficial since it may prevent antisperm antibodies induction or at least eliminate them in the post-operative period.

Effect of testis nondescent or orchidopexy on antisperm antibodies and testis histology in rats.

OBJECTIVE: To examine effects of nondescent of normal testis and of various orchidopexy techniques on antisperm antibody (ASA) production and histologic testicular lesions. DESIGN: Experimental cohort study. SETTING: Laboratories of surgical research and biology of reproduction, academic medical centers. PATIENT(S): Lewis rats, immature and adult. INTERVENTION(S): Eighteen-day-old rats (6 groups): intra-abdominal stay of testis after closure of inguinal canal, classic dartos pouch orchidopexy, orchidopexy by testis fixation through tunica albuginea, orchidopexy by transparenchymal testicular fixation, sham operation, and bilateral vasectomy. Adult rats (1 group): transparenchymal testicular fixation. MAIN OUTCOME MEASURE(S): The ASA--antiacrosome and antitail--were measured by indirect immunofluorescence in sera collected preoperatively, on 50th and 120th day in immature rats, and 90 days after surgery in adult rats. Testicular histology was also examined at the end of sera collection. RESULT(S): Neither intra-abdominal testicular localization nor orchidopexies induced significant ASA. Testicular nondescent and fixation (transparenchymal or transtunical) caused hypospermatogenesis; dartos pouch was harmless. Bilateral vasectomy produced significantly increased ASA, but no significant testicular lesions. Contralateral testes were unaffected. CONCLUSION(S): Intra-abdominal testicular stay and orchidopexy do not elicit autoimmune response to sperm; histologic testicular lesions might not be associated with ASA. In operated cryptorchids, ASA are probably due to other reason than testicular heat or orchidopexy trauma.

[Testicular perfusion injury. Cytokines and cell adhesion molecules in humans]. / Testikuläre Ischämie. Verlauf von Zytokinen und Zelladhäsionsmolekülen beim Menschen.

BACKGROUND: Ischemia and reperfusion (I/R) lead to cellular damage. A disturbance of testicular perfusion occurs during the therapy of cryptorchidism and in cases of testicular torsion. This results in the activation of mediator cells with an increasing synthesis of mediators of infection like TNF-alpha and the expression of cell adhesion molecules like ICAM (intercellular adhesion molecule) and VCAM (vascular cell adhesion molecule) at the cellular surface. METHODS: The expression of the cytokines IL-10 and TNF-alpha and the adhesion molecules ICAM and VCAM after defined testicular I/R injury in nine male transsexuals was evaluated with rt-PCR. Furthermore we examined lactate and the diameter of the testicular tubulus under ischemic conditions. RESULTS: During ischemia ICAM, IL-10, and VCAM do not show significant changes on the side of testicular ischemia and the contralateral side; the same was seen for the tubulus diameter. TNF-alpha and the testicular lactate values showed a significant change of the expression pattern. DISCUSSION: The statistical changes of TNF-alpha and testicular lactate are the expression of leukocyte migration, infectious reaction, and immune response. To what extent the TNF-alpha expression represents a severe immunological reaction remains undefined. This human study shows primary results for the immunological understanding of and cellular response to testicular ischemia.

Antisperm antibodies in prepubertal boys with cryptorchidism.

The presence of antisperm antibodies in male individuals before puberty is controversial due to the lack of finally differentiated male germ cells. It was questioned whether the pathologic conditions of the male gonad may influence antisperm antibody formation in individuals before puberty. Sera samples of 76 individuals and 10 healthy boys with testicular failure (mainly uni- or bilateral cryptorchidism) were examined by means of indirect immunobead-binding test (IDIBT). The presence of antisperm antibodies was found in 3.95% of the studied subjects. Antibodies recognizing antigenic determinants present on the surface of mature sperm cells may be produced before puberty in individuals suffering from cryptorchidism or the other gonadal disorders. Antisperm antibodies that did develop in a minority of the studied male population may be proof for individual predispositions to autoimmune reactions.

Increased IL-18 levels in seminal plasma of infertile men with genital tract infections.

PROBLEM: Interleukin (IL)-18 is a novel cytokine, previously known as interferon (IFN)-gamma inducing factor. We evaluated the levels of IL-18 and IFN-gamma in seminal plasma (SP) of fertile and infertile men. METHOD OF STUDY: Semen samples were obtained by masturbation from 80 men, and were examined for the levels of IL-18 and IFN-gamma by enzyme-linked immunosorbent assay. Seven groups were included: (i) fertile men (n = 18), (i) infertile men with genital tract infections (n = 17), (iii) with varicocele (n = 15), (iv) with Klinefelter syndrome (n = 6), (v) with cryptorchidism (n = 7), (vi) with mumps orchitis (n = 7), and (vii) with idiopathic testicular lesions (n = 10). RESULTS: Mean levels of IL-18 were higher in SP from infertile men with genital tract infections compared with SP from other groups except Klinefelter syndrome (P < 0.05). However, no significant differences could be detected for IFN-gamma. A significant positive correlations was found between IL-18 and IFN-gamma in total patient population (P < 0.001). Moreover, a negative correlation was observed between IL-18 and sperm concentrations, and motility (P < 0.01 and < 0.03, respectively). Furthermore, there was a positive and statistically significant association between IL-18 and IFN-gamma levels in SP of infertile men with genital tract infections (P < 0.0001). However, there was no relationship between IL-18 and IFN-gamma, and semen parameters in the same group. CONCLUSION: SP IL-18 levels were increased in men with urogenital infections. Thus, the elevated expression of IL-18 in SP may be used as a diagnostic marker in the male genital tract infections.

