Sperm Antibody Testing in Infertile Men
Syst Biol Reprod Med Downloaded from informahealthcare.com by Flinders University of South Australia on 01/14/15 For personal use only.
C. SCHOENFELD, R. D. AMELAR, and L. DUBIN Six hundred male patients were examined for circulating spermagglutinating antibodies by the Kibrick sperm-agglutination test and 20% demonstrated autoantibodies. The high incidence of positive findings may be explained by the select population studied. Of 80 men tested who demonstrated more than 10% agglutination in their semen, but were otherwise normospermic, 41% demonstrated positive titers by the Kibrick method. Of 300 men examined for circulating sperm-immobilizing antibodies by the Isojima sperm-immobilizing test, 6% had autoantibodies. This high incidence of positive findings demonstrates the need for these tests in males who otherwise appear normospermic or demonstrate an unexplained infertility. Key Words: Autoantibodies; Sperm agglutination; Sperm immobilization; Kibrick spermagglutination test; lsojima sperm-immobilization test.
INTRODUCTION
Although the ability for spermatozoa to invoke an immunological response has been well established [7,8], it was not until recently that this immunological process was demonstrated to play a major role in reproduction. While these factors occur in both males and females, only their role in males will be discussed. Implicit in the immunologic processes is the ability of a host to recognize foreign antigens. Since the various parts of the male reproductive system do not attain functional maturity until puberty, are unique, and are released outside the body, the individual has no opportunity to recognize these components as self-antigens. Thus, any antigen, not present or sequestered when tolerance to self-antigens is being developed, may at a later time evoke an immunological response. Sperm fulfill these criteria since they are sequestered within the blood-testis barrier. This investigation was undertaken in order to determine the occurrence of sperm autoantibodies among infertile men. MATERIALS AND METHODS The serum of 600 male patients was examined for the presence of circulating spermagglutinating antibodies using the Shulman modification[ 121 of the macroscopic gelatin sperm agglutination test of Kibrick et al. [6]. The patients were selected from couples demonstrating infertility for at least two years. Serum was inactivated at 56°C for 30 minutes and routinely tested in dilutions of 1:4 with at least two different semen samples. All were tested in the presence of positive and negative control sera. Sera positive at 1:4 were tested by two-fold serial dilutions, 114-1:4,096. Received August 1977. From the Fertility Laboratory, Inc. and the Department of Urology, New York University School of Medicine, New York. Reprint requests to Fertility Laboratory, Inc., 137 East 36 Street, New York, N.Y. 10016 U.S.A. (Dr. Cy Schoenfeld). @ ARCHIVES OF ANDROLOGY I (January 1978): 1 1 1-1 14
111
112
C. Schoenfeld et al.
Syst Biol Reprod Med Downloaded from informahealthcare.com by Flinders University of South Australia on 01/14/15 For personal use only.
The serum of 300 male patients was examined for the presence of circulating spermimmobilizing antibodies using the Isojima sperm-immobilization test [ 5 ] . Sera were inactivated as above and routinely tested undiluted in the presence of hamster or rabbit complement. Positive samples were tested in two-fold dilutions, 1: I- 1: 1.024. Specimens were tested in the presence of both positive and negative control sera with at least two different semen samples.
RESULTS Positive circulating sperm-agglutination titers occurred in 120 males (20%) of those tested (Table I). Titers ranged from 1 :4 to more than 1 : 1,024. Of the 600 male patients tested, 225 had no organic or apparent cause for their infertility since the physical examinations, endocrine profile, and semen analyses were all within normal limits (Table 2). An additional 80 men were completely normal except for their semen analyses which demonstrated that 10% or more of their spermatozoa agglutinated. Of the 300 male patients tested, 19 (6%) demonstrated positive circulating spermimmobilizing antibodies with titers ranging from I : 4 to 1 : 128 (Table 1). Patients with circulating sperm-agglutinating antibodies did not necessarily have positive spermimmobilizing antibodies. The converse was true, although seven men had both types of antibodies in their sera.
DISCUSSION Although the relationship between circulating sperm-agglutinating antibodies and infertility is not an absolute one, a correlation is made apparent through the use of the Kibrick sperm-agglutination test. Rumke et al. [ 101 demonstrated the fertility status of 254 men who had been unable to have children and who had sperm-agglutination titers ranging from I :4 to greater than 1 : 1,024. All of the titers were detected using the Kibrick test (61. Of the men studied by Rumke, 36 became fertile following the testing.
