FERTILITY AND STERILITY Copyright < 1975 The American Fertility Society
Vol. 26, No. 10, October 1975 Printed in U.SA.
IMMUNOLOGIC STUDIES OF MALE INFERTILITY JACK R. WALL, M.D.,* M.R.A.C.P., D.H.M.S.A., JITKA STEDRONSKA, RAPHAEL D. DAVID, M.D., ROBBIE F. HARRISON, F.R.C.S.(E)., M.R.C.P.(I)., M.R.C.O.G., DOROTHY GORIUP, A.I.M.L.T., AND MAURICE H. LESSOF, M.A., M.D., F.R.C.P. Department of Medicine, Guy's Hospital Medical School, London Bridge SEl 9RT, United Kingdom
A large proportion of infertile men has no apparent underlying disorder. Testicular biopsy often reveals germinal cell aplasia, arrest of spermatogenesis at various stages, or shedding defects. 1, 2 The interstitium is usually normal. In most cases, plasma testosterone levels are within the normal range, although often lower than in fertile men. 3 ,4 Levels of luteinizing hormone (LH) are usually normal, 3 and follicle-stimulating hormone (FSH) levels are normal or high. 1, 2, 5, 6 Clinically, the only abnormality is the presence of small, soft testes, although in some cases they may be of normal size and consistency. Because of the possible role of testicular germinal cell antibodies and cellmediated immunity to testis in the orchitis of lepromatous leprosy/,8 immunologic studies of infertile men with testicular disorders of unknown cause were carried out. Evidence was obtained that primary immunologic abnormalities may play a role in a small number of cases of male infertility. EXPERTIMENTALPROCEDURE
who were attending the subfertility clinics at Chelsea and Guy's Hospitals. All had been infertile for at least 1 year and had fewer than 10 million spermatozoa/ml on repeated seminal analyses. Men with possible causes for the infertility, such as varicocele, mumps orchitis, cryptorchidism, reflux ejaculation, or trauma, were excluded from the study. The men appeared normal on examination, except for small testes (50% of cases), normalsized but soft testes (6% of cases), unilateral testicular atrophy (6% of cases), or mild hypogonadism (one case). Men with more than 10 million spermatozoa/ml but low motility or an increased proportion of abnormal spermatozoa were also excluded from the study. Following establishment of true oligospermia, the minimal investigations carried out were measurement of serum LH and FSH and plasma testosterone, and performance of testicular biopsy. The control group consisted of 100 male blood donors and laboratory staff, including 18 healthy men ages 23 to 57 (mean, 33 years) on whom lymphocyte cultures were carried out.
Clinical Subjects
The investigation concerned 63 men with azo- or oligospermia of unknown cause, ages 23 to 45 (mean, 31 years), Received December 6, 1974. *Reprint requests: Dr. J. R. Wall, Repatriation Hospital, Greenslopes, Brisbane, Australia.
Methods
Immunologic reaction to testicular antigens was assessed in infertile patients and normal subjects by measuring serum testicular antibodies and culturing peripheral lymphocytes in the presence of testis extracts.
1035
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WALL ET AL.
Lymphocyte Cultures. Peripheral blood lymphocytes were separated from 20 ml of heparinized blood, using a Triosil-Ficoll gradient and following the method of Boyum. 9 The cells (0.3 to 1 x 106 ) were added to culture medium composed of 0.9 ml of TC199 (Wellcome Laboratories, Beckenham, England), 10% fetal calf serum, 0.1 ml of autologous serum, 50 U of penicillin/ml, and 100 p,g of streptomycin/ ml. Testis extract (0.1 ml) was added to test cultures in the dose range of 100 to 800 p.,g. Cultures were set up in triplicate. Control cultures consisted of (1) 0.1 ml of saline instead of antigen (unstimulated culture), (2) medium and 400 p.,g of testis but no cells (medium control), and (3) medium as for test cultures, but with antigen (400 p.,g) added at the end of the period of culture, before dialysis (macrophage inhibitory factor (MIF) control). Lymphocytes were cultured for 6 days at 37° C, without medium change. Lymphocyte cultures were carried out on 22 patients and 18 normal men. Lymphocyte Transformation. In the presence of specific antigen, sensitized thymus-derived (T) lymphocytes undergo blastoid transformation, commence cell division, and incorporate 3H-thymidine into newly synthesized DNA. This series of events correlates with cell-mediated immunity (delayed hypersensitivity) to the antigen, as determined by skin testing. tO Lymphocyte transformation in response to testis was estimated in 10 of the patients and in 10 normal subjects by adding 1 p.,Ci of 3H-thymidine (specific activity, 5 CiJmmole; Radiochemical Centre, Amersham, England) to the cultures 18 hours before termination. The cells were harvested, and the DNA content was precipitated in trichloroacetic acid and collected onto Whatman glass fiber filter discs. The oven-dried discs were dispersed in 12 ml of a toluene-based scintillation fluid and counted for 10 minutes in a
October 1975
Packard Tri-Carb liquid scintillation counter. Results were expressed as transformation ratios (TR), i.e., the ratio of counts per minute in antigen-stimulated cultures to counts per minute in unstimulated cultures. For the purposes of this study, a TR > 2.0 was considered positive. The mean maximal TR for infertile patients was compared with that for normal subjects. Macrophage Inhibitory Factor Tests. Sensitized T lymphocytes exert their effect primarily by releasing soluble mediators called lymphokines, of which the migration inhibitory factor has been most studied. Sensitized T lymphocytes release detectable amounts ofMIF in vitro within 8 hours of culturing in the presence of specific antigen. 10 The culture supernatants from infertile patients and normal subjects were tested for MIF production by the indirect method,l1, t2 using macrophages from unsensitized guinea pigs. The supernatants were dialyzed for 48 hours before testing. The tests were read with a projection microscope after 24 hours, and the migration areas were calculated with a planimeter. Quadruplicate estimations were made of each supernatant and the means were calculated. Results were expressed as migration indices (MI), i.e., the ratio of the mean area of macrophage migration with supernatants from stimulated cultures to migration with the supernatants from control (unstimulated) cultures. A migration index < 0.8 for any antigen concentration was considered positive for MIF. The mean minimal value (maximal inhibition) for infertile patients was compared with that for normal subjects.
