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Testicular tumors as a possible cause of antisperm autoimmune response
Testicular tumors as a possible cause of antisperm autoimmune response Donatella Paoli, B.Sc., Ph.D., Barbara Gilio, M.D., Ph.D., Emanuela Piroli, M.D., Mariagrazia Gallo, B.Sc., Francesco Lombardo, M.D., Ph.D., Franco Dondero, M.D., Andrea Lenzi, M.D., and Loredana Gandini, B.Sc. Laboratory of Seminology-Sperm Bank, Department of Medical Pathophysiology, University of Rome ‘‘La Sapienza,’’ Rome, Italy
Objective: To evaluate the presence of antisperm antibodies in testicular cancer patients 1 month after orchiectomy and before radiotherapy or chemotherapy. Design: Clinical study. Setting: Department of andrology and seminology at a university hospital. Patient(s): One hundred ninety patients with testicular cancer. Intervention(s): Determination of semen parameters and autoimmune reaction evaluated on the sperm surface and in blood serum. Main Outcome Measure(s): Autoimmune reaction on the sperm surface by the direct immunobead test (IBT), and in blood serum by the indirect IBT and the gelatin agglutination test (GAT), was evaluated 1 month after orchiectomy and before beginning chemotherapy or radiotherapy. Result(s): Of the 190 patients, 11 (5.8%) were positive for antisperm antibody by GAT. On indirect IBT, 3 of the 11 GAT-positive patients were positive to IgG class only, with values of 22%, 24%, and 40%. Of the 11 GAT-positive patients, 4 showed no antibody bound to the sperm surface, and 3 were positive to IgG class only (28%, 21%, and 38%), with binding exclusively on the tail. Direct IBT could not be performed in the remaining 4 patients. Conclusion(s): Our data support the hypothesis that testicular cancer might not be a possible cause of antisperm autoimmunization and infertility. (Fertil Steril 2009;91:414–9. 2009 by American Society for Reproductive Medicine.) Key Words: Testicular cancer, ASA, infertility, semen analysis
Antisperm antibodies (ASA) are found in approximately 10% of infertile men (1, 2). Knowledge of the antigen systems involved in antisperm immune reactions is not yet well established. Various theories have been proposed to explain the sperm cell’s protection within the seminiferous tubules, because from an antigenic point of view it is partially foreign to the organism producing it. The biological, anatomical, and functional mechanisms underlying the prevention of the antisperm immune response take place in the testis. A number of andrologic diseases can affect the blood–testis barrier, inducing antisperm autoimmunization. There may be numerous triggers: inflammation, trauma, torsion of the spermatic cord, complete or incomplete obstructions, and testicular neoplasia. Testicular cancer constitutes approximately 1% of all cancers in men but is the most frequent cancer in men aged 15 to 39 years. Advances in chemotherapy and radiotherapy in combination with surgical techniques over the last 10 years mean that 90% of patients can now be cured.
Received August 3, 2007; revised and accepted November 27, 2007. Supported by a grant from the Italian Ministry of Education and Research (MIUR-COFIN) and the University of Rome ‘‘La Sapienza.’’ Reprint requests: Loredana Gandini, B.Sc., Laboratory of SeminologySperm Bank, Department of Medical Pathophysiology, University of Rome ‘‘La Sapienza,’’ Viale del Policlinico 155, Rome 00161, Italy (FAX: þ39-06-4997-0717; E-mail: [email protected]).
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Germ-cell tumors originate from the germinal epithelium of the seminiferous tubules and have various histologic types (seminoma, embryonal carcinoma, choriocarcinoma, teratoma, and mixed tumors, consisting of more than one of these histotypes), with differing degrees of invasiveness and a high level of metastasis. Because testicular cancer directly affects the gamete production site, it has been postulated as a trigger for an autoimmune response. The presence of ASA may in fact be explained by local effects caused by the cancer, such as raised scrotal temperature connected with blood flow alterations or disruption of the blood–testis barrier, with a massive release of sperm antigens that stimulate antisperm immunization. For this reason, some investigators (3–5) have considered autoimmunity to be closely correlated with testicular cancer. The aim of our study was to evaluate the presence of antisperm antibodies in testicular cancer patients after orchiectomy and before radiotherapy or chemotherapy.
MATERIALS AND METHODS Patients The study was approved by the institutional review board of our university hospital. We studied 190 consecutive patients with testicular cancer, with a mean (SD) age of 27.9 5.4
Fertility and Sterility Vol. 91, No. 2, February 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2007.11.084
years, who cryobanked sperm between 2001 and 2005 at the Seminology Laboratory-Sperm Bank, Department of Medical Pathophysiology, University of Rome ‘‘La Sapienza’’ before beginning chemotherapy or radiotherapy. All patients were evaluated approximately 1 month after orchiectomy, to give them the necessary time for postoperative physical recovery. Patients were grouped according to their histotype— seminoma, embryonal carcinoma, or mixed tumor (variable combination of seminoma, teratocarcinoma, choriocarcinoma, and yolk sac tumor)—and clinical stage (pT1, pT2, pT3, pT4). They were also classified on the basis of normal testicular descent into the scrotal sac at birth. Seminal fluid examination and ASA detection were performed for all patients. We compared the ASA results for testicular cancer patients with data from a control group of 580 consecutive patients diagnosed with infertility from 2003 to 2005 and a second control group of 60 fertile subjects. Both control groups had a mean age similar to that of the study group.
Seminal Fluid Examination Semen samples were collected by masturbation after 2–7 days’ abstinence. All samples were allowed to liquefy at 37 C for 60 minutes and were then assessed according to World Health Organization (WHO) guidelines (6, 7). The following variables were taken into consideration: ejaculate volume, sperm concentration, total sperm count, forward motility, and morphology (percentage of abnormal forms). Because of the urgent need for patients to begin cancer therapy, semen analysis was performed on only one sample for each patient. However, the examination was performed twice for azoospermia cases, and diagnosis was made only after having carefully evaluated the pellet after centrifugation. The pellet was also stained (May-Gr€ unwald-Giemsa) to evaluate the presence of germline cells. All semen samples were examined by the same biologist (L.G.).