TABLE 1 Titers of Cirrulating Sperm-Agglutinatingand Sperm-Immobilizing Antibodies
SPERM AGGLUTINATING ANTIBODIES (600 MEN) TITER
% POSITIVt
ANTIBODIES (300 M E N ) TITER % POSITIVE
undiluted
1 :4 1 :8 1.16
1:1 1:4 1 :8 1:16 1 :32
I :32 1 :64 I : 128 1 :?56
1
:a
I : I28 51 :256
1:512 41:1,024
Total
SPERM IMMOBILIZING
20
Total
6
Sperm Antibody Testing in Infertile Men
113
TABLE 2 Infertile Males Examined for Circulating SpermagglutinatingAntibodies Using the Kibrick Test
Syst Biol Reprod Med Downloaded from informahealthcare.com by Flinders University of South Australia on 01/14/15 For personal use only.
DIAGNOSE
NUMBER PATIENTS
% POSITIVE
Normal Normal (except for agglutination in semen) Varicocele left right bilateral Hypogonadotropic oligospermia Hydrocele Post vasovasostomy Azoospermia bilateral absence of vasa post vasectomy atrophic testis hypogonadotropism epididymal obstruction (bilateral)
225 80
41
100 2 24
I 0 12
10 2 28
20 0 25
56 20 14 12
63 65 0 0
21
30
Total
600
20
5
There was an inverse relationship between the circulating sperm-agglutinating titer and the probability of becoming fertile in these normospermic men. It seems that serum titers up to 1:16 have no clinical significance but that the significance increases when the titers are higher; only 7 of 91 normospermic men who had titers of 1 :256 or higher eventually became fathers. The greatest decrease in the probability of becoming fertile occurred between the titers of 1 : 16 and 1:32. The finding of positive circulating sperm-agglutinating antibodies in 20% of the patients tested confirms a previous report [11] that these patients were a highly selected group. Of the 225 normospermic males in this study, only 12 (5%) had circulating sperm-agglutinating antibodies in their sera. This percentage compares favorably with a previous report in women of 4.4% [13]. Among the 80 patients in this series that demonstrated more than 10% agglutination of their semen specimens, over 4 1% demonstrated circulating sperm-agglutinins. In addition, this current group of patients included persons with a high incidence of sperm-agglutinating antibody, namely men with bilateral absence of the vasa and vasectomized men [I]. The Kibrick test appears to be a more reliable indicator of true circulating spermagglutinins than the Franklin-Dukes Sperm Agglutination test [3] since the Kibrick test measures an antibody which is a true antibody found in the immunoglobulin G fraction [2]. In addition, the Franklin-Dukes test yields a high percentage of positive results for sperm-agglutinating activity in the sera of women who were pregnant at the time of testing [9].
Syst Biol Reprod Med Downloaded from informahealthcare.com by Flinders University of South Australia on 01/14/15 For personal use only.
114
C. Schoenfeld et al.
Of the 300 male patients examined for circulating sperm-immobilizing antibody by the Isojima sperm-immobilizing test, 19 (6%) demonstrated positive titers ranging from 1:4 to 1:128. This percentage differs greatly from Isojima's initial study of women with unexplained infertility, where 12% demonstrated positive reactions in the test [ 5 ] . Isojima also reported that 1% of the women studied demonstrared spermimmobilizing antibodies in their sera [4] and 6Wo demonstrated positive spermagglutinating antibodies when the Kibrick test was used. In a previous report by the authors, 6% of a group of male patients demonstrated positive sperm-immobilizing antibodies in their sera [ 113. The differences in the percentages of positive patients for both the Kibrick sperm-agglutinating test and the Isojima sperm-immobilizing test appears to be due to an unknown factor in the selection of patients. The Isojima sperm-immobilization test is an excellent method for measuring the antibody which causes immobilization of spermatozoa. The activity of this antibody occurs in the immunoglobulin G fraction of serum and the reaction is complement dependent, affording assurance that one is measuring a true antibody. This test is best utilized when studying men whose semen demonstrates necrospermia or asthenospermia and when all other causes have been ruled out. REFERENCES