Antigen Preparation. Because the testicular antigens which may be involved in infertility are species-nonspecific,7 rat testis, which is more readily available than human testis, was used as the antigen for the lymphocyte cultures in these studies. Human testis, however, was
Vol. 26, No. 10
IMMUNOLOGIC STUDIES OF MALE INFERTILITY
used for the immunofluorescence studies. Fresh rat testis was homogenized in 0.25 M sucrose, filtered, and extracted overnight at 4° C in an equal volume of phosphate-buffered saline (pH 7.15). The extract was centrifuged at 800 x g and the supernatant, containing subcellular elements and soluble proteins, was used for the lymphocyte cultures. The protein concentration of the supernatant was measured by the optical density method. Aliquots (0.1 ml) containing 100, 200, 400, or 800 ILg of protein were stored at - 20° C until used. Immunofluorescent Antibody Technique. Serum testicular germinal cell antibodies were tested for by the indirect immunofluorescent technique, using human testis as substrate, as previously described. 7 Antibodies against thyroid cytoplasm, gastric parietal cells, nuclear material, mitochondria, smooth muscle, and spermatozoa were also tested for by this technique, using human thyrotoxic thyroid, rat stomach and kidney, and human donor spermatozoa with >40 million motile spermatozoa/ml, respectively, as substrates. Serum dilution was 1:10 for all tests. A Reichert ultraviolet microscope with an SP3 exciting filter and an ultraviolet pass-absorbing filter was used. Serum Immunoglobulins. Serum immunoglobulins were measured in 15 patients by the radial diffusion method 13
1037
using Immunoplates (Hyland Laboratories, Los Angeles, Calif.). The normal ranges for this laboratory are: IgG, 500 to 1500 mg/100 ml; IgM, 47 to 170 mg/100 ml; and IgA, 160 to 420 mg/100 ml. Testicular Biopsies. The method of Johnsen1 for rating seminiferous tubular maturation was used. The tubular basement membranes, Sertoli cells, Leydig cells, and interstitium were also examined. In 20 cases, direct immunofluorescent tests using polyvalent and monovalent conjugates and anti-/He-/HA conjugates were carried out on frozen sections of testis for deposition of antibody or complement in testicular tissues which would indicate an immunologic reaction in vivo. Hormonal Estimations. Plasma testosterone and serum LH and FSH were measured by radioimmunoassays14. 15 in 27 randomly selected cases. For men, normal ranges in this laboratory are: testosterone, 320 to 835 ng/100 ml; LH, 0.7 to 13.2 mIUlml; and FSH, 0.1 to 3.2 mIU/ml. RESULTS
Serum antibodies against testicular germinal cells were found in 9 of 63 patients, compared with 5 of 100 normal men; however, the difference was not significant (Table 1). No staining of Sertoli cells16 or Leydig cells17 was found
TABLE 1. Prevalence of Serum Immunofluorescent Antibodies in Infertile Men with Azo- or Oligospermia of Unknown Cause Positive immunofluorescencea
Group Testisb
Infertile men (n = 63) 9 (14%) Normal men (n = 100) 5 (5%) pd NS
SpennatozoaC"
13 (21%) 5 (5%) 0.
--------------
_&L ______ I>
0 0
normal
l
infertile
normal
FIG. 1. In vitro Lymphocyte transformation to testis extract in infertile men with azoor oligospermia of unknown cause and in normal subjects. Values above the dotted line (TR > 2.0) indicate significant transformation. Testis concentrations over the range of 100 to 800 JLglml were used; maximal values for each culture are shown. There was no significant difference between the means of the two groups (3.07 ± 1.4 SEM and 1.41 ± 0.49 SEM, respectively).
