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.

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.

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.

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.

[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.

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.

Focal orchitis in undescended testes: discussion of pathogenetic mechanisms of tubular atrophy.

OBJECTIVE: To evaluate seminiferous epithelium lesions in adult cryptorchid testes showing lymphoid infiltrates in seminiferous tubules and interstitium (i.e., focal orchitis). Also, to consider the possible role of this lesion in the etiology of tubular atrophy. METHODS: We performed a histopathologic study of the cryptorchid testes and adjacent epididymides removed from 50 adult men who had not been previously treated for cryptorchidism. The study included morphologic and semiquantitative evaluation of seminiferous tubule pathology (according to germ cell numbers), Sertoli cell morphology, tubular lumen dilation, rete testis pattern (normal, hypoplastic, or cystic), and epididymal pattern (normal or epididymal duct hypoplasia). The study also included immunohistochemical evaluation of immune cell markers. The results were compared with clinical and laboratory findings. RESULTS: Focal lymphoid infiltrates (mainly lymphocytes) in seminiferous tubules and interstitium were found in 22 patients (44%), all of whom had unilateral cryptorchidism. The course of orchitis was asymptomatic, and laboratory data were normal. According to the seminiferous tubule pathology, a variety of histopathologic diagnoses, were made: (1) mixed atrophy consisting of Sertoli cell-only tubules intermingled with tubules showing maturation arrest of spermatogonia (11 testes, 4 of which also showed hyalinized tubules); (2) Sertoli cell-only tubules plus hyalinized tubules (4 testes); (3) Sertoli cell-only tubules (3 testes); (4) intratubular germ cell neoplasia (2 testes, 1 of which also showed hyalinized tubules); (5) complete tubular hyalinization (1 testis); and (6) tubular hyalinization plus some groups of tubules with hypospermatogenesis (all germ cell types were present although in lower numbers, 1 testis). Dysgenetic Sertoli cells, that is, Sertoli cells that had undergone anomalous, incomplete maturation, were observed in all nonhyalinized seminiferous tubules with inflammatory infiltrates. Tubular ectasia was observed in 13 cases. The rete testis was hypoplastic and showed cystic transformation in 18 testes, and the epididymis was hypoplastic in 15 testes. CONCLUSIONS: The causes of these focal inflammatory infiltrates are unknown. It is possible that tubular ectasia and Sertoli cell dysgenesis are involved and that these alterations cause a disruption of the blood-testis barrier and allow antigens to enter the testicular interstitium, giving rise to an autoimmune process.

Absence of antisperm surface antibodies in prepubertal boys with cryptorchidism and other anomalies of the inguinoscrotal region before and after surgery.

PURPOSE: Although the prepubertal immune system cannot recognize postmeiotic germ cell antigens, an overall 21 to 28% incidence of antisperm antibodies directed at these antigens has been reported preoperatively in prepubertal children with cryptorchidism and other inguinoscrotal anomalies. We investigated the prevalence of antisperm antibodies in these prepubertal patients before and after surgery. MATERIALS AND METHODS: We examined 82 prepubertal boys 0.6 to 13.2 years old, including 33 with unilateral cryptorchidism, 21 with inguinoscrotal anomalies and 28 who were normal. IgG, IgM and IgA antisperm antibodies were determined by the indirect Immunobead test. Serum testing was repeated 1 and 2 years postoperatively and annually for 2 more years in the normal children. Also sera from 183 infertile men 21 to 47 years old with a history of cryptorchidism and/or inguinal hernia operated on in childhood were similarly studied. RESULTS: Of the adults 70 (39%) tested IgG positive, including 12 (7%) who were also IgA positive, and all tested IgM negative. Repeat measurements were negative for all IgG, IgA and IgM isotypes in all children, patients and controls. CONCLUSIONS: We conclude that there are no antibodies to sperm surface antigens in prepubertal children with cryptorchidism and inguinoscrotal anomalies before and within 2 years after surgery. Autoimmunity against postmeiotic sperm membrane antigens is apparent in adults only.

Fertility and unilateral undescended testis in the rat model II.

Continuing experimental work on the effect of experimentally created unilateral undescended testis (UL-UDT) in neonatal rats, this study examined the fertility and correlated it to contralateral (CL) testicular morphology, seminiferous tubular diameter (STD), DNA flowcytometry, and the presence of serum anti-sperm antibodies (ASA) at 120-135 days of age. In our previous reported work, the fertility of rats with UL-UDT at 65-80 days of age was the same as that of controls. In the present study the rats with UL-UDT had significantly reduced fertility (P < 0.01) compared to controls, even though the Johnsen scores and mean STD of the CL testicular tissue were comparable. DNA flowcytometry demonstrated a significant decrease (P < 0.001) in haploid cell population in the CL testicular tissue of rats with UL-UDT. Furthermore, the rats with UL-UDT who either received an immunosuppressive or in whom the UDT was excised early showed almost normal fertility and DNA histograms like those of controls. Significantly high titres of serum ASA were detected only in the group with UL-UDT when tested at 135 days of age. From these results, in combination with earlier results on similar work, it may be inferred that UL-UDT causes immunologically-mediated, progressive damage to the CL descended testis, leading to a decrease in fertility in rats.

Stage-specific localization of basigin, a member of the immunoglobulin superfamily, during mouse spermatogenesis.