zoa. 11. A gelatin agglutination test. Frrtif I . Amelar. R. D.. Dubin, L.. and SchoenStrril 3, 430-438. feld, C. ( 1975): Circulating spermagglutin7. Landsteiner, K. (1899): Kenntnis der speating antibodies in azoospermic men with congenital bilateral absence of the vasa zifisch auf Blukorpechen wirkende Sera. Zrntralbl Bakteriol 25, 546-549. diferentia. Fertil Steril 26, 228-23 1 . 8. Metchnikoff. E. I. (1899): ktudes sur la 2. Boettcher. B., Kay, D. J . , Rumke, P.. resorption des cellules. Ann lnsr Pasteirr and Wright, L. E. (1971): Human sera 13, 737-769. containing immunoglobulin and nonimmu9. Pacheco-Romero, J . C., Gleich, G. J.. noglobu 1in spe rmagglu t i n i n s . Biol R ryro d Loegering, D. A . , and Johnson, C . (1973): 5 , 236-245. Spermagglutinating activity and female in3. Franklin, R. R. and Dukes, C . D. (1964): fertility: a study of serum and genital tract Antispermatozoal antibody and unexplained infertility. Anzrr J Obstet G~t7~'('0/ secretions. JAMA 224,849-852. 10. Rumke, P.. Van Amstel, N . , Messer, E. 89, 6-9. N.. and Benzemer. P. D. (1974): Prog4. Isojima, S . (1969): Relationship between nosis of fertility of men with sperm aggluantibodies to spermatozoa and sterility in tinins in the serum. Fprtil Srpvil 25, 393females. In: Jnimrinology iind Rqwotlric~398. tion, P. G . Edwards. ed., International Planned Parenthood Federation, London. 1 1 . Schoenfeld. C., Amelar, R . D.. and Dubin, L. (1976): Clinical experience with pp. 267-279. sperm antibody testing. Ftjrtil Strril 27, 5 . Isojima. S.. Li. T. S.. and Ashitaka. Y. 1199-1203. (1968): Immunologic analysis of spermimmobilizing factor found in sera of 12. Shulman. S. (1971): Sperm antibodies as a cause of infertility. CRC' Crit Rev C'liji \cornen with unexplained sterility. Amcr J OhstcJt GyricJt.ol101, 677-683. Lab S t i 2, 393-460. 6. Kibrick, S.. Belding, D. L.. and Merrill. 13. Shulman. S. (1972): Immunologic barriers t o fertility. Obstct Gynecol Sitrvty 27, €3. (1952): Methods for the detection of .. 5 5 3- 606. antibodies against mammalian spermato-
Syst Biol Reprod Med Downloaded from informahealthcare.com by Flinders University of South Australia on 01/14/15 For personal use only.
C. SCHOENFELD, R. D. AMELAR, and L. DUBIN Six hundred male patients were examined for circulating spermagglutinating antibodies by the Kibrick sperm-agglutination test and 20% demonstrated autoantibodies. The high incidence of positive findings may be explained by the select population studied. Of 80 men tested who demonstrated more than 10% agglutination in their semen, but were otherwise normospermic, 41% demonstrated positive titers by the Kibrick method. Of 300 men examined for circulating sperm-immobilizing antibodies by the Isojima sperm-immobilizing test, 6% had autoantibodies. This high incidence of positive findings demonstrates the need for these tests in males who otherwise appear normospermic or demonstrate an unexplained infertility. Key Words: Autoantibodies; Sperm agglutination; Sperm immobilization; Kibrick spermagglutination test; lsojima sperm-immobilization test.
INTRODUCTION
Although the ability for spermatozoa to invoke an immunological response has been well established [7,8], it was not until recently that this immunological process was demonstrated to play a major role in reproduction. While these factors occur in both males and females, only their role in males will be discussed. Implicit in the immunologic processes is the ability of a host to recognize foreign antigens. Since the various parts of the male reproductive system do not attain functional maturity until puberty, are unique, and are released outside the body, the individual has no opportunity to recognize these components as self-antigens. Thus, any antigen, not present or sequestered when tolerance to self-antigens is being developed, may at a later time evoke an immunological response. Sperm fulfill these criteria since they are sequestered within the blood-testis barrier. This investigation was undertaken in order to determine the occurrence of sperm autoantibodies among infertile men. MATERIALS AND METHODS The serum of 600 male patients was examined for the presence of circulating spermagglutinating antibodies using the Shulman modification[ 121 of the macroscopic gelatin sperm agglutination test of Kibrick et al. [6]. The patients were selected from couples demonstrating infertility for at least two years. Serum was inactivated at 56°C for 30 minutes and routinely tested in dilutions of 1:4 with at least two different semen samples. All were tested in the presence of positive and negative control sera. Sera positive at 1:4 were tested by two-fold serial dilutions, 114-1:4,096. Received August 1977. From the Fertility Laboratory, Inc. and the Department of Urology, New York University School of Medicine, New York. Reprint requests to Fertility Laboratory, Inc., 137 East 36 Street, New York, N.Y. 10016 U.S.A. (Dr. Cy Schoenfeld). @ ARCHIVES OF ANDROLOGY I (January 1978): 1 1 1-1 14
111
112
C. Schoenfeld et al.
Syst Biol Reprod Med Downloaded from informahealthcare.com by Flinders University of South Australia on 01/14/15 For personal use only.