FIG. 2. In vitro macrophage inhibitory factor production in infertile men with azo- or oligospermia of unknown cause and in normal subjects. Patients' lymphocytes were cultured in the presence of testis extract and the supernatants were tested for MIF by the indirect method. Values below the dotted line (MI < 0.8) indicate significant inhibition. Testis concentrations over the range 100 to 800 JLglml were used; maximal inhibition for each culture is shown. There was no significant difference between the means of the two groups (0.98 ± 0.37 SEM and 0.98 ± 0.26 SEM, respectively).
in this study. Spermatozoal antibodies were found in 13 of 63 patients, compared with 5 of 100 control subjects. This difference was significant (X 2 test;P < 0.001). Thirteen patients had one or more other serum autoantibodies: viz., against thyroid cytoplasm (seven patients), gastric parietal cells (three patients), smooth muscle (two patients), and nuclear material and mitochondria (one patient each). Such antibodies were found in 4 of 100 normal men: viz., thyroid cytoplasmic antibody (1 man; P < 0.001), gastric parietal cell antibody (2 men), and antinuclear antibody (1 man). These latter differences were not significant.
of the normal subjects (1.41 ± 0.49 SEM) (Fig. 1). Significant macrophage inhibition (MI < 0.8) was found in 7 of 22 infertile patients (32%), compared with 1 of 18 normal men (Fig. 2). This difference was significant (X 2 test; P < 0.05). There was no significant difference, however, between the means of the two groups (0.98 ± 0.37 SEM and 0.98 ± 0.26 SEM, respectively). Serum immunoglobulin levels were normal in all 15 patients tested.
Significant lymphocyte transformation to testis (TR > 2) was found in 2 of 10 infertile men tested, compared with 1 of10 normal men. The mean maximal value for the infertile group (3.07 ± 1.4 SEM) was not significantly different from that
Histologic examination of testicular biopsies provided little evidence for an immunologic disorder. None of the biopsies demonstrated increased interstitial lymphoid tissue. The most common findings were germinal cell aplasia, spermatozoal maturation arrests, shedding defects, and, occasionally, complete disorganization and fibrosis. Direct immunofluorescent tests on sections of the patients'
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IMMUNOLOGIC STUDIES OF MALE INFERTILITY
own testis with polyvalent (IgG/IgM), monovalent (lgG, IgM, and IgA), and anti-/He-/nA conjugates showed no evidence of antibody or complement deposition in the testicular tissue. Plasma testosterone levels were measured in 27 cases. Nine patients (33%) had levels below the lower limit of normal, while the mean for the group (461 ng/100 ml ± 35.7 SEM) was lower than that for a control group of normal men of comparable ages (522 ng/100 ml ± 35 SEM), although the difference was not significant. FSH levels were normal in 24 cases and undetectable in 2 cases. The latter two patients had biochemical evidence of pituitary deficiency, viz., low levels ofLH and FSH and borderline low levels of testosterone. Other pituitary function tests are presently being carried out. The mean testosterone level of patients with evidence of cell-mediated immunity to testis and/or testicular germinal cell antibodies was not significantly lower than that of patients with no such evidence of testicular autoimmunity. Similarly, the presence of spermatozoal antibodies did not correlate with testosterone levels. Finally, there was no close correlation between the histologic lesions found on biopsy and the presence of germinal cell antibodies or cell-mediated immunity to testis. However, patients with complete testicular fibrosis and disorganization tended to have these findings more often than those with maturation or shedding defects, although the difference was not significant. DISCUSSION
Of 63 men with azo- or oligospermia, 2 were found to have hypogonadotropic hypogonadism and an additional 7 had evidence of a failure of androgen production as well as of spermatogenesis. No distinctive features were found in the remainder. In contrast to that for lepromatous
1039
orchitis,7. 8 the evidence for an immunologic role in the pathogenesis of male infertility is not impressive. Only 14% of infertile men with primary testicular disorders tested had antibodies against testicular germinal cells, compared with 70% of lepromatous patients. None of the patients tested had elevated serum immunoglobulin levels, and there was no excess of lymphoid tissue on testicular biopsy. Although 21% of the patients had spermatozoal antibodies, it is likely that this was secondary to the underlying testicular lesion since there is no good evidence that spermatozoal antibodies are a cause of oligospermia. 18. 19 However, onethird had evidence of cell-mediated immunity to testis. It has been postulated7. 8 that leprous orchitis may be a model for an organ-specific autoimmune disease in which infection with Mycobacterium leprae induces the production of antibodies and sensitized T lymphocytes and leads to progressive testicular damage. The present study was initiated to test the hypothesis that some cases of infertility due to primary testicular disorders may be due to such organspecific autoimmunity. Although the over-all evidence is unimpressive and lymphoid tissue was absent on biopsy, one-third of this group had evidence of cell-mediated hypersensitivity to testis on in vitro testing. Since cell-mediated immunity is thought to be more important than antibodies in the pathogenesis of autoimmune disease,2()"22 the inability to detect germinal cell antibodies does not exclude this etiology, especially in advanced cases with fibrosed, disorganized testes. However, this explanation seems inadequate in view of the high prevalence of antibodies in leprosy, since leprosy is associated with both the autoantibodies and the cellular reaction of an autoimmune orchitis. Therefore, it must be postulated either that male infertility has different causes or that more than
WALLETAL.