Ablation of the transmembrane glycoprotein basigin leads to azoospermic mice, indicating that this gene is essential for spermatogenesis. To examine the functions of basigin in the testis, the precise localization of basigin during spermatogenesis was examined immunohistochemically. In the adult mouse testis, basigin immunoreactivity appeared on the cell surface of leptotene spermatocytes and gradually increased in intensity during the meiotic prophase. Cytoplasmic staining, as well as cell surface staining, was detected in spermatids. The most conspicuous reactivity was found in the spermatids at steps 9-11 and in the flagella of spermatids. Immuno-electron microscopic analysis demonstrated that basigin was localized not only on the plasma membranes of spermatocytes and spermatids, but also on the plasma membrane of the Sertoli cell processes which contact the spermatocytes and spermatids. Basigin immunoreactivity was also detected during postnatal development in spermatocytes and spermatids but not in spermatogonia. Experimental cryptorchid testes which contain only spermatogonia and Sertoli cells in the seminiferous epithelium showed no basigin immunoreactivity. Seven days after surgical reversal of the cryptorchid testis, spermatocytes reappeared in the tubules, along with basigin immunoreactivity. Furthermore, in sterile mutant mice, in which neither spermatocytes nor spermatids were generated, no basigin immunoreactivity was detected in the seminiferous tubules. These findings indicate that expression of basigin is concomitant with appearance of spermatocytes in the seminiferous tubule, and suggest that basigin is involved in the interaction between Sertoli cells and germ cells at specific stages of spermatogenesis.

Anticuerpos antiesperma en niños con criptorquidia / Antisperm antibodies in children with cryptorchidism

Se investigó mediante inmunofluorescencia la presencia de anticuerpos antiesperma en un grupo de 20 niños con edades de 2 a 14 años y antecedentes de criptorquidia. Se utilizó un grupo testigo de 20 niños sanos con edades similares. Los anticuerpos fueron positivos en cinco de los pacientes con antecedentes de criptorquidia y en dos del grupo testigo

Effects of testicular cytokines on proliferation of rat T-leukaemic lymphoblasts in vitro.

A rat acute lymphoblastic leukaemia (ALL) model was used to study the mechanisms involved in the tendency to testicular relapse of ALL in boys. Previous studies have indicated that the infiltration and growth of leukaemic lymphoblasts in the testis are influenced by the same endocrine and paracrine control systems that regulate normal testicular function. In the present study the effects of aqueous extracts of scrotal, abdominal and estrogen-treated postpubertal rat testes on rat-leukaemic lymphoblast proliferation were evaluated. The effects of recombinant cytokines analogous to those observed in the testis on leukaemic cell DNA-synthesis were also evaluated since changes in the levels of these factors have been observed in association with cryptorchidism and low levels of gonadotropins. Transforming growth factor-beta 1 (TGF-beta1), significantly inhibited the proliferation of leukaemic rat lymphoblasts after 24 h of culture, whereas TGF-beta 2, interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6 or combinations of them were inactive. Extracts of estrogen-treated testes and abdominal testes of unilaterally cryptorchid animals inhibited leukaemic T-cell proliferation significantly more than extracts of normal testes. The inhibitory activity in abdominal testes could be neutralized by anti-TGF-beta 1 antibodies. These results suggest that testicular TGF-beta 1 may influence growth of leukaemic lymphoblasts in the testis but also that other as yet unknown, testicular factors are involved in the regulation of leukaemic cell function in the testis.

[Early or late orchidopexy? An evaluation of germ cell proliferation by PCNA immune expression]. / Orquidopexia precoz o tardía? Valoración de la proliferación de las células germinales mediante inmunoexpresión de PCNA.

A immunocytochemical study for detection of proliferating cell nuclear antigen (PCNA) in order to quantify the number of PCNA-positive spermatogonia, and cytophotometric determination of spermatogonial DNA were performed in cryptorchid and control testes. The number of PCNA-positive spermatogonia, and the average DNA content of spermatogonia in the cryptorchid testes were altered from first years of age. These precocious spermatogonial alterations suggest that the early surgical testicular descent doesn't prevent lesions of germ cells.

Role of transforming growth factor beta in testicular immunosuppression.

The potential role of transforming growth factor beta (TGF beta) in the regulation of the immunological milieu of the testis was investigated. Antibodies neutralizing TGF beta reversed the previously observed suppression of rat peripheral blood lymphocyte proliferation induced by rat abdominal testis extract. Recombinant TGF beta 1 dose-dependently inhibited testicular interleukin-1-like factor-driven proliferation of murine thymocytes and ConA-stimulated rat peripheral blood mononuclear cells. Extracts of seminiferous tubules contained a M(r) approximately 25 K TGF beta-like growth inhibitor of the CLL-64 mink lung epithelial cell line. The present findings suggest an important role for TGF beta in testicular immunosuppression.

Antisperm antibodies in cryptorchid boys.

To explore whether the development of antisperm antibodies might play a role in inhibiting the future fertility of cryptorchid boys, 32 boys who underwent orchiopexy and 21 boys who underwent other inguinal surgeries were examined for the presence of antibodies to donor sperm. This group was compared to 23 boys who underwent noninguinal surgery or who had unrelated medical conditions. Using the indirect immunobead assay, 28% of the boys in the study group were found to have antisperm antibodies. These were predominantly of the IgG isotype. In the comparison group, only 1 boy (4%) had antisperm antibodies (P < 0.05).