The serum of 300 male patients was examined for the presence of circulating spermimmobilizing antibodies using the Isojima sperm-immobilization test [ 5 ] . Sera were inactivated as above and routinely tested undiluted in the presence of hamster or rabbit complement. Positive samples were tested in two-fold dilutions, 1: I- 1: 1.024. Specimens were tested in the presence of both positive and negative control sera with at least two different semen samples.
RESULTS Positive circulating sperm-agglutination titers occurred in 120 males (20%) of those tested (Table I). Titers ranged from 1 :4 to more than 1 : 1,024. Of the 600 male patients tested, 225 had no organic or apparent cause for their infertility since the physical examinations, endocrine profile, and semen analyses were all within normal limits (Table 2). An additional 80 men were completely normal except for their semen analyses which demonstrated that 10% or more of their spermatozoa agglutinated. Of the 300 male patients tested, 19 (6%) demonstrated positive circulating spermimmobilizing antibodies with titers ranging from I : 4 to 1 : 128 (Table 1). Patients with circulating sperm-agglutinating antibodies did not necessarily have positive spermimmobilizing antibodies. The converse was true, although seven men had both types of antibodies in their sera.
DISCUSSION Although the relationship between circulating sperm-agglutinating antibodies and infertility is not an absolute one, a correlation is made apparent through the use of the Kibrick sperm-agglutination test. Rumke et al. [ 101 demonstrated the fertility status of 254 men who had been unable to have children and who had sperm-agglutination titers ranging from I :4 to greater than 1 : 1,024. All of the titers were detected using the Kibrick test (61. Of the men studied by Rumke, 36 became fertile following the testing.
TABLE 1 Titers of Cirrulating Sperm-Agglutinatingand Sperm-Immobilizing Antibodies
SPERM AGGLUTINATING ANTIBODIES (600 MEN) TITER
% POSITIVt
ANTIBODIES (300 M E N ) TITER % POSITIVE
undiluted
1 :4 1 :8 1.16
1:1 1:4 1 :8 1:16 1 :32
I :32 1 :64 I : 128 1 :?56
1
:a
I : I28 51 :256
1:512 41:1,024
Total
SPERM IMMOBILIZING
20
Total
6
Sperm Antibody Testing in Infertile Men
113
TABLE 2 Infertile Males Examined for Circulating SpermagglutinatingAntibodies Using the Kibrick Test
Syst Biol Reprod Med Downloaded from informahealthcare.com by Flinders University of South Australia on 01/14/15 For personal use only.
DIAGNOSE
NUMBER PATIENTS
% POSITIVE
Normal Normal (except for agglutination in semen) Varicocele left right bilateral Hypogonadotropic oligospermia Hydrocele Post vasovasostomy Azoospermia bilateral absence of vasa post vasectomy atrophic testis hypogonadotropism epididymal obstruction (bilateral)
225 80
41
100 2 24
I 0 12
10 2 28
20 0 25
56 20 14 12
63 65 0 0
21
30
Total
600
20
5
There was an inverse relationship between the circulating sperm-agglutinating titer and the probability of becoming fertile in these normospermic men. It seems that serum titers up to 1:16 have no clinical significance but that the significance increases when the titers are higher; only 7 of 91 normospermic men who had titers of 1 :256 or higher eventually became fathers. The greatest decrease in the probability of becoming fertile occurred between the titers of 1 : 16 and 1:32. The finding of positive circulating sperm-agglutinating antibodies in 20% of the patients tested confirms a previous report [11] that these patients were a highly selected group. Of the 225 normospermic males in this study, only 12 (5%) had circulating sperm-agglutinating antibodies in their sera. This percentage compares favorably with a previous report in women of 4.4% [13]. Among the 80 patients in this series that demonstrated more than 10% agglutination of their semen specimens, over 4 1% demonstrated circulating sperm-agglutinins. In addition, this current group of patients included persons with a high incidence of sperm-agglutinating antibody, namely men with bilateral absence of the vasa and vasectomized men [I]. The Kibrick test appears to be a more reliable indicator of true circulating spermagglutinins than the Franklin-Dukes Sperm Agglutination test [3] since the Kibrick test measures an antibody which is a true antibody found in the immunoglobulin G fraction [2]. In addition, the Franklin-Dukes test yields a high percentage of positive results for sperm-agglutinating activity in the sera of women who were pregnant at the time of testing [9].