ASA Detection Autoimmune reaction was evaluated on the sperm surface by the direct immunobead test (IBT) (8–11) (Irvine Scientific, Santa Ana, CA), and in blood serum by the indirect IBT and the gelatin agglutination test (GAT) (12). For direct and indirect IBT, samples showing binding of >20% were considered positive. A binding percentage of >50% was considered clinically significant. In GAT, an antibody titre of 1:32 or more in blood serum was considered clinically significant. All indirect tests (GAT and indirect IBT) were performed twice with different antigens. Indirect tests can be carried out in blood serum and seminal plasma. In our study, ASA were evaluated in blood serum only because the semen sample was used for sperm cryopreservation. Direct tests were performed only for cases whose semen parameters enabled the test to be performed with small aliquots of seminal fluid. Fertility and Sterility
Statistical Analysis Means and SDs were computed for interval variables; for nominal variables, frequencies and percentages are reported. Prevalence of ASA positivity in the three groups (testicular cancer patients, infertile patients, and fertile subjects) were compared by odds ratio and chi-square test. RESULTS The 190 testicular cancer patients were divided into three groups by histotype: seminoma (n ¼ 113), embryonal carcinoma (n ¼ 38), and mixed tumor (n ¼ 39). Clinical staging was available for 146 of 190 patients only: stage I: 99 patients; stage II: 42; stage III: 5. Data documenting normal testicular descent into the scrotum at the time of birth were available for 171 of 190 patients only: 149 of 171 patients (87.1%) were normal. Eighteen patients (10.5%) had unilateral cryptorchidism: 9 on the right, of whom 1 had contralateral testicular cancer, and 9 on the left, of whom 1 had contralateral testicular cancer. Four patients had retractile testis (2 in the right and 2 in the left testis); all 4 had testicular cancer in the retractile testis. Of 18 patients with cryptorchidism, 12 were affected by seminoma, 4 by embryonal carcinoma, and 2 by mixed tumor. Means and SDs for age and semen parameters (volume, sperm concentration, total sperm count, percentage forward motility, and percentage abnormal forms) for the three groups are reported in Table 1. In the seminoma group, 7 patients were azoospermic at the time of the semen analysis and 1 was cryptozoospermic. In the embryonal carcinoma group, only 1 patient was azoospermic. These patients were excluded from the statistical analysis, and therefore semen data refer to 181 patients. GAT and Indirect IBT The presence of ASA in blood serum by GAT and indirect IBT was evaluated in testicular cancer patients. Of the 190 patients, 11 (5.8%) were found positive for ASA by GAT, of whom 7 were affected by seminoma, 3 by embryonal carcinoma, and 1 by mixed tumor, as shown in Table 2. Of the 11 ASA-positive patients, 10 presented with clinical stage I, and 1 presented with stage II. Five patients had an antibody titre of 1:4, 3 of 1:16, and 3 of 1:32 (Table 3); thus only 3 of these 190 patients (1.6%) presented with clinically significant positivity (1:32). On indirect IBT, 3 of the 11 GAT-positive patients were found positive to IgG class only, with values of 22%, 24%, and 40% (Table 3). There was no clinically significant positivity. Blood serum ASA was evaluated by GAT in both control groups. Of the 580 infertile patients, 64 (11.0%) were positive for ASA, of whom 19 had an antibody titre of 1:4–1:16 and 45 had an antibody titre of R1:32 (range, 1:32–1:512). Thus 7.7% of infertile patients (45 of 580) presented with clinically 415
TABLE 1 Semen parameters by testicular cancer histotype.
Histotype Seminoma Embryonal carcinoma Mixed tumor Testicular cancer
Patients (n)
Age (y)
Volume (mL)
Sperm concentration (3106/mL)
Total sperm count (3106)
Forward Abnormal motility (%) forms (%) 35.2 16.7 69.7 12.2 31.1 17.4 71.1 12.3
105 37
29.5 5.48 3.8 1.8 25.3 4.91 2.8 1.2
36.8 37.0 26.5 24.6
124.9 136.1 72.0 77.4
39 181
26.2 4.42 3.7 2.1 27.9 5.5 3.6 1.8
35.2 33.2 34.3 34.1
116.2 138.4 35.5 17.2 69.8 13.0 112.2 128.0 34.4 16.9 70.0 12.4
Note: Values are mean SD. Paoli. Testicular tumors and ASA. Fertil Steril 2009.
significant positivity. Only 2 of the 60 fertile patients (3.3%) were positive for ASA, both with a titre of 1:4. None of the fertile patients showed clinically significant positivity. There was no statistically significant difference in the percentage positivity of the testicular cancer group and the control group of fertile subjects, whereas the difference was statistically significant (P<.05) when compared with the infertile control group. Of the testicular cancer group, 1.6% were ASA positive with a titre of R1:32, compared with 7.7% of the infertile control group. This difference was statistically significant (P<.01) (Table 4).
Direct IBT Direct IBT was carried out in 96 (of 181) semen samples from the testicular cancer group, from which it was possible to take a small aliquot of seminal fluid before using the sample for cryopreservation. Of the 11 GAT-positive patients, 4 showed no antibody bound to the sperm surface, and 3 were positive to IgG class only (28%, 21%, and 38%), with binding exclusively on the tail. The test could not be performed in the remaining 4 patients because 2 were azoospermic and 2 had severe oligoasthenozoospermia (Table 3).