Identification and characterization of an antigen recognized by monoclonal antibody TRA 54 in mouse epididymis and vas deferens.

Spermatozoa in testicular fluid are known to have weak forward motility and cannot fertilize eggs. The epididymis is known to participate in sperm maturation leading fertilization, but little is known about the specific epididymal molecules involved in the modification of sperm. In this study, we characterized the new pattern of expression of an antigen previously identified in testicular germ cells by monoclonal antibody (mAb) TRA 54. This antigen is expressed in epididymal and vas deferens epithelial cells in mice older than 24 days but not during younger developmental stages. Evaluation by immunohistochemistry shows that antigen expression is limited to the cytoplasm of a specific cell population of epithelia along the epididymal regions and vas deferens of adult mice. The molecules synthesized and released by epididymal and vas deferens epithelia into their lumen seem to bind on spermatozoa moving down through the ducts. Immunoblot analysis showed that the molecules recognized by mAb TRA 54 in testis and epididymis were similar and share a common epitope involving carbohydrate domains. Interestingly, the antigens identified in epididymal and vas deferens epithelial cells were expressed independently of testicular germ cells and are produced in an androgen-dependent manner. Finally, the molecules recognized by mAb TRA 54 seem to play an important role in spermatogenesis, as well as in epididymal function related to spermatozoa maturation and ability to fertilize.

Median raphe cyst in the scrotum, mimicking a serous borderline tumor, associated with cryptorchidism after orchiopecxy.

Median raphe cyst (MRC) is a benign lesion occurring predominantly in the ventral surface of the penises of young men and is an embryological developmental anomaly of the male genitalia. Serous borderline tumors (SBT) are found most frequently in the female ovary and only several cases with SBT of the male genitalia have been reported. We describe a case of MRC with features of SBT, which appeared in the scrotum of a 9-year-old boy after orchiopexy and was associated with surgery for cryptorchidism. The cyst arose on the right testicular tunica and consisted of cystic components with intracystic papillae lined by stratified epithelial cells, some of which showed mild cytological atypia and sporadic mitosis. These epithelial cells expressed CA 125, CA 19-9, carcinoembryonic antigen, estrogen receptor and progesterone receptor. Although no cases of MRC with characteristics of SBT in association with the rete testis has been described, the current report gives additional information for follow-up of cryptorchidism.

Puberty does not induce serum antisperm surface antibodies in patients with previously operated cryptorchidism.

PURPOSE: We investigated serum antisperm surface antibody (ASA) prevalence at puberty, which is reported to be as high as 38% in the sera of males with cryptorchidism operated on before puberty. Operative technique impact, dartos pouch orchiopexy or testis fixation, was also examined. MATERIALS AND METHODS: We examined a total of 61 pubertal males (Tanner stage 2 or greater) divided into 3 groups. Group 1 consisted of 24 males with cryptorchidism 10 to 17.9 years old who underwent unilateral dartos pouch orchiopexy before puberty (median age 5.85). All of these cases were known to be negative for ASA preoperatively, and 20 before puberty. Group 2 consisted of 22 males with cryptorchidism 12.1 to 17.7 years old operated on previously (median age 10.35) by testicular fixation among other techniques. Group 3 consisted of 15 healthy males 12.2 to 17.3 years old. Prepubertal ASA status was unknown for groups 2 and 3. Operated testis was compared with counterpart before serum collection in group 1 and during operation in group 2. IgG IgM and IgA ASA were studied by the indirect Immunobead (BioRad, Clinisciences S.A., Montrouge, France) test. RESULTS: All sera tested were found negative in the 3 groups. Dartos pouch operation, testis fixation or even consecutive operations did not induce ASA production. Alterations in size or consistency were observed in operated testes in 10 patients in group 1, and in 8 patients in group 2. CONCLUSIONS: Our results suggest that dartos pouch orchiopexy, testicular fixation and/or intrinsic developmental alterations of the cryptorchid testis does not elicit an autoimmune response against sperm surface antigens at puberty.

[Expression of FasL in rat cryptorchidism].

