- Email: [email protected]
Sperm Antibodies in Vasectomized Men*
Vol. 23. No.9, September 1972 Printed in U.S.A.
FERTIUTY AND STERILITY
Copyright © 1972 by The Williams & Wilkins Co.
SPERM ANTIBODIES IN VASECTOMIZED MEN* Rum ANSBACHER, M.D., M.S., KWOK KEUNG-YEUNG, M.B.B.S., t AND JOHN C. WURSTER, M.D.
Department of Obstetrics and Gynecology and Urology Service, Brooke General Hospital, Fort Sam Houston, Texas 78234, and Department of Obstetrics and Gynecology, The University of Michigan Medical Center, Ann Arbor, Michigan 48104
Immunologic and followup studies on bilaterally vasectomized men have recently been advocated to determine the onset and incidence of sperm antibody production and to evaluate possible sequelae. 1 - 3 With the demand increasing for vasectomy throughout the United States and abroad, with the introduction of the use of plugs of various materials and removable clips to temporarily interrupt the function of the vas deferens, and with the application of a vas deferens valve that can be turned off and on at will to allow sperm passage, further studies must be done to delineate long-term medical problems that may arise and to determine whether reversibility as evidenced by fertility is obtainable. In 1959, Riimke and Hellinga 4 suggested that the initiation of sperm antibody production could occur from the extravasation of spermatozoa into the interstitium, lymph vessels, or blood capillaries of the epididymis with subsequent transfer to the regional lymph nodes. Subsequently, Phadke 5 demonstrated intraluminal phagocytosis as one mechanism involved in the disposal of spermatozoa in patients with obstructive azoospermia or with ligated vas deferens. In this study, we compared the findings at the 12-month examination with those previously reported for the 6-month postvasectomy visit. 1 In addition, a new Received March 2, 1972; revised April 26, 1972. * Presented at the 28th Annual Meeting of The American Fertility Society, New York, N. Y., 1972. t China Medical Board Research Fellow.
640
group of men undergoing vasectomy were studied to determine the onset of sperm antibody production with special attention to serum uric acid values prior to and 6 weeks after vasectomy, due to the recent report of elevated serum uric acids in vasectomized men. 6 MATERIALS AND METHODS
Ten milliliters of blood were obtained from 27 men returning to our vasectomy clinic 1 year following surgery. The serum was removed after centrifugation of the clotted blood and complement was destroyed by applying heat at 56° C. for 30 min. The macroscopic gelatin sperm-agglutination test of Kibrick, Belding, and Merrill 7 and the sperm-immobilization procedure of Isojima, Li, and Ashitaka 8 were used to determine the presence of sperm antibody activity in the sera. Pooled untreated rabbit serum served as the complement source. Ejaculates were collected from 24 vasectomized men and examined for spermatozoa. The seminal plasmas were centrifuged at 2500 r.p.m. for 15 min., the supernatant removed, the complement destroyed, and the sperm antibody activity determined. Fresh semen was obtained from 2 donors whose counts were consistently above 60 million spermatozoa per milliliter coupled with motility greater than 70%. A health questionnaire was completed by each patient. Blood was also obtained prior to vasectomy from 51 men whose average age was
September 1972
641
SPERM ANTIBODIES
TABLE 1. Sperm-Agglutiootion Tests (Serum) Negative
Positive
1 (3.5%) (Titer 1 :4)
Postvasectomy (1 yr.)
Postvasectomy (6 mos.)
Prevasectomy
26
Total
Positive
Negative
Total
13
27
14 (51. 9%) (Titers 1: 2-1 : 64)
27
Negative
Positive
15 (55.5%) 12 (Titers 1: 2-1 : 1024)
Total
27
TABLE 2. Sperm-Immobilization Tests (Serum) Postvasectomy (6 mos.)