TABLE 2 Histotype and ASA percentage positivity with GAT. Histotype Seminoma Embryonal carcinoma Mixed tumour Total
GAT-positive GAT-negative % patients (n) patients (n) 6.2 7.9
7 3
106 35
2.6 5.8
1 11
38 179
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With respect to the correlation between semen parameters and ASA, 5 patients were normozoospermic, 2 presented with severe oligoasthenoteratozoospermia (OAT), 2 with teratozoospermia, and 2 with azoospermia. None of the 18 patients with cryptorchidism was positive for ASA. DISCUSSION Studies have demonstrated that ASA can interfere with the fertilizing ability of spermatozoa, affecting both sperm motility in the semen and their ability to pass through female genital secretions, the fusion of the gametes (a key event of fertilization), and possibly the first step of embryo development (13–15). Sperm in the seminiferous epithelium and cells in the immune system are kept separate by the tight junctions between Sertoli cells, which form a blood–testis barrier, defined as the organism’s most efficient tissue barrier (16). This barrier both prevents contact between sperm antigens and immunocompetent cells and blocks the access of lymphocytes to the seminiferous tubules. Various andrologic diseases modify the normal testis structure and thus alter this barrier’s integrity, resulting in contact between sperm antigens and the immune system, which triggers an antisperm autoimmune response. Testicular cancer is considered one of the diseases that may trigger this autoimmune reaction. Testicular carcinoma in situ is considered an initial condition that is followed by testicular cancer (17, 18). Progression to testicular cancer may be associated with an alteration in the blood–testis barrier and local immune dysregulation, with the consequent production of ASAs. Few published studies have evaluated the presence of ASA in testicular cancer patients. Guazzieri et al. (3) studied 48 patients with different cancer histotypes and clinical stages (15 before orchiectomy and 18 1 month after orchiectomy). Antisperm antibody positivity of 73.3% and 38.8%, respectively, was revealed Vol. 91, No. 2, February 2009
TABLE 3 Methods and intensity of ASA positivity. GAT
Indirect IBT
Direct IBT
Patient
Titre
IgG
IgA
IgM
IgG
1 2 3 4 5 6 7 8 9 10 11
1:4 1:4 1:32 1:4 1:32 1:4 1:16 1:16 1:32 1:16 1:16
Neg Neg 22% (T) Neg 24% (T) Neg Neg Neg 40% (T) Neg Neg
Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
Neg Neg 28% (T) Neg Neg 21% (T) 38% (T)
IgA Neg Neg Neg Neg Not carried out Neg Neg Not carried out Neg Not carried out Not carried out
IgM Neg Neg Neg Neg Neg Neg Neg
Note: Neg ¼ negative, T ¼ tail. Paoli. Testicular tumors and ASA. Fertil Steril 2009.
by ELISA, with a high antibody titre. The investigators noted that patients in a more advanced clinical stage presented a higher percentage of ASA positivity and that the percentage of patients with antibodies dropped after suitable cancer therapy. Foster et al. (4) found 21% ASA positivity using immunofluorescence. These investigators evaluated 52 patients, after orchiectomy but before further therapy, with nonseminomatous low-stage testicular cancer (with no metastases or with small retroperitoneal metastases after orchiectomy). H€obarth et al. (5) found 18% ASA in 22 testicular cancer patients after orchiectomy, again using immunofluorescence. The ASA positivity found in these studies is undoubtedly high in comparison with that generally found in infertile men. Leonhartsberger et al. (19) recently published a comparative study of two surgical techniques, orchiectomy and organsparing surgery, and their effect on the development of ASA. Fifty-four testicular tumor patients were operated, and ASA positivity was measured with an ELISA plate coated with a mixture of spermatozoa proteins. The investigators reported no statistically significant difference between ASA levels in the two groups.
Our data are in contrast with the published reports: we found a much lower ASA positivity—just 5.8%—in testicular cancer patients. We also found no statistically significant difference between the testicular cancer group and the fertile control group, whereas there was a statistically significant difference in comparison with the infertile control group. Furthermore, in our caseload the semen parameters of patients with seminoma, embryonal carcinoma, and mixed tumor fell within the normal range (WHO 1992–1999), as already demonstrated by Gandini et al. (20), who reported that only 35.5% of testicular cancer patients presented a sperm count of <40 106/ejaculate. Guazzieri et al. (3) not only defined 72.2% of their patient group as oligozoospermic or azoospermic but also found that most of these were ASA positive. In contrast, we found an incidence of only 4.7% azoospermia (9 of 190); approximately half of our ASA-positive patients (5 of 11) were normozoospermic, 2 were teratozoospermic, 2 had severe OAT, and 2 were azoospermic. It should be stressed that in our caseload most patients attending for sperm cryopreservation presented with a low stage of cancer. Sixty-eight percent (99 of 146) were stage I, 29% (42 of 146) stage II, and only 3% (5 of 146) stage III. This
TABLE 4 Percentage ASA in testicular cancer. Authors (year) (reference) Guazzieri et al. (1985) (3) Foster et al. (1991) (4) € barth et al. (1994) (5) Ho
Patients (n)
ASA positivity (%)
Methods
48 52 22
73.3 21 18
ELISA Immunofluorescence Immunofluorescence
Paoli. Testicular tumors and ASA. Fertil Steril 2009.