1040
one disease group was represented by the parents we studied. The commonly found histologic lesions of spermatozoal maturation arrest, germinal cell aplasia, shedding defects, and gross fibrosis and disorganization, however, do not resemble classic immunologic disorders such as are found in Hashimoto's thyroiditis or idiopathic adrenalitis. Again, this does not exclude an immunologic pathogenesis, since it is well known that in experimental auto allergic orchitis the lesion is restricted to the germinal epithelium and there is no interstitial infiltration by lymphoid cells. 23 ,24 However, like human patients with leprosy, these animals had serum testicular antibodies which presumably were cytotoxic to germinal cells. For the main group of case studies we have thus failed to obtain convincing evidence for immunologic factors in the pathogenesis of idiopathic azo- or oligospermia. However, significant immunologic abnormalities have been detected in a minority of cases. One possible hypothesis is that antibodies may be directed against factors necessary for orderly spermatogenesis and that such antibodies may play a role in some cases of maturation arrest and shedding defects. In this study as in others,5, 25 the testicular lesions could not be correlated closely with the hormonal findings. However, a relationship between FSH levels and the products of testicular immunologic reaction, e.g., MIF, has not been excluded in the present study. SUMMARY
Infertile men with azo- or oligospermia of unknown cause were investigated for evidence oftesticular autoimmunity. Testicular germinal cell antibodies were found in 14% of the patients, compared with 5% of normal men, and 21% had spermatozoal antibodies, compared with 5% of the normal subjects. One-third had
October 1975
positive macrophage inhibitory factor tests, compared with 5% of normal subjects. However, of autoantibodies against thyroid, stomach, and nuclear material, only the prevalence of thyroid cytoplasmic antibodies was significantly greater than in normal subjects; serum IgG, IgM, and IgA levels were normal in all cases tested. Furthermore, there was no excess of lymphoid tissue on biopsy and no evidence of antibody deposition in the testicular tissue. The evidence for autoimmunity is less impressive than that for leprous orchitis, which has been proposed as a model for testicular organ-specific autoimmunity. Nevertheless, it is possible that certain germinal cell or spermatozoal antibodies may be directed against factors necessary for orderly spermatogenesis. If so, they may playa role in some cases of maturation arrest and shedding defects.
Acknowledgments. The authors wish to thank Mr. W. F. Hendry and Mr. H. Kinder for allowing us to study their patients, Mr. D. Newling for performance of the testicular biopsies, Dr. Missen and the pathology department for histologic examination of the biopsy specimens, Miss Marilyn Bailes for the hormonal estimations, and Mr. J. Gibbs for the seminal analyses.
REFERENCES 1. Johnsen SG: Testicular biopsies score count -
a method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormones 1:2, 1970 2. Meinhard E, McRae CH, Chisholm GD: Testicular biopsies in evaluation of male infertility. Br Med J 3:577, 1973 3. Franchimont P: In Reproductive Endocrinology, Edited by J Irvine. Edinburgh, Livingstone, 1970, p 1 4. deKretser DM, Burger HG, Fortune D, Hudson B, Long AR, Paulsen CA, Taft HP: Hormonal, histological and chromosomal studies in adult males with testicular disorders. J Clin Endocrinol Metab 35:392, 1972
•
,
t
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IMMUNOLOGIC STUDIES OF MALE INFERTILITY
5. Franchimont P, Miller D, Vendrely E, Setawe J, Legros JJ, Netter A: Relationship between spermatogenesis and serum gonadotrophin levels in azoospermia and oligospermia. J Clin Endocrinol Metab 34:1003,1972 6. deKretser DM, Burger HG, Hudson B: The relationship between germinal cells and serum FSH levels in males with infertility. J Clin Endocrinol Metab 38:787,1974 7. Wall JR, Wright DJM: Antibodies against testicular germinal cells in lepromatous leprosy. J Clin Immuno117:51, 1974 8. Wall JR, Stedronska J, Lessof MH: Cell-mediated immunity to testis in lepromatous leprosy. Submitted for publication, 1975 9. Boyum A: Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 21(SuppI97):77, 1968 10. Bloom BR, Glade PR (Editors): In Vitro Methods in Cell-Mediated Immunity. New York, Academic Press, 1971 11. David JR, AI-Askai S, Lawrence HS, Thomas L: Delayed hypersensitivity in vitro. 1. The specificity of inhibition of cell migration by antigens. J Immunol 93:264, 1964 12. Bendixen G, SIlborg M: A leucocyte migration technique for in vitro detection of cellular (delayed type) hypersensitivity in man. Danish Med Bull 16:1, 1969 13. Mancini G, Carbonara AO, Heremans JF: Immunochemical quantitations of antigens by single radial diffusion. Immunochemistry 2: 235, 1965 14. Collins WP, Mansfield MD, Alladina NS, Sommerville IF: Radioimmunoassay of plasma testosterone. J Steroid Biochem 3:333,1972 15. Besser GM, McNeilly AS, Anderson DC, Marshall JD, Harsoulis P, Hall R, Ormston BJ, Alexander L, Collins WP: Hormone response to synthetic luteinizing hormone and follicle
16. 17.