Prevasectomy
Positive
o
Negative
27
Total
Positive
Negative
10 (37%) (Titers 1: 2-1: 128)
27
37 years and who had fathered an average of 3 children. Thirty-seven (72%) returned for the 6-week postoperative examination, at which time blood and semen samples were collected, processed, and tested as described above. Uric acids were determined on the pre- and 6-week postvasectomy sera by the manual method as adapted by Technicon. 9 RESULTS
Comparable data for sperm-agglutinating and sperm-immobilizing antibodies prevasectomy, 6 months postvasectomy, and 1 year following vasectomy for the 27 men are given in Tables 1 and 2, respectively. No significant difference between the incidence of sperm antibodies at 6 months and 1 year postvasectomy was apparent. Immobilizing antibodies were observed only when agglutinating antibodies were present. The sperm antibody titers are compared in Table 3. Increased titers were demonstrated in 5 men (greater than a two tube dilution increase). Microscopic examination of the ejaculates revealed that only 1 of the 24 specimens contained a few sluggishly motile spermatozoa. Agglutinating and immobilizing antibodies could not be found in the seminal plasmas examined. Eight of the 15 men with circulating sperm antibodies 1 year following bilateral
Postvasectomy (1 yr.)
---Total
17
Positive
27
Negative
Total
16
27
11 (40.7%) (Titers 1: 2-1: 1024)
TABLE 3. Serum Sperm Antibody Titers Postvasectomy Titer
Agglutination
Immobilization
6 mos.
1 yr.
1 5 4 0 3 0 0 0 0
2 0 5 2 0 2 1 1 0 2
2 3 0 3 1 0 1 0 0 0
1 3 4 0 0 0 0 1 0 2
14
15
10
11
6 mos ..
1 vr.
--------
1:2 1:4 1:8 1: 16 1:32 1:64 1:128 1:256 1:512 1:1024
1
Total
TABLE 4. Sperm-Agglutiootion Tests (Serum) Postvasectomy (6 wk.)
Prevasectomy Positive
Nega-
0
51
tive
Total
Positive
51
19 (51.3%) (Titers 1: 2-1: 128)
Negative
Total
18
37
vasectomy gave a positive allergy history, whereas only 3 of the 12 men with no detectable sperm antibodies had a positive allergy history. No other pertinent history could be correlated from this group and no adverse sequelae following vasectomy were documented. Sperm-agglutinating antibodies were present in 19 sera (51.3%) 6 weeks after vasectomy (Table 4). Fourteen (37.8%) men had circulating sperm-immobilizing antibodies (Table 5) although 4 of them
642
ANSBACHER ET AL.
Vol. 23
showed a negative sperm-agglutination test. The serum sperm antibody titers ranged from 1: 2-1: 128 (Table 6). None of these men had demonstrable sperm antibodies prior to vasectomy. Only 1 of the 37 semen samples 6 weeks
postvasectomy contained motile spermatozoa, and none had sperm antibodies. Serum uric acid levels were not statistically different after vasectomy (Table 7, t test).
TABLE 5. Sperm-Immobilization Tests (Serum)
The incidence of sperm antibodies in vasectomized men 1 year postoperatively was comparable to that of 6 months postoperatively. However, definite sperm antibody titer increases occurred in 5 of the men indicative of a continued antigenic stimulus with subsequent antibody production. This finding was evident for both sperm-agglutinating and spermimmobilizing circulating antibodies. A long-term followup study on this group of men is planned. The incidence of sperm antibodies in men 6 weeks after vasectomy parallels that at 6 and 12 months postvasectomy, but the titers at the earlier date were lower than at the later ones. Studies are in progress to ascertain whether the antibody response is immediate or delayed. Since spermatogenesis occurs at least for some time following bilateral vasectomy,10 destruction and subsequent phagocytosis of spermatozoa may add significantly to the nucleic acid pool, as possibly reflected by an increase in the serum uric acid. No change in serum uric acid concentration 6 weeks following vasectomy had occurred in our series, but this observation does not exclude a delayed response which we are now investigating. The necessity of followup studies on technics for temporary interruption of the vas deferens is evident from the findings in a man who had undergone vasovasostomy 8 years after vasectomy. Prior to the vasovasostomy, a serum sperm-agglutination titer of 1: 4 was recorded. Two months following vasovasostomy, his ejaculate contained motile spermatozoa with a sperm count of 30 million/ml., although
Prevasectomy
Postvasectomy (6 wk.)