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may partly explain the normal semen parameters observed in these patients and the lower percentage of antisperm antibodies detected. Guazzieri et al. (3) reported 30% (3 of 10) ASA positivity in stage I and II patients, and 50% (4 of 8) for the more advanced stages. Foster et al. (4) reported that 36% of ASA-positive patients (4 of 11) presented small retroperitoneal metastases. According to these investigators, therefore, the higher percentage of ASA in patients with a more advanced stage supports the hypothesis that neoplasia triggers antisperm autoimmunization. In our study, almost all ASApositive patients (10 of 11) presented with stage I disease, and only 1 presented with stage II. With respect to the intensity of the autoimmune reaction, most of our ASA-positive patients were found to have a clinically nonsignificant antibody titre on GAT, with 5 patients having a titre of 1:4, 3 of 1:16, and only 3 (1.6%) showing a clinically significant antibody titre of 1:32. The difference between this result and that for the infertile patient group (7.7%) is statistically significant. These data suggest that testicular cancer may not be an important cause of antisperm autoimmune response, given not only the low number of ASA-positive testicular cancer patients but also the low autoimmune reaction intensity. It should be remembered that the antibody titre must be evaluated for every assay found positive in order to quantify the immune reaction. This is also essential in immunology of reproduction, because the finding of a low antibody titre in fertile controls would seem to indicate the existence of a titre threshold under which autoantibodies do not cause infertility but may delay the time to pregnancy. In fact, it is clear that ASA-associated infertility is not absolute but depends on concentration, avidity, biological activity, and the antibodies’ Ig class. It should also be stressed that Guazzieri et al. (3), Foster et al (4), H€ obarth et al. (5), and Leonhartsberger et al. (19) used immunofluorescence and immunoenzymatic assays, which can give numerous false-positive and falsenegative results and are therefore not considered as reference methods. Furthermore, the numerous studies of ASA demonstrate that the results achieved with radioimmunologic and immunoenzymatic methods are characterized by a low sensitivity and specificity, owing to the use of extractive antigens (21, 22). These were proposed in the past to simplify routine evaluation of commercial ASA kits, with unreliable results; Gandini et al. (23) tested reference sera by comparing GAT with some RIA and ELISA kits. Neither of the kits used in this study provided results consistent with those obtained by traditional ASA detection methods. To date, our attempts and those of other groups to optimize these methods have been inapplicable to commercial production; in fact, only the use of the entire sperm cell as antigen, rather than extractive antigens, has enabled ASA detection methods to become reproducible. Moreover, to avoid false-positive results each sample must be analyzed with more than one method, using reference methods. The most common protocol involves the
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use of GAT and indirect IBT in blood serum and seminal plasma and direct IBT to study ASA bound to the sperm surface (24–26). Furthermore, use of the greatest possible variety of antigen systems is necessary; all samples must therefore be tested using sperm from a number of donors as the antigen. The use of unreliable methods for ASA detection may therefore be the reason for the disagreement between our data and results reported in the literature. Our study found that patients in the first stages of testicular cancer present with a low percentage of ASA (5.8%). Our patients also showed mean semen parameters above WHO reference values (6, 7). The relationship between testicular cancer and infertility is in fact controversial: according to some investigators more than half of these patients are subfertile on diagnosis, and cancer treatment can further affect fertility. These patients present with more or less severe oligozoospermia before beginning therapy (27–30), and in some cases infertility becomes almost a ‘‘symptom’’ of testicular cancer. Skakkebaek et al. (31) reported that patients with OAT may be at risk of developing testicular cancer. This discrepancy in results can be explained by considering genetic, environmental, and ethnic differences that may have a decisive impact on the control of spermatogenesis. In conclusion, our data support the hypothesis that testicular cancer may not be a possible cause of antisperm autoimmunization and infertility. This is a reassuring conclusion because testicular cancer patients who bank their semen not only have a good chance of recovering spermatogenesis approximately 2 years after therapy (32) but have a minimal ASA autoimmune response, which will enable their return to future fertility. Acknowledgements: We thank Marie-Helene Hayles for her assistance in the English translation of the manuscript and Professor Franco Culasso for the statistical analysis.
REFERENCES 1. Haas GG Jr, Cines DB, Schreiber AD. Immunologic infertility: identification of patients with antisperm antibody. N Engl J Med 1980;303: 722–7. 2. Clarke GN, Stojanoff A, Cauchi MN, Johnston WI. The immunoglobulin class of antispermatozoal antibodies in serum. Am J Reprod Immunol Microbiol 1985;7:143–7. 3. Guazzieri S, Lembo A, Ferro G, Artibani W, Merlo F, Zanchetta R, et al. Sperm antibody and infertility in patients with testicular cancer. Urology 1985;26:139–42. 4. Foster RS, Rubin LR, McNulty A, Bihrle R, Donohue JP. Detection of antisperm- antibodies in patients with primary testicular cancer. Int Androl 1991;14:179–85. 5. H€obarth K, Klinger HC, Maier U, Kollaritsch H. Incidence of antisperm antibodies in patients with carcinoma of the testis and in subfertile men with normogonadotropic oligoasthenoteratozoospermia. Urol Int 1994;52:162–5. 6. World Health Organization. Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 3rd ed. New York: Cambridge University Press, 1992. 7. World Health Organization. Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 4th ed. New York: Cambridge University Press, 1999.
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8. Bronson RA, Cooper GW, Rosenfeld DL. Membrane-bound sperm specific antibodies: their role in infertility. In: Vogel H, Jagiello G Jr, eds. Bioregulators of reproduction. New York: Academic Press, 1981: 526–7. 9. Bronson RA. Antisperm antibodies: a critical evaluation and clinical guidelines. J Reprod Immunol 1999;45:159–83. 10. Lenzi A, Familiari G, Caggiati A, Gandini L, Claroni F, Dondero F. Antisperm antibodies detection with the immunobead test as viewed by TEM. Arch Androl 1988;20:261–5. 11. Dondero F, Lenzi A, Gandini L, Lombardo F, Culasso FA. A comparison of the direct immunobead test and other tests for sperm antibodies detection. J End Inv 1991;14:443–9. 12. Kibrick S, Belding DL, Merrill B. Methods for the detection of antibodies against mammalian spermatozoa. II. A gelatin agglutination test. Fertil Steril 1952;3:430–8. 13. Shushan A, Schenker JG. Immunological factors in infertility. Am J Reprod Immunol 1992;28:285–7. 14. Vazquez Levin MH, Notrica JA, Polak de Fried E. Male immunologic infertility: sperm performance on in vitro fertilization. Fertil Steril 1997;68:675–81. 15. Koide SS, Wang L, Kamada M. Antisperm antibodies associated with infertility: properties and encoding genes of target antigens. Proc Soc Exp Biol Med 2000;224:123–32. 16. Tung KS, Okada A, Yanagimachi R. Sperm autoantigens and fertilization. I. Effects of antisperm autoantibodies on Rouleaux formation, viability and acrosome reaction of guinea pig spermatozoa. Biol Reprod 1980;23:877–86. 17. Skakkebaek NE, Berthelsen JG, Giwercman A, M€uller J. Carcinoma in situ of the testis: possible origin from gonocytes and precursor of all types of germ cell tumors except spermatocytoma. Int J Androl 1987;10:19–28. 18. Dauggaard G, von der Masse H, Olsen J, Rø´rth M, Skakkebaek NE. Carcinoma in situ testis in patients with assumed extragonadal germ cell tumours. Lancet 1987;2:528–30. 19. Leonhartsberger N, Gozzi C, Akkad T, Springer-Stoehr B, Bartsch G, Steiner H. Organ-sparing surgery does not lead to greater antisperm antibody levels than orchidectomy. BJU Int 2007;100:371–4.