18.
19.
20.
21.
22. 23.
24.
25.
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stimulating hormone-releasing hormone in man. Br Med J 3:267, 1972 Wall JR, Stedronska J, Lessof MH: Antibodies against Sertoli cells in human infertility. Clin Endocrinol 3:187, 1974 Irvine WJ, Chan MM, North L, Kolb FO, Hartog M, Bayliss RS, Drury MI: Immunological aspects of premature ovarian failure associated with idiopathic Addison's disease. Lancet 2: 883, 1968 RUmke P: Autoantibody formation against spermatozoa caused by extravasation of spermatozoa within the interstitium of the epididymus of aged men. Int J Fertil 17:86, 1972 Rumke P, Hellinga G: Autoantibodies against spermatozoa in sterile men. Am J Clin Pathol 32:357,1959 Rose NR, Kite JH, Doebbler TK, Spier R, Skelton FR, Witebsky E: Studies on experimental thyroiditis. Ann NY Acad Sci 124:201, 1965 Volpe R, Edmonds M, Lamki L, Clarke PV, Row VV: The pathogenesis of Graves' disease: a disorder of delayed hypersensitivity. Mayo Clin Proc 47:824, 1972 Edmonds M, Lamki L, Kilbinger DW, Volpe R: Autoimmune thyroiditis, adrenalitis and oophritis. Am J Med 54:782, 1973 Bishop DW, Narbaitz R, Lessof MH: Induced aspermatogenesis in adult guinea pigs injected with testicular antigen and adjuvant in neonatal states. Dev BioI 3:444, 1961 Laurence KA, Carpuk 0, Lefense A, Mauldin D: Allergic aspermatogenesis in the guinea pig: induction and recovery studies. Int J Fertil 10:341, 1965 Paulsen CA, Leonard JM, deKretser DM: Interrelationship between spermatogenesis and FSH levIes in gonadotrophins, Edited by BB Saxena, GC Belling, HM Gandy. New York, WileyInterscience, 1972, p 628
Vol. 26, No. 10, October 1975 Printed in U.SA.
IMMUNOLOGIC STUDIES OF MALE INFERTILITY JACK R. WALL, M.D.,* M.R.A.C.P., D.H.M.S.A., JITKA STEDRONSKA, RAPHAEL D. DAVID, M.D., ROBBIE F. HARRISON, F.R.C.S.(E)., M.R.C.P.(I)., M.R.C.O.G., DOROTHY GORIUP, A.I.M.L.T., AND MAURICE H. LESSOF, M.A., M.D., F.R.C.P. Department of Medicine, Guy's Hospital Medical School, London Bridge SEl 9RT, United Kingdom
A large proportion of infertile men has no apparent underlying disorder. Testicular biopsy often reveals germinal cell aplasia, arrest of spermatogenesis at various stages, or shedding defects. 1, 2 The interstitium is usually normal. In most cases, plasma testosterone levels are within the normal range, although often lower than in fertile men. 3 ,4 Levels of luteinizing hormone (LH) are usually normal, 3 and follicle-stimulating hormone (FSH) levels are normal or high. 1, 2, 5, 6 Clinically, the only abnormality is the presence of small, soft testes, although in some cases they may be of normal size and consistency. Because of the possible role of testicular germinal cell antibodies and cellmediated immunity to testis in the orchitis of lepromatous leprosy/,8 immunologic studies of infertile men with testicular disorders of unknown cause were carried out. Evidence was obtained that primary immunologic abnormalities may play a role in a small number of cases of male infertility. EXPERTIMENTALPROCEDURE
who were attending the subfertility clinics at Chelsea and Guy's Hospitals. All had been infertile for at least 1 year and had fewer than 10 million spermatozoa/ml on repeated seminal analyses. Men with possible causes for the infertility, such as varicocele, mumps orchitis, cryptorchidism, reflux ejaculation, or trauma, were excluded from the study. The men appeared normal on examination, except for small testes (50% of cases), normalsized but soft testes (6% of cases), unilateral testicular atrophy (6% of cases), or mild hypogonadism (one case). Men with more than 10 million spermatozoa/ml but low motility or an increased proportion of abnormal spermatozoa were also excluded from the study. Following establishment of true oligospermia, the minimal investigations carried out were measurement of serum LH and FSH and plasma testosterone, and performance of testicular biopsy. The control group consisted of 100 male blood donors and laboratory staff, including 18 healthy men ages 23 to 57 (mean, 33 years) on whom lymphocyte cultures were carried out.
Clinical Subjects
The investigation concerned 63 men with azo- or oligospermia of unknown cause, ages 23 to 45 (mean, 31 years), Received December 6, 1974. *Reprint requests: Dr. J. R. Wall, Repatriation Hospital, Greenslopes, Brisbane, Australia.