Posi· tive
Nega· tive
Total
Positive
0
51
51
14 (37.8%) (Titers 1:2-1:64)
Nega· tive
Total
23
37
TABLE 6. Serum Sperm Antibody Titers 6 Weeks Postvasectomy Titer
Agglutination
Immobilization
1:2 1:4 1:8 1:16 1:32 1:64 1:128
1 3 5 5 3 1 1
2 5 2 4 0 1 0
Total
19
14
TABLE 7. Serum Uric Acid (mg. %) Name
Age
Prevasectomy
P08tvasectomy (6 wk.)
BEN MET REE ERK BUR HES CLA SYL BRO CAR KRI SNI TOU GON BUT CAS HED MON TAY
36 31 43 33 42 49 32 30 43 43 28 49 41 38 32 34 38 32 43
5.6 6.7 6.6 6.7 7.2 5.8 7.0 7.9 8.6 6.7 7.5 5.5 6.1 6.8 5.4 5.6 5.7 7.4 8.2
5.5 6.4 5.5 6.3 7.6 5.5 5.8 7.2 8.3 6.6 6.9 4.5 5.4 5.7 5.2 5.6 6.0 7.5 8.3
6.68 ±0.96
6.30 ±1.07
Mean S.D.
37.8
DISCUSSION
September 1972
643
SPERM ANTIBODIES
his serum findings were unchanged. Nine months after vasovasostomy, the serum sperm-agglutination titer had risen to 1: 16 and the sperm count had increased to 50 million/ml. Examination of his seminal plasma for sperm antibody activity revealed a 1: 16 agglutination titer, suggesting that the antibody was being transferred to the seminal plasma postvasovasostomy, since sperm antibodies could not be demonstrated in his seminal plasma by these technics previously. Sperm-immobilization tests on both his sera and seminal plasmas were consistently negative. This single observation must be confirmed by examining a significant number of vasovasostomy patients. Similar studies must be performed on specimens obtained from men utilizing temporary methods of vas deferens occlusion for contraceptive purposes. These procedures may be irreversible when sperm antibodies are formed. SUMMARY
Circulation sperm-agglutinating antibodies were demonstrated in 15 of 27 men (55.5%) who returned for examination 1 year postvasectomy. Sperm-immobilizing antibodies were present in 11 cases (40.7%), and were found only when spermagglutinating antibodies were present. Although the incidence of sperm antibodies in vasectomized men 1 year postoperatively remained comparable with that at 6 months, definite sperm antibody titer increases had occurred in 5 men. Circulating sperm-agglutinating antibodies were demonstrated in 19 of 37 men
(51.3%) 6 weeks following bilateral vasectomy. Sperm-immobilizing antibodies were present in 14 cases (37.8%), although 4 had no sperm-agglutinating antibodies. No statistical difference was noted in the serum uric acid following vasectomy at the 6-week examination. Acknowledgment. The health questionnaire was kindly supplied by H. J. Roberts, M.D., Palm Beach Institute for Medical Research, West Palm Beach, Florida 33407. REFERENCES 1. ANSBACHER, R. Sperm-agglutinating and spermimmobilizing antibodies in vasectomized men. Fertil Steril 22:629, 1971. 2. ROBERTS, H. J. Letter to the Editor. Perspect Bioi Med 14:176, 1970. 3. ROBERTS, H. J. Thrombophlebitis after vasectomy. New Eng J Med 284:1330, 1971. 4. RUMKE, P., AND HELLINGA, G. Autoantibodies against spermatozoa in sterile men. Amer J Clin Path 32:357, 1959. 5. PHADKE, A. M. Fate of spermatozoa in cases of obstructive azoospermia and after ligation of vas deferens in man. J Reprod Fertil 7: 1, 1964. 6. JOHNSON, D. S. Reversible male sterilization: current status and future directions. Contraception 5:327, 1972. 7. KIBRICK, S., BELDING, D. L., AND MERRILL, B. Methods for the detection of antibodies against mammalian spermatozoa. II. A gelatin agglutination test. Fertil Steril 3:430, 1952. 8. ISOJIMA, S., LI, T. S., AND ASHITAKA, Y. Immunologic analysis of sperm-immobilizing factor found in sera of women with unexplained sterility. Amer J Obstet Gynec 101:677, 1968. 9. OSER, B. L., Ed. Hawk's Physiological Chemistry (ed. 14). McGraw-Hill, New York, 1965, pp. 1044-1048. 10. PHADKE, G. M., AND PHADKE, A. G. Experiences in the reanastomosis of the vas deferens. J Urol 97:888, 1967.