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20. Gandini L, Lombardo F, Salacone P, Paoli D, Anselmo AP, Culasso F, et al. Testicular cancer and Hodgkin’s disease: evaluation of semen quality. Hum Reprod 2003;18:796–801. 21. Mettler L, Czuppon AB, Alexander N, D’Almeida M, Haas GG Jr, Hjort T, et al. Antibodies to spermatozoa and seminal plasma antigens detected by various enzyme-linked immunosorbent (ELISA) assays. J Reprod Immunol 1985;8:301–12. 22. Clarke GN. Lack of correlation between the immunobead test and the enzyme-linked immunosorbent assay for sperm antibody detection. Am J Reprod Immunol Microbiol 1988;18:44–6. 23. Gandini L, Lenzi A, Lombardo F, Dondero F. Radio- immuno binding test for antisperm antibody detection: analysis and critical revision of various methodological steps. Andrologia 1991;23:61–8. 24. Shulman S. Antisperm antibodies in serum and genital tract secretions. Fertil Steril 1991;56:1208–10. 25. Dondero F, Gandini L, Lombardo F, Salacone P, Caponecchia L, Lenzi A. Antisperm antibody detection: 1. Methods and standard protocol. Am J Reprod Immunol 1997;38:218–23. 26. Lenzi A, Gandini L, Lombardo F, Rago R, Paoli D, Dondero F. Antisperm antibody detection: 2. Clinical, biological, and statistical correlation between methods. Am J Reprod Immunol 1997;38:224–30. 27. Berthelsen JC, Skakkebaek NE. Gonadal function in men with testis cancer. Fertil Steril 1983;39:68–75. 28. Agarwal A, Tolentino MV Jr, Sidhu RS, Ayzman I, Lee JC, Thomas AJ Jr, et al. Effect of cryopreservation on semen quality in patients with testicular cancer. Urology 1995;46:382–9. 29. Lange PH, Chang WY, Fraley E. Fertility issues in the therapy of nonseminomatous testicular tumors. Urol Clin North Am 1987;14:731–47. 30. Meirow D, Schenker JG. Cancer and male infertility. Hum Reprod 1995;10:2017–22. 31. Skakkebaek NE, Rajpert-De Meyts E, Main KM. Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Hum Reprod 2001;16:972–8. 32. Gandini L, Sgro P, Lombardo F, Paoli D, Culasso F, Toselli L, et al. Effect of chemo- or radiotherapy on sperm parameters of testicular cancer patients. Hum Reprod 2006;21:2882–9.
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Objective: To evaluate the presence of antisperm antibodies in testicular cancer patients 1 month after orchiectomy and before radiotherapy or chemotherapy. Design: Clinical study. Setting: Department of andrology and seminology at a university hospital. Patient(s): One hundred ninety patients with testicular cancer. Intervention(s): Determination of semen parameters and autoimmune reaction evaluated on the sperm surface and in blood serum. Main Outcome Measure(s): Autoimmune reaction on the sperm surface by the direct immunobead test (IBT), and in blood serum by the indirect IBT and the gelatin agglutination test (GAT), was evaluated 1 month after orchiectomy and before beginning chemotherapy or radiotherapy. Result(s): Of the 190 patients, 11 (5.8%) were positive for antisperm antibody by GAT. On indirect IBT, 3 of the 11 GAT-positive patients were positive to IgG class only, with values of 22%, 24%, and 40%. Of the 11 GAT-positive patients, 4 showed no antibody bound to the sperm surface, and 3 were positive to IgG class only (28%, 21%, and 38%), with binding exclusively on the tail. Direct IBT could not be performed in the remaining 4 patients. Conclusion(s): Our data support the hypothesis that testicular cancer might not be a possible cause of antisperm autoimmunization and infertility. (Fertil Steril 2009;91:414–9. 2009 by American Society for Reproductive Medicine.) Key Words: Testicular cancer, ASA, infertility, semen analysis
Antisperm antibodies (ASA) are found in approximately 10% of infertile men (1, 2). Knowledge of the antigen systems involved in antisperm immune reactions is not yet well established. Various theories have been proposed to explain the sperm cell’s protection within the seminiferous tubules, because from an antigenic point of view it is partially foreign to the organism producing it. The biological, anatomical, and functional mechanisms underlying the prevention of the antisperm immune response take place in the testis. A number of andrologic diseases can affect the blood–testis barrier, inducing antisperm autoimmunization. There may be numerous triggers: inflammation, trauma, torsion of the spermatic cord, complete or incomplete obstructions, and testicular neoplasia. Testicular cancer constitutes approximately 1% of all cancers in men but is the most frequent cancer in men aged 15 to 39 years. Advances in chemotherapy and radiotherapy in combination with surgical techniques over the last 10 years mean that 90% of patients can now be cured.
Received August 3, 2007; revised and accepted November 27, 2007. Supported by a grant from the Italian Ministry of Education and Research (MIUR-COFIN) and the University of Rome ‘‘La Sapienza.’’ Reprint requests: Loredana Gandini, B.Sc., Laboratory of SeminologySperm Bank, Department of Medical Pathophysiology, University of Rome ‘‘La Sapienza,’’ Viale del Policlinico 155, Rome 00161, Italy (FAX: þ39-06-4997-0717; E-mail: [email protected]).