Methods
Immunologic reaction to testicular antigens was assessed in infertile patients and normal subjects by measuring serum testicular antibodies and culturing peripheral lymphocytes in the presence of testis extracts.
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WALL ET AL.
Lymphocyte Cultures. Peripheral blood lymphocytes were separated from 20 ml of heparinized blood, using a Triosil-Ficoll gradient and following the method of Boyum. 9 The cells (0.3 to 1 x 106 ) were added to culture medium composed of 0.9 ml of TC199 (Wellcome Laboratories, Beckenham, England), 10% fetal calf serum, 0.1 ml of autologous serum, 50 U of penicillin/ml, and 100 p,g of streptomycin/ ml. Testis extract (0.1 ml) was added to test cultures in the dose range of 100 to 800 p.,g. Cultures were set up in triplicate. Control cultures consisted of (1) 0.1 ml of saline instead of antigen (unstimulated culture), (2) medium and 400 p.,g of testis but no cells (medium control), and (3) medium as for test cultures, but with antigen (400 p.,g) added at the end of the period of culture, before dialysis (macrophage inhibitory factor (MIF) control). Lymphocytes were cultured for 6 days at 37° C, without medium change. Lymphocyte cultures were carried out on 22 patients and 18 normal men. Lymphocyte Transformation. In the presence of specific antigen, sensitized thymus-derived (T) lymphocytes undergo blastoid transformation, commence cell division, and incorporate 3H-thymidine into newly synthesized DNA. This series of events correlates with cell-mediated immunity (delayed hypersensitivity) to the antigen, as determined by skin testing. tO Lymphocyte transformation in response to testis was estimated in 10 of the patients and in 10 normal subjects by adding 1 p.,Ci of 3H-thymidine (specific activity, 5 CiJmmole; Radiochemical Centre, Amersham, England) to the cultures 18 hours before termination. The cells were harvested, and the DNA content was precipitated in trichloroacetic acid and collected onto Whatman glass fiber filter discs. The oven-dried discs were dispersed in 12 ml of a toluene-based scintillation fluid and counted for 10 minutes in a
October 1975
Packard Tri-Carb liquid scintillation counter. Results were expressed as transformation ratios (TR), i.e., the ratio of counts per minute in antigen-stimulated cultures to counts per minute in unstimulated cultures. For the purposes of this study, a TR > 2.0 was considered positive. The mean maximal TR for infertile patients was compared with that for normal subjects. Macrophage Inhibitory Factor Tests. Sensitized T lymphocytes exert their effect primarily by releasing soluble mediators called lymphokines, of which the migration inhibitory factor has been most studied. Sensitized T lymphocytes release detectable amounts ofMIF in vitro within 8 hours of culturing in the presence of specific antigen. 10 The culture supernatants from infertile patients and normal subjects were tested for MIF production by the indirect method,l1, t2 using macrophages from unsensitized guinea pigs. The supernatants were dialyzed for 48 hours before testing. The tests were read with a projection microscope after 24 hours, and the migration areas were calculated with a planimeter. Quadruplicate estimations were made of each supernatant and the means were calculated. Results were expressed as migration indices (MI), i.e., the ratio of the mean area of macrophage migration with supernatants from stimulated cultures to migration with the supernatants from control (unstimulated) cultures. A migration index < 0.8 for any antigen concentration was considered positive for MIF. The mean minimal value (maximal inhibition) for infertile patients was compared with that for normal subjects.
Antigen Preparation. Because the testicular antigens which may be involved in infertility are species-nonspecific,7 rat testis, which is more readily available than human testis, was used as the antigen for the lymphocyte cultures in these studies. Human testis, however, was
Vol. 26, No. 10
IMMUNOLOGIC STUDIES OF MALE INFERTILITY