FERTIUTY AND STERILITY
Copyright © 1972 by The Williams & Wilkins Co.
SPERM ANTIBODIES IN VASECTOMIZED MEN* Rum ANSBACHER, M.D., M.S., KWOK KEUNG-YEUNG, M.B.B.S., t AND JOHN C. WURSTER, M.D.
Department of Obstetrics and Gynecology and Urology Service, Brooke General Hospital, Fort Sam Houston, Texas 78234, and Department of Obstetrics and Gynecology, The University of Michigan Medical Center, Ann Arbor, Michigan 48104
Immunologic and followup studies on bilaterally vasectomized men have recently been advocated to determine the onset and incidence of sperm antibody production and to evaluate possible sequelae. 1 - 3 With the demand increasing for vasectomy throughout the United States and abroad, with the introduction of the use of plugs of various materials and removable clips to temporarily interrupt the function of the vas deferens, and with the application of a vas deferens valve that can be turned off and on at will to allow sperm passage, further studies must be done to delineate long-term medical problems that may arise and to determine whether reversibility as evidenced by fertility is obtainable. In 1959, Riimke and Hellinga 4 suggested that the initiation of sperm antibody production could occur from the extravasation of spermatozoa into the interstitium, lymph vessels, or blood capillaries of the epididymis with subsequent transfer to the regional lymph nodes. Subsequently, Phadke 5 demonstrated intraluminal phagocytosis as one mechanism involved in the disposal of spermatozoa in patients with obstructive azoospermia or with ligated vas deferens. In this study, we compared the findings at the 12-month examination with those previously reported for the 6-month postvasectomy visit. 1 In addition, a new Received March 2, 1972; revised April 26, 1972. * Presented at the 28th Annual Meeting of The American Fertility Society, New York, N. Y., 1972. t China Medical Board Research Fellow.
640
group of men undergoing vasectomy were studied to determine the onset of sperm antibody production with special attention to serum uric acid values prior to and 6 weeks after vasectomy, due to the recent report of elevated serum uric acids in vasectomized men. 6 MATERIALS AND METHODS
Ten milliliters of blood were obtained from 27 men returning to our vasectomy clinic 1 year following surgery. The serum was removed after centrifugation of the clotted blood and complement was destroyed by applying heat at 56° C. for 30 min. The macroscopic gelatin sperm-agglutination test of Kibrick, Belding, and Merrill 7 and the sperm-immobilization procedure of Isojima, Li, and Ashitaka 8 were used to determine the presence of sperm antibody activity in the sera. Pooled untreated rabbit serum served as the complement source. Ejaculates were collected from 24 vasectomized men and examined for spermatozoa. The seminal plasmas were centrifuged at 2500 r.p.m. for 15 min., the supernatant removed, the complement destroyed, and the sperm antibody activity determined. Fresh semen was obtained from 2 donors whose counts were consistently above 60 million spermatozoa per milliliter coupled with motility greater than 70%. A health questionnaire was completed by each patient. Blood was also obtained prior to vasectomy from 51 men whose average age was
September 1972
641
SPERM ANTIBODIES
TABLE 1. Sperm-Agglutiootion Tests (Serum) Negative
Positive
1 (3.5%) (Titer 1 :4)
Postvasectomy (1 yr.)
Postvasectomy (6 mos.)
Prevasectomy
26
Total
Positive
Negative
Total
13
27
14 (51. 9%) (Titers 1: 2-1 : 64)
27
Negative
Positive
15 (55.5%) 12 (Titers 1: 2-1 : 1024)
Total
27
TABLE 2. Sperm-Immobilization Tests (Serum) Postvasectomy (6 mos.)