414
Germ-cell tumors originate from the germinal epithelium of the seminiferous tubules and have various histologic types (seminoma, embryonal carcinoma, choriocarcinoma, teratoma, and mixed tumors, consisting of more than one of these histotypes), with differing degrees of invasiveness and a high level of metastasis. Because testicular cancer directly affects the gamete production site, it has been postulated as a trigger for an autoimmune response. The presence of ASA may in fact be explained by local effects caused by the cancer, such as raised scrotal temperature connected with blood flow alterations or disruption of the blood–testis barrier, with a massive release of sperm antigens that stimulate antisperm immunization. For this reason, some investigators (3–5) have considered autoimmunity to be closely correlated with testicular cancer. The aim of our study was to evaluate the presence of antisperm antibodies in testicular cancer patients after orchiectomy and before radiotherapy or chemotherapy.
MATERIALS AND METHODS Patients The study was approved by the institutional review board of our university hospital. We studied 190 consecutive patients with testicular cancer, with a mean (SD) age of 27.9 5.4
Fertility and Sterility Vol. 91, No. 2, February 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2007.11.084
years, who cryobanked sperm between 2001 and 2005 at the Seminology Laboratory-Sperm Bank, Department of Medical Pathophysiology, University of Rome ‘‘La Sapienza’’ before beginning chemotherapy or radiotherapy. All patients were evaluated approximately 1 month after orchiectomy, to give them the necessary time for postoperative physical recovery. Patients were grouped according to their histotype— seminoma, embryonal carcinoma, or mixed tumor (variable combination of seminoma, teratocarcinoma, choriocarcinoma, and yolk sac tumor)—and clinical stage (pT1, pT2, pT3, pT4). They were also classified on the basis of normal testicular descent into the scrotal sac at birth. Seminal fluid examination and ASA detection were performed for all patients. We compared the ASA results for testicular cancer patients with data from a control group of 580 consecutive patients diagnosed with infertility from 2003 to 2005 and a second control group of 60 fertile subjects. Both control groups had a mean age similar to that of the study group.
Seminal Fluid Examination Semen samples were collected by masturbation after 2–7 days’ abstinence. All samples were allowed to liquefy at 37 C for 60 minutes and were then assessed according to World Health Organization (WHO) guidelines (6, 7). The following variables were taken into consideration: ejaculate volume, sperm concentration, total sperm count, forward motility, and morphology (percentage of abnormal forms). Because of the urgent need for patients to begin cancer therapy, semen analysis was performed on only one sample for each patient. However, the examination was performed twice for azoospermia cases, and diagnosis was made only after having carefully evaluated the pellet after centrifugation. The pellet was also stained (May-Gr€ unwald-Giemsa) to evaluate the presence of germline cells. All semen samples were examined by the same biologist (L.G.).
ASA Detection Autoimmune reaction was evaluated on the sperm surface by the direct immunobead test (IBT) (8–11) (Irvine Scientific, Santa Ana, CA), and in blood serum by the indirect IBT and the gelatin agglutination test (GAT) (12). For direct and indirect IBT, samples showing binding of >20% were considered positive. A binding percentage of >50% was considered clinically significant. In GAT, an antibody titre of 1:32 or more in blood serum was considered clinically significant. All indirect tests (GAT and indirect IBT) were performed twice with different antigens. Indirect tests can be carried out in blood serum and seminal plasma. In our study, ASA were evaluated in blood serum only because the semen sample was used for sperm cryopreservation. Direct tests were performed only for cases whose semen parameters enabled the test to be performed with small aliquots of seminal fluid. Fertility and Sterility
Statistical Analysis Means and SDs were computed for interval variables; for nominal variables, frequencies and percentages are reported. Prevalence of ASA positivity in the three groups (testicular cancer patients, infertile patients, and fertile subjects) were compared by odds ratio and chi-square test. RESULTS The 190 testicular cancer patients were divided into three groups by histotype: seminoma (n ¼ 113), embryonal carcinoma (n ¼ 38), and mixed tumor (n ¼ 39). Clinical staging was available for 146 of 190 patients only: stage I: 99 patients; stage II: 42; stage III: 5. Data documenting normal testicular descent into the scrotum at the time of birth were available for 171 of 190 patients only: 149 of 171 patients (87.1%) were normal. Eighteen patients (10.5%) had unilateral cryptorchidism: 9 on the right, of whom 1 had contralateral testicular cancer, and 9 on the left, of whom 1 had contralateral testicular cancer. Four patients had retractile testis (2 in the right and 2 in the left testis); all 4 had testicular cancer in the retractile testis. Of 18 patients with cryptorchidism, 12 were affected by seminoma, 4 by embryonal carcinoma, and 2 by mixed tumor. Means and SDs for age and semen parameters (volume, sperm concentration, total sperm count, percentage forward motility, and percentage abnormal forms) for the three groups are reported in Table 1. In the seminoma group, 7 patients were azoospermic at the time of the semen analysis and 1 was cryptozoospermic. In the embryonal carcinoma group, only 1 patient was azoospermic. These patients were excluded from the statistical analysis, and therefore semen data refer to 181 patients. GAT and Indirect IBT The presence of ASA in blood serum by GAT and indirect IBT was evaluated in testicular cancer patients. Of the 190 patients, 11 (5.8%) were found positive for ASA by GAT, of whom 7 were affected by seminoma, 3 by embryonal carcinoma, and 1 by mixed tumor, as shown in Table 2. Of the 11 ASA-positive patients, 10 presented with clinical stage I, and 1 presented with stage II. Five patients had an antibody titre of 1:4, 3 of 1:16, and 3 of 1:32 (Table 3); thus only 3 of these 190 patients (1.6%) presented with clinically significant positivity (1:32). On indirect IBT, 3 of the 11 GAT-positive patients were found positive to IgG class only, with values of 22%, 24%, and 40% (Table 3). There was no clinically significant positivity. Blood serum ASA was evaluated by GAT in both control groups. Of the 580 infertile patients, 64 (11.0%) were positive for ASA, of whom 19 had an antibody titre of 1:4–1:16 and 45 had an antibody titre of R1:32 (range, 1:32–1:512). Thus 7.7% of infertile patients (45 of 580) presented with clinically 415
TABLE 1 Semen parameters by testicular cancer histotype.