used for the immunofluorescence studies. Fresh rat testis was homogenized in 0.25 M sucrose, filtered, and extracted overnight at 4° C in an equal volume of phosphate-buffered saline (pH 7.15). The extract was centrifuged at 800 x g and the supernatant, containing subcellular elements and soluble proteins, was used for the lymphocyte cultures. The protein concentration of the supernatant was measured by the optical density method. Aliquots (0.1 ml) containing 100, 200, 400, or 800 ILg of protein were stored at - 20° C until used. Immunofluorescent Antibody Technique. Serum testicular germinal cell antibodies were tested for by the indirect immunofluorescent technique, using human testis as substrate, as previously described. 7 Antibodies against thyroid cytoplasm, gastric parietal cells, nuclear material, mitochondria, smooth muscle, and spermatozoa were also tested for by this technique, using human thyrotoxic thyroid, rat stomach and kidney, and human donor spermatozoa with >40 million motile spermatozoa/ml, respectively, as substrates. Serum dilution was 1:10 for all tests. A Reichert ultraviolet microscope with an SP3 exciting filter and an ultraviolet pass-absorbing filter was used. Serum Immunoglobulins. Serum immunoglobulins were measured in 15 patients by the radial diffusion method 13
1037
using Immunoplates (Hyland Laboratories, Los Angeles, Calif.). The normal ranges for this laboratory are: IgG, 500 to 1500 mg/100 ml; IgM, 47 to 170 mg/100 ml; and IgA, 160 to 420 mg/100 ml. Testicular Biopsies. The method of Johnsen1 for rating seminiferous tubular maturation was used. The tubular basement membranes, Sertoli cells, Leydig cells, and interstitium were also examined. In 20 cases, direct immunofluorescent tests using polyvalent and monovalent conjugates and anti-/He-/HA conjugates were carried out on frozen sections of testis for deposition of antibody or complement in testicular tissues which would indicate an immunologic reaction in vivo. Hormonal Estimations. Plasma testosterone and serum LH and FSH were measured by radioimmunoassays14. 15 in 27 randomly selected cases. For men, normal ranges in this laboratory are: testosterone, 320 to 835 ng/100 ml; LH, 0.7 to 13.2 mIUlml; and FSH, 0.1 to 3.2 mIU/ml. RESULTS
Serum antibodies against testicular germinal cells were found in 9 of 63 patients, compared with 5 of 100 normal men; however, the difference was not significant (Table 1). No staining of Sertoli cells16 or Leydig cells17 was found
TABLE 1. Prevalence of Serum Immunofluorescent Antibodies in Infertile Men with Azo- or Oligospermia of Unknown Cause Positive immunofluorescencea
Group Testisb
Infertile men (n = 63) 9 (14%) Normal men (n = 100) 5 (5%) pd NS
SpennatozoaC"
13 (21%) 5 (5%) 0.
--------------
_&L ______ I>
0 0
normal
l
infertile
normal
FIG. 1. In vitro Lymphocyte transformation to testis extract in infertile men with azoor oligospermia of unknown cause and in normal subjects. Values above the dotted line (TR > 2.0) indicate significant transformation. Testis concentrations over the range of 100 to 800 JLglml were used; maximal values for each culture are shown. There was no significant difference between the means of the two groups (3.07 ± 1.4 SEM and 1.41 ± 0.49 SEM, respectively).
FIG. 2. In vitro macrophage inhibitory factor production in infertile men with azo- or oligospermia of unknown cause and in normal subjects. Patients' lymphocytes were cultured in the presence of testis extract and the supernatants were tested for MIF by the indirect method. Values below the dotted line (MI < 0.8) indicate significant inhibition. Testis concentrations over the range 100 to 800 JLglml were used; maximal inhibition for each culture is shown. There was no significant difference between the means of the two groups (0.98 ± 0.37 SEM and 0.98 ± 0.26 SEM, respectively).
in this study. Spermatozoal antibodies were found in 13 of 63 patients, compared with 5 of 100 control subjects. This difference was significant (X 2 test;P < 0.001). Thirteen patients had one or more other serum autoantibodies: viz., against thyroid cytoplasm (seven patients), gastric parietal cells (three patients), smooth muscle (two patients), and nuclear material and mitochondria (one patient each). Such antibodies were found in 4 of 100 normal men: viz., thyroid cytoplasmic antibody (1 man; P < 0.001), gastric parietal cell antibody (2 men), and antinuclear antibody (1 man). These latter differences were not significant.
of the normal subjects (1.41 ± 0.49 SEM) (Fig. 1). Significant macrophage inhibition (MI < 0.8) was found in 7 of 22 infertile patients (32%), compared with 1 of 18 normal men (Fig. 2). This difference was significant (X 2 test; P < 0.05). There was no significant difference, however, between the means of the two groups (0.98 ± 0.37 SEM and 0.98 ± 0.26 SEM, respectively). Serum immunoglobulin levels were normal in all 15 patients tested.