Prevasectomy
Positive
o
Negative
27
Total
Positive
Negative
10 (37%) (Titers 1: 2-1: 128)
27
37 years and who had fathered an average of 3 children. Thirty-seven (72%) returned for the 6-week postoperative examination, at which time blood and semen samples were collected, processed, and tested as described above. Uric acids were determined on the pre- and 6-week postvasectomy sera by the manual method as adapted by Technicon. 9 RESULTS
Comparable data for sperm-agglutinating and sperm-immobilizing antibodies prevasectomy, 6 months postvasectomy, and 1 year following vasectomy for the 27 men are given in Tables 1 and 2, respectively. No significant difference between the incidence of sperm antibodies at 6 months and 1 year postvasectomy was apparent. Immobilizing antibodies were observed only when agglutinating antibodies were present. The sperm antibody titers are compared in Table 3. Increased titers were demonstrated in 5 men (greater than a two tube dilution increase). Microscopic examination of the ejaculates revealed that only 1 of the 24 specimens contained a few sluggishly motile spermatozoa. Agglutinating and immobilizing antibodies could not be found in the seminal plasmas examined. Eight of the 15 men with circulating sperm antibodies 1 year following bilateral
Postvasectomy (1 yr.)
---Total
17
Positive
27
Negative
Total
16
27
11 (40.7%) (Titers 1: 2-1: 1024)
TABLE 3. Serum Sperm Antibody Titers Postvasectomy Titer
Agglutination
Immobilization
6 mos.
1 yr.
1 5 4 0 3 0 0 0 0
2 0 5 2 0 2 1 1 0 2
2 3 0 3 1 0 1 0 0 0
1 3 4 0 0 0 0 1 0 2
14
15
10
11
6 mos ..
1 vr.
--------
1:2 1:4 1:8 1: 16 1:32 1:64 1:128 1:256 1:512 1:1024
1
Total
TABLE 4. Sperm-Agglutiootion Tests (Serum) Postvasectomy (6 wk.)
Prevasectomy Positive
Nega-
0
51
tive
Total
Positive
51
19 (51.3%) (Titers 1: 2-1: 128)
Negative
Total
18
37
vasectomy gave a positive allergy history, whereas only 3 of the 12 men with no detectable sperm antibodies had a positive allergy history. No other pertinent history could be correlated from this group and no adverse sequelae following vasectomy were documented. Sperm-agglutinating antibodies were present in 19 sera (51.3%) 6 weeks after vasectomy (Table 4). Fourteen (37.8%) men had circulating sperm-immobilizing antibodies (Table 5) although 4 of them
642
ANSBACHER ET AL.
Vol. 23
showed a negative sperm-agglutination test. The serum sperm antibody titers ranged from 1: 2-1: 128 (Table 6). None of these men had demonstrable sperm antibodies prior to vasectomy. Only 1 of the 37 semen samples 6 weeks
postvasectomy contained motile spermatozoa, and none had sperm antibodies. Serum uric acid levels were not statistically different after vasectomy (Table 7, t test).
TABLE 5. Sperm-Immobilization Tests (Serum)
The incidence of sperm antibodies in vasectomized men 1 year postoperatively was comparable to that of 6 months postoperatively. However, definite sperm antibody titer increases occurred in 5 of the men indicative of a continued antigenic stimulus with subsequent antibody production. This finding was evident for both sperm-agglutinating and spermimmobilizing circulating antibodies. A long-term followup study on this group of men is planned. The incidence of sperm antibodies in men 6 weeks after vasectomy parallels that at 6 and 12 months postvasectomy, but the titers at the earlier date were lower than at the later ones. Studies are in progress to ascertain whether the antibody response is immediate or delayed. Since spermatogenesis occurs at least for some time following bilateral vasectomy,10 destruction and subsequent phagocytosis of spermatozoa may add significantly to the nucleic acid pool, as possibly reflected by an increase in the serum uric acid. No change in serum uric acid concentration 6 weeks following vasectomy had occurred in our series, but this observation does not exclude a delayed response which we are now investigating. The necessity of followup studies on technics for temporary interruption of the vas deferens is evident from the findings in a man who had undergone vasovasostomy 8 years after vasectomy. Prior to the vasovasostomy, a serum sperm-agglutination titer of 1: 4 was recorded. Two months following vasovasostomy, his ejaculate contained motile spermatozoa with a sperm count of 30 million/ml., although
Prevasectomy
Postvasectomy (6 wk.)
Posi· tive
Nega· tive
Total
Positive
0
51
51
14 (37.8%) (Titers 1:2-1:64)
Nega· tive
Total
23
37
TABLE 6. Serum Sperm Antibody Titers 6 Weeks Postvasectomy Titer
Agglutination
Immobilization
1:2 1:4 1:8 1:16 1:32 1:64 1:128
1 3 5 5 3 1 1
2 5 2 4 0 1 0
Total
19
14
TABLE 7. Serum Uric Acid (mg. %) Name
Age
Prevasectomy
P08tvasectomy (6 wk.)