Histotype Seminoma Embryonal carcinoma Mixed tumor Testicular cancer
Patients (n)
Age (y)
Volume (mL)
Sperm concentration (3106/mL)
Total sperm count (3106)
Forward Abnormal motility (%) forms (%) 35.2 16.7 69.7 12.2 31.1 17.4 71.1 12.3
105 37
29.5 5.48 3.8 1.8 25.3 4.91 2.8 1.2
36.8 37.0 26.5 24.6
124.9 136.1 72.0 77.4
39 181
26.2 4.42 3.7 2.1 27.9 5.5 3.6 1.8
35.2 33.2 34.3 34.1
116.2 138.4 35.5 17.2 69.8 13.0 112.2 128.0 34.4 16.9 70.0 12.4
Note: Values are mean SD. Paoli. Testicular tumors and ASA. Fertil Steril 2009.
significant positivity. Only 2 of the 60 fertile patients (3.3%) were positive for ASA, both with a titre of 1:4. None of the fertile patients showed clinically significant positivity. There was no statistically significant difference in the percentage positivity of the testicular cancer group and the control group of fertile subjects, whereas the difference was statistically significant (P<.05) when compared with the infertile control group. Of the testicular cancer group, 1.6% were ASA positive with a titre of R1:32, compared with 7.7% of the infertile control group. This difference was statistically significant (P<.01) (Table 4).
Direct IBT Direct IBT was carried out in 96 (of 181) semen samples from the testicular cancer group, from which it was possible to take a small aliquot of seminal fluid before using the sample for cryopreservation. Of the 11 GAT-positive patients, 4 showed no antibody bound to the sperm surface, and 3 were positive to IgG class only (28%, 21%, and 38%), with binding exclusively on the tail. The test could not be performed in the remaining 4 patients because 2 were azoospermic and 2 had severe oligoasthenozoospermia (Table 3).
TABLE 2 Histotype and ASA percentage positivity with GAT. Histotype Seminoma Embryonal carcinoma Mixed tumour Total
GAT-positive GAT-negative % patients (n) patients (n) 6.2 7.9
7 3
106 35
2.6 5.8
1 11
38 179
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With respect to the correlation between semen parameters and ASA, 5 patients were normozoospermic, 2 presented with severe oligoasthenoteratozoospermia (OAT), 2 with teratozoospermia, and 2 with azoospermia. None of the 18 patients with cryptorchidism was positive for ASA. DISCUSSION Studies have demonstrated that ASA can interfere with the fertilizing ability of spermatozoa, affecting both sperm motility in the semen and their ability to pass through female genital secretions, the fusion of the gametes (a key event of fertilization), and possibly the first step of embryo development (13–15). Sperm in the seminiferous epithelium and cells in the immune system are kept separate by the tight junctions between Sertoli cells, which form a blood–testis barrier, defined as the organism’s most efficient tissue barrier (16). This barrier both prevents contact between sperm antigens and immunocompetent cells and blocks the access of lymphocytes to the seminiferous tubules. Various andrologic diseases modify the normal testis structure and thus alter this barrier’s integrity, resulting in contact between sperm antigens and the immune system, which triggers an antisperm autoimmune response. Testicular cancer is considered one of the diseases that may trigger this autoimmune reaction. Testicular carcinoma in situ is considered an initial condition that is followed by testicular cancer (17, 18). Progression to testicular cancer may be associated with an alteration in the blood–testis barrier and local immune dysregulation, with the consequent production of ASAs. Few published studies have evaluated the presence of ASA in testicular cancer patients. Guazzieri et al. (3) studied 48 patients with different cancer histotypes and clinical stages (15 before orchiectomy and 18 1 month after orchiectomy). Antisperm antibody positivity of 73.3% and 38.8%, respectively, was revealed Vol. 91, No. 2, February 2009
TABLE 3 Methods and intensity of ASA positivity. GAT
Indirect IBT
Direct IBT
Patient
Titre
IgG
IgA
IgM
IgG
1 2 3 4 5 6 7 8 9 10 11
1:4 1:4 1:32 1:4 1:32 1:4 1:16 1:16 1:32 1:16 1:16
Neg Neg 22% (T) Neg 24% (T) Neg Neg Neg 40% (T) Neg Neg
Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
Neg Neg 28% (T) Neg Neg 21% (T) 38% (T)
IgA Neg Neg Neg Neg Not carried out Neg Neg Not carried out Neg Not carried out Not carried out
IgM Neg Neg Neg Neg Neg Neg Neg
Note: Neg ¼ negative, T ¼ tail. Paoli. Testicular tumors and ASA. Fertil Steril 2009.
by ELISA, with a high antibody titre. The investigators noted that patients in a more advanced clinical stage presented a higher percentage of ASA positivity and that the percentage of patients with antibodies dropped after suitable cancer therapy. Foster et al. (4) found 21% ASA positivity using immunofluorescence. These investigators evaluated 52 patients, after orchiectomy but before further therapy, with nonseminomatous low-stage testicular cancer (with no metastases or with small retroperitoneal metastases after orchiectomy). H€obarth et al. (5) found 18% ASA in 22 testicular cancer patients after orchiectomy, again using immunofluorescence. The ASA positivity found in these studies is undoubtedly high in comparison with that generally found in infertile men. Leonhartsberger et al. (19) recently published a comparative study of two surgical techniques, orchiectomy and organsparing surgery, and their effect on the development of ASA. Fifty-four testicular tumor patients were operated, and ASA positivity was measured with an ELISA plate coated with a mixture of spermatozoa proteins. The investigators reported no statistically significant difference between ASA levels in the two groups.
Our data are in contrast with the published reports: we found a much lower ASA positivity—just 5.8%—in testicular cancer patients. We also found no statistically significant difference between the testicular cancer group and the fertile control group, whereas there was a statistically significant difference in comparison with the infertile control group. Furthermore, in our caseload the semen parameters of patients with seminoma, embryonal carcinoma, and mixed tumor fell within the normal range (WHO 1992–1999), as already demonstrated by Gandini et al. (20), who reported that only 35.5% of testicular cancer patients presented a sperm count of <40 106/ejaculate. Guazzieri et al. (3) not only defined 72.2% of their patient group as oligozoospermic or azoospermic but also found that most of these were ASA positive. In contrast, we found an incidence of only 4.7% azoospermia (9 of 190); approximately half of our ASA-positive patients (5 of 11) were normozoospermic, 2 were teratozoospermic, 2 had severe OAT, and 2 were azoospermic. It should be stressed that in our caseload most patients attending for sperm cryopreservation presented with a low stage of cancer. Sixty-eight percent (99 of 146) were stage I, 29% (42 of 146) stage II, and only 3% (5 of 146) stage III. This
TABLE 4 Percentage ASA in testicular cancer. Authors (year) (reference) Guazzieri et al. (1985) (3) Foster et al. (1991) (4) € barth et al. (1994) (5) Ho
Patients (n)
ASA positivity (%)
Methods
48 52 22
73.3 21 18
ELISA Immunofluorescence Immunofluorescence
Paoli. Testicular tumors and ASA. Fertil Steril 2009.
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may partly explain the normal semen parameters observed in these patients and the lower percentage of antisperm antibodies detected. Guazzieri et al. (3) reported 30% (3 of 10) ASA positivity in stage I and II patients, and 50% (4 of 8) for the more advanced stages. Foster et al. (4) reported that 36% of ASA-positive patients (4 of 11) presented small retroperitoneal metastases. According to these investigators, therefore, the higher percentage of ASA in patients with a more advanced stage supports the hypothesis that neoplasia triggers antisperm autoimmunization. In our study, almost all ASApositive patients (10 of 11) presented with stage I disease, and only 1 presented with stage II. With respect to the intensity of the autoimmune reaction, most of our ASA-positive patients were found to have a clinically nonsignificant antibody titre on GAT, with 5 patients having a titre of 1:4, 3 of 1:16, and only 3 (1.6%) showing a clinically significant antibody titre of 1:32. The difference between this result and that for the infertile patient group (7.7%) is statistically significant. These data suggest that testicular cancer may not be an important cause of antisperm autoimmune response, given not only the low number of ASA-positive testicular cancer patients but also the low autoimmune reaction intensity. It should be remembered that the antibody titre must be evaluated for every assay found positive in order to quantify the immune reaction. This is also essential in immunology of reproduction, because the finding of a low antibody titre in fertile controls would seem to indicate the existence of a titre threshold under which autoantibodies do not cause infertility but may delay the time to pregnancy. In fact, it is clear that ASA-associated infertility is not absolute but depends on concentration, avidity, biological activity, and the antibodies’ Ig class. It should also be stressed that Guazzieri et al. (3), Foster et al (4), H€ obarth et al. (5), and Leonhartsberger et al. (19) used immunofluorescence and immunoenzymatic assays, which can give numerous false-positive and falsenegative results and are therefore not considered as reference methods. Furthermore, the numerous studies of ASA demonstrate that the results achieved with radioimmunologic and immunoenzymatic methods are characterized by a low sensitivity and specificity, owing to the use of extractive antigens (21, 22). These were proposed in the past to simplify routine evaluation of commercial ASA kits, with unreliable results; Gandini et al. (23) tested reference sera by comparing GAT with some RIA and ELISA kits. Neither of the kits used in this study provided results consistent with those obtained by traditional ASA detection methods. To date, our attempts and those of other groups to optimize these methods have been inapplicable to commercial production; in fact, only the use of the entire sperm cell as antigen, rather than extractive antigens, has enabled ASA detection methods to become reproducible. Moreover, to avoid false-positive results each sample must be analyzed with more than one method, using reference methods. The most common protocol involves the
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use of GAT and indirect IBT in blood serum and seminal plasma and direct IBT to study ASA bound to the sperm surface (24–26). Furthermore, use of the greatest possible variety of antigen systems is necessary; all samples must therefore be tested using sperm from a number of donors as the antigen. The use of unreliable methods for ASA detection may therefore be the reason for the disagreement between our data and results reported in the literature. Our study found that patients in the first stages of testicular cancer present with a low percentage of ASA (5.8%). Our patients also showed mean semen parameters above WHO reference values (6, 7). The relationship between testicular cancer and infertility is in fact controversial: according to some investigators more than half of these patients are subfertile on diagnosis, and cancer treatment can further affect fertility. These patients present with more or less severe oligozoospermia before beginning therapy (27–30), and in some cases infertility becomes almost a ‘‘symptom’’ of testicular cancer. Skakkebaek et al. (31) reported that patients with OAT may be at risk of developing testicular cancer. This discrepancy in results can be explained by considering genetic, environmental, and ethnic differences that may have a decisive impact on the control of spermatogenesis. In conclusion, our data support the hypothesis that testicular cancer may not be a possible cause of antisperm autoimmunization and infertility. This is a reassuring conclusion because testicular cancer patients who bank their semen not only have a good chance of recovering spermatogenesis approximately 2 years after therapy (32) but have a minimal ASA autoimmune response, which will enable their return to future fertility. Acknowledgements: We thank Marie-Helene Hayles for her assistance in the English translation of the manuscript and Professor Franco Culasso for the statistical analysis.
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