OBJECTIVE: To investigate the expression of FasL in rat cryptorchidism and its significance. METHODS: Twenty-four male SD rats (22-day old) were randomly divided into two groups: unilateral cryptorchid group (n = 12) and pseudo-operation group (n = 12). When the rats were 110-day old, blood samples were taken and the rats were killed for analysis. Immunohistochemical method (SP) was used to detect FasL expression in testes and ELISA method to detect serum antisperm antibody (AsAb). RESULTS: The positive FasL expression rates in cryptorchid and contralateral testes were significantly higher than those in pseudo-operation group (P < 0.001). The serum AsAb positive rates in the cryptorchid group and the pseudo-operation group were 41.7% and 0, respectively, with significant difference(P < 0.01). CONCLUSIONS: FasL expression upregulating in both testes of the unilateral cryptorchid rat may be a protective response of the testis to autoimmunity.

Valoración de las lesiones en el testículo criptorquídico: estudio anatomopatológico y ultrastructural / Assessment of the lesions in the cryptorchid testis: pathological and ultrastructural studies

Numerosos estudios han llegado a la conclusión de que el testículo criptorquídico es anormal con alteración de la espermatogénesis primaria. El mecanismo que produce las lesiones está sujeto a controversia. Se realizó biopsia en 20 pacientes con criptorquidia unilateral tomada durante el descenso testicular, y se llevó a cabo estudio ultrastructural con microscopia electrónica, valorando las lesiones que ocurren en el testículo no descendido. La degeneración tubular y celular fue focalizada dentro de un mismo testículo y túbulo, respectivamente. Las lesiones degenerativas fueron, fundamentalmente, vacuolización (índice de degeneración metabólica), edema y apoptosis celular o muerte celular programada. Esta fue la lesión más constante y llamativa. Al participar la apoptosis en procesos mediados inmunológicamente, hizo que nos planteáramos si la lesión en el testículo criptorquídico está mediada por un mecanismo inmune (AU)

IL-10 is highly expressed in the cryptorchid cryptepididymal epithelium: a probable mechanism preventing immune responses against autoantigenic spermatozoa in the epididymal tubule.

The expression of several immunoregulatory adhesion proteins and cytokines was studied in the normal epididymis, cryptorchid cryptepididymis, the epididymis of oestrogen-treated mice and the epididymis of non-obese diabetic (NOD) mice at the protein level to see which of these immunoregulatory proteins may be involved in lymphocyte regulation in the normal or pathological epididymis and if cytokine balance in this organ is on the cellular or humoral side. The aim of the study was to characterize the immunological microenvironment of the epididymis to explain the survival of the autoantigenic spermatozoa in this site. In the 6-week-old BALB/c or NOD mouse epididymis there were some CD18 and CD44 expressing cells in the interstitial tissue. There were no differences between these strains in the expression of the studied antigens, except that some CD4 positive cells were present in the interstitial tissue of BALB/c mice. In the cryptorchid cryptepididymis CD4, CD8, CD18, CD44, CD54 and CD106 expressing cells were occasionally present in the connective tissue surrounding the epididymal tubule. In the epididymis of the oestrogen-treated mice these antigens were not expressed. In the cryptorchid cryptepididymis the epithelial cells expressed IL-10 highly and the myoid peritubular cells IL-6. The present results suggest that the epididymal epithelial IL-10 suppressing TH0, TH1 and TH2 immune responses may be involved in the protection of autoantigenic spermatozoa from immune destruction.

Cytotoxic phenotype of intra-epithelial lymphocytes in normal and cryptorchid human testicular excurrent ducts.

BACKGROUND: Most testicular and epididymal lymphocytes express T-cell markers, but their cytotoxic potential and activation status have not been reported. In this study, distribution of the cytotoxic cells was compared between normal and cryptorchid testes stratified into two groups: the first with complete absence of germ cells [Sertoli cell-only (SCO)] and the second with arrested spermatogenesis (SCA). METHODS: Immunohistochemistry for the T-lymphocyte marker CD3 and cytotoxic markers CD8, TIA-1 and granzyme B was performed on paraffin-embedded sections. RESULTS: The number of CD8+ and CD3+ intra-epithelial lymphocytes (IELs) increased distally throughout the normal epididymis. TIA-1 immunostaining revealed that a significant proportion of IELs exhibited cytotoxic potential, whereas granzyme B staining disclosed a subpopulation of activated cytotoxic lymphocytes (CTLs). TIA-1/CD8 and granzyme B/CD8 double immunostaining revealed that the vast majority of TIA-1+ and granzyme B+ cells were CD8+. The proportion of activated granzyme B+ lymphocytes increased distally throughout the normal epididymis. The number of TIA-1+ and granzyme B+ intra-epithelial and stromal lymphocytes was significantly increased in the normal as opposed to the SCO cryptorchid epididymis and proximal vas deferens. CONCLUSIONS: These results suggest that exposure of the testicular excurrent ducts to spermatozoa or immature germ cells triggers the activation and recruitment of CTLs. Cytotoxic granule effector mechanisms may contribute to the immunological barrier preventing the immune response to spermatozoa in testicular ducts.