BEN MET REE ERK BUR HES CLA SYL BRO CAR KRI SNI TOU GON BUT CAS HED MON TAY
36 31 43 33 42 49 32 30 43 43 28 49 41 38 32 34 38 32 43
5.6 6.7 6.6 6.7 7.2 5.8 7.0 7.9 8.6 6.7 7.5 5.5 6.1 6.8 5.4 5.6 5.7 7.4 8.2
5.5 6.4 5.5 6.3 7.6 5.5 5.8 7.2 8.3 6.6 6.9 4.5 5.4 5.7 5.2 5.6 6.0 7.5 8.3
6.68 ±0.96
6.30 ±1.07
Mean S.D.
37.8
DISCUSSION
September 1972
643
SPERM ANTIBODIES
his serum findings were unchanged. Nine months after vasovasostomy, the serum sperm-agglutination titer had risen to 1: 16 and the sperm count had increased to 50 million/ml. Examination of his seminal plasma for sperm antibody activity revealed a 1: 16 agglutination titer, suggesting that the antibody was being transferred to the seminal plasma postvasovasostomy, since sperm antibodies could not be demonstrated in his seminal plasma by these technics previously. Sperm-immobilization tests on both his sera and seminal plasmas were consistently negative. This single observation must be confirmed by examining a significant number of vasovasostomy patients. Similar studies must be performed on specimens obtained from men utilizing temporary methods of vas deferens occlusion for contraceptive purposes. These procedures may be irreversible when sperm antibodies are formed. SUMMARY
Circulation sperm-agglutinating antibodies were demonstrated in 15 of 27 men (55.5%) who returned for examination 1 year postvasectomy. Sperm-immobilizing antibodies were present in 11 cases (40.7%), and were found only when spermagglutinating antibodies were present. Although the incidence of sperm antibodies in vasectomized men 1 year postoperatively remained comparable with that at 6 months, definite sperm antibody titer increases had occurred in 5 men. Circulating sperm-agglutinating antibodies were demonstrated in 19 of 37 men
(51.3%) 6 weeks following bilateral vasectomy. Sperm-immobilizing antibodies were present in 14 cases (37.8%), although 4 had no sperm-agglutinating antibodies. No statistical difference was noted in the serum uric acid following vasectomy at the 6-week examination. Acknowledgment. The health questionnaire was kindly supplied by H. J. Roberts, M.D., Palm Beach Institute for Medical Research, West Palm Beach, Florida 33407. REFERENCES 1. ANSBACHER, R. Sperm-agglutinating and spermimmobilizing antibodies in vasectomized men. Fertil Steril 22:629, 1971. 2. ROBERTS, H. J. Letter to the Editor. Perspect Bioi Med 14:176, 1970. 3. ROBERTS, H. J. Thrombophlebitis after vasectomy. New Eng J Med 284:1330, 1971. 4. RUMKE, P., AND HELLINGA, G. Autoantibodies against spermatozoa in sterile men. Amer J Clin Path 32:357, 1959. 5. PHADKE, A. M. Fate of spermatozoa in cases of obstructive azoospermia and after ligation of vas deferens in man. J Reprod Fertil 7: 1, 1964. 6. JOHNSON, D. S. Reversible male sterilization: current status and future directions. Contraception 5:327, 1972. 7. KIBRICK, S., BELDING, D. L., AND MERRILL, B. Methods for the detection of antibodies against mammalian spermatozoa. II. A gelatin agglutination test. Fertil Steril 3:430, 1952. 8. ISOJIMA, S., LI, T. S., AND ASHITAKA, Y. Immunologic analysis of sperm-immobilizing factor found in sera of women with unexplained sterility. Amer J Obstet Gynec 101:677, 1968. 9. OSER, B. L., Ed. Hawk's Physiological Chemistry (ed. 14). McGraw-Hill, New York, 1965, pp. 1044-1048. 10. PHADKE, G. M., AND PHADKE, A. G. Experiences in the reanastomosis of the vas deferens. J Urol 97:888, 1967.