Significant lymphocyte transformation to testis (TR > 2) was found in 2 of 10 infertile men tested, compared with 1 of10 normal men. The mean maximal value for the infertile group (3.07 ± 1.4 SEM) was not significantly different from that
Histologic examination of testicular biopsies provided little evidence for an immunologic disorder. None of the biopsies demonstrated increased interstitial lymphoid tissue. The most common findings were germinal cell aplasia, spermatozoal maturation arrests, shedding defects, and, occasionally, complete disorganization and fibrosis. Direct immunofluorescent tests on sections of the patients'
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IMMUNOLOGIC STUDIES OF MALE INFERTILITY
own testis with polyvalent (IgG/IgM), monovalent (lgG, IgM, and IgA), and anti-/He-/nA conjugates showed no evidence of antibody or complement deposition in the testicular tissue. Plasma testosterone levels were measured in 27 cases. Nine patients (33%) had levels below the lower limit of normal, while the mean for the group (461 ng/100 ml ± 35.7 SEM) was lower than that for a control group of normal men of comparable ages (522 ng/100 ml ± 35 SEM), although the difference was not significant. FSH levels were normal in 24 cases and undetectable in 2 cases. The latter two patients had biochemical evidence of pituitary deficiency, viz., low levels ofLH and FSH and borderline low levels of testosterone. Other pituitary function tests are presently being carried out. The mean testosterone level of patients with evidence of cell-mediated immunity to testis and/or testicular germinal cell antibodies was not significantly lower than that of patients with no such evidence of testicular autoimmunity. Similarly, the presence of spermatozoal antibodies did not correlate with testosterone levels. Finally, there was no close correlation between the histologic lesions found on biopsy and the presence of germinal cell antibodies or cell-mediated immunity to testis. However, patients with complete testicular fibrosis and disorganization tended to have these findings more often than those with maturation or shedding defects, although the difference was not significant. DISCUSSION
Of 63 men with azo- or oligospermia, 2 were found to have hypogonadotropic hypogonadism and an additional 7 had evidence of a failure of androgen production as well as of spermatogenesis. No distinctive features were found in the remainder. In contrast to that for lepromatous
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orchitis,7. 8 the evidence for an immunologic role in the pathogenesis of male infertility is not impressive. Only 14% of infertile men with primary testicular disorders tested had antibodies against testicular germinal cells, compared with 70% of lepromatous patients. None of the patients tested had elevated serum immunoglobulin levels, and there was no excess of lymphoid tissue on testicular biopsy. Although 21% of the patients had spermatozoal antibodies, it is likely that this was secondary to the underlying testicular lesion since there is no good evidence that spermatozoal antibodies are a cause of oligospermia. 18. 19 However, onethird had evidence of cell-mediated immunity to testis. It has been postulated7. 8 that leprous orchitis may be a model for an organ-specific autoimmune disease in which infection with Mycobacterium leprae induces the production of antibodies and sensitized T lymphocytes and leads to progressive testicular damage. The present study was initiated to test the hypothesis that some cases of infertility due to primary testicular disorders may be due to such organspecific autoimmunity. Although the over-all evidence is unimpressive and lymphoid tissue was absent on biopsy, one-third of this group had evidence of cell-mediated hypersensitivity to testis on in vitro testing. Since cell-mediated immunity is thought to be more important than antibodies in the pathogenesis of autoimmune disease,2()"22 the inability to detect germinal cell antibodies does not exclude this etiology, especially in advanced cases with fibrosed, disorganized testes. However, this explanation seems inadequate in view of the high prevalence of antibodies in leprosy, since leprosy is associated with both the autoantibodies and the cellular reaction of an autoimmune orchitis. Therefore, it must be postulated either that male infertility has different causes or that more than
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one disease group was represented by the parents we studied. The commonly found histologic lesions of spermatozoal maturation arrest, germinal cell aplasia, shedding defects, and gross fibrosis and disorganization, however, do not resemble classic immunologic disorders such as are found in Hashimoto's thyroiditis or idiopathic adrenalitis. Again, this does not exclude an immunologic pathogenesis, since it is well known that in experimental auto allergic orchitis the lesion is restricted to the germinal epithelium and there is no interstitial infiltration by lymphoid cells. 23 ,24 However, like human patients with leprosy, these animals had serum testicular antibodies which presumably were cytotoxic to germinal cells. For the main group of case studies we have thus failed to obtain convincing evidence for immunologic factors in the pathogenesis of idiopathic azo- or oligospermia. However, significant immunologic abnormalities have been detected in a minority of cases. One possible hypothesis is that antibodies may be directed against factors necessary for orderly spermatogenesis and that such antibodies may play a role in some cases of maturation arrest and shedding defects. In this study as in others,5, 25 the testicular lesions could not be correlated closely with the hormonal findings. However, a relationship between FSH levels and the products of testicular immunologic reaction, e.g., MIF, has not been excluded in the present study. SUMMARY
Infertile men with azo- or oligospermia of unknown cause were investigated for evidence oftesticular autoimmunity. Testicular germinal cell antibodies were found in 14% of the patients, compared with 5% of normal men, and 21% had spermatozoal antibodies, compared with 5% of the normal subjects. One-third had
October 1975
positive macrophage inhibitory factor tests, compared with 5% of normal subjects. However, of autoantibodies against thyroid, stomach, and nuclear material, only the prevalence of thyroid cytoplasmic antibodies was significantly greater than in normal subjects; serum IgG, IgM, and IgA levels were normal in all cases tested. Furthermore, there was no excess of lymphoid tissue on biopsy and no evidence of antibody deposition in the testicular tissue. The evidence for autoimmunity is less impressive than that for leprous orchitis, which has been proposed as a model for testicular organ-specific autoimmunity. Nevertheless, it is possible that certain germinal cell or spermatozoal antibodies may be directed against factors necessary for orderly spermatogenesis. If so, they may playa role in some cases of maturation arrest and shedding defects.
Acknowledgments. The authors wish to thank Mr. W. F. Hendry and Mr. H. Kinder for allowing us to study their patients, Mr. D. Newling for performance of the testicular biopsies, Dr. Missen and the pathology department for histologic examination of the biopsy specimens, Miss Marilyn Bailes for the hormonal estimations, and Mr. J. Gibbs for the seminal analyses.
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