Syst Biol Reprod Med Downloaded from informahealthcare.com by Flinders University of South Australia on 01/14/15 For personal use only.
114
C. Schoenfeld et al.
Of the 300 male patients examined for circulating sperm-immobilizing antibody by the Isojima sperm-immobilizing test, 19 (6%) demonstrated positive titers ranging from 1:4 to 1:128. This percentage differs greatly from Isojima's initial study of women with unexplained infertility, where 12% demonstrated positive reactions in the test [ 5 ] . Isojima also reported that 1% of the women studied demonstrared spermimmobilizing antibodies in their sera [4] and 6Wo demonstrated positive spermagglutinating antibodies when the Kibrick test was used. In a previous report by the authors, 6% of a group of male patients demonstrated positive sperm-immobilizing antibodies in their sera [ 113. The differences in the percentages of positive patients for both the Kibrick sperm-agglutinating test and the Isojima sperm-immobilizing test appears to be due to an unknown factor in the selection of patients. The Isojima sperm-immobilization test is an excellent method for measuring the antibody which causes immobilization of spermatozoa. The activity of this antibody occurs in the immunoglobulin G fraction of serum and the reaction is complement dependent, affording assurance that one is measuring a true antibody. This test is best utilized when studying men whose semen demonstrates necrospermia or asthenospermia and when all other causes have been ruled out. REFERENCES
zoa. 11. A gelatin agglutination test. Frrtif I . Amelar. R. D.. Dubin, L.. and SchoenStrril 3, 430-438. feld, C. ( 1975): Circulating spermagglutin7. Landsteiner, K. (1899): Kenntnis der speating antibodies in azoospermic men with congenital bilateral absence of the vasa zifisch auf Blukorpechen wirkende Sera. Zrntralbl Bakteriol 25, 546-549. diferentia. Fertil Steril 26, 228-23 1 . 8. Metchnikoff. E. I. (1899): ktudes sur la 2. Boettcher. B., Kay, D. J . , Rumke, P.. resorption des cellules. Ann lnsr Pasteirr and Wright, L. E. (1971): Human sera 13, 737-769. containing immunoglobulin and nonimmu9. Pacheco-Romero, J . C., Gleich, G. J.. noglobu 1in spe rmagglu t i n i n s . Biol R ryro d Loegering, D. A . , and Johnson, C . (1973): 5 , 236-245. Spermagglutinating activity and female in3. Franklin, R. R. and Dukes, C . D. (1964): fertility: a study of serum and genital tract Antispermatozoal antibody and unexplained infertility. Anzrr J Obstet G~t7~'('0/ secretions. JAMA 224,849-852. 10. Rumke, P.. Van Amstel, N . , Messer, E. 89, 6-9. N.. and Benzemer. P. D. (1974): Prog4. Isojima, S . (1969): Relationship between nosis of fertility of men with sperm aggluantibodies to spermatozoa and sterility in tinins in the serum. Fprtil Srpvil 25, 393females. In: Jnimrinology iind Rqwotlric~398. tion, P. G . Edwards. ed., International Planned Parenthood Federation, London. 1 1 . Schoenfeld. C., Amelar, R . D.. and Dubin, L. (1976): Clinical experience with pp. 267-279. sperm antibody testing. Ftjrtil Strril 27, 5 . Isojima. S.. Li. T. S.. and Ashitaka. Y. 1199-1203. (1968): Immunologic analysis of spermimmobilizing factor found in sera of 12. Shulman. S. (1971): Sperm antibodies as a cause of infertility. CRC' Crit Rev C'liji \cornen with unexplained sterility. Amcr J OhstcJt GyricJt.ol101, 677-683. Lab S t i 2, 393-460. 6. Kibrick, S.. Belding, D. L.. and Merrill. 13. Shulman. S. (1972): Immunologic barriers t o fertility. Obstct Gynecol Sitrvty 27, €3. (1952): Methods for the detection of .. 5 5 3- 606. antibodies against mammalian spermato-
