Fertilizing capacity and sperm antibodies in vasovasostomized men*

FERTILITY AND STERILITY Copyright © 1983 The American Fertility Society

Vol. 39, No.2, February 1983

Printed in U.8A.

Fertilizing capacity and sperm antibodies in vasovasostomized men*

Elisabeth Requeda, M.D. t Jules Charron, M.D.:j: Kenneth D. Roberts, Ph.D.§ Alcide Chapdelaine, M.D.§ Gilles Bleau, Ph.D.II~ Maisonneuve-Rosemont Research Center, University of Montreal, Montreal, Quebec, Canada

In order to explain the discrepancy between the patency rate (80%) and the pregnancy rate (46%) in a series of vaso vasostomies, attention was focused on a group of patients who became normospermic. The mean age at vasectomy, the duration of vasobstruction, and the parameters of semen analysis were not different for those couples who achieved a pregnancy (n = 8), compared with those couples without pregnancy (n = 7). In the group with pregnancy, six of the eight patients had low titers of serum agglutinins (absent to 1 :32), and the fertilizing capacity of their spermatozoa was normal. None had immobilizing antibodies. In the group without pregnancy, six of the seven patients had elevated serum agglutinins (> 1 :256), and four had agglutinating antibodies in their seminal plasma as well as serum immobilizing antibodies. The spermatozoa of seven patients failed to fertilize zona-free hamster ova. It is concluded that a loss of fertilizing ability of the spermatozoa due to sperm antibodies is an important cause of infertility in vasovasostomized men. Fertil Steril 39:197, 1983

An increasing number of vasectomized men request vasovasostomy in an attempt to recover their fertility. The rate of surgical success, measured in terms of reappearance of spermatozoa in Received July 7, 1982; revised and accepted October 25, 1982. *Supported by the Medical Research Council of Canada (PG-14) and the Fonds de Recherche en Sante du Quebec. tPresent address: Service d'Endocrinologie du Professeur J. Hazard, Hopital Henri Mondor, Creteil, France. :j:Department of Urology. §Department of Biochemistry and of Medicine. IIDepartment of Obstetrics and Gynecology. ~Reprint requests: Dr. Gilles Bleau, Centre de Recherches Maisonneuve-Rosemont, 5415 boulevard de l'Assomption, Montreal, Quebec, Canada HIT 2M4. Vol. 39, No.2, February 1983

the ejaculate can be as high as 60% to 95%; however, the reported pregnancy rates are lower, ranging between 10% and 68%.1 Several reasons have been proposed that could account for the marked discrepancy between patency and pregnancy rates,2 such as semen quality at the time of vasectomy, the technique of vasectomy, the duration of time the vas deferens has been occluded, the technique of reanastomosis, the presence of a granuloma at the site of vasectomy, and the fertility of the partner. These reasons cannot be invoked, however, in the many cases where vasovasostomized patients, in spite of a return to normal semen, are unable to impregnate their apparently normal sex partner. It is mostly in these cases that sperm antiRequeda et al. Fertilizing capacity after vasovasostomy

197

bodies have been implicated as a possible cause of the infertility.2-6 The development of sperm antibodies in about two thirds of vasectomized men is a well-known face; however, their effect on fertility after vasovasostomy remains controversial. In recent years, a method using zona-free hamster ova has been developed and found to be useful in the assessment of the fertilizing capacity of human spermatozoa. 8 This test has also been proven to be sensitive to sperm antibodies. 9-11 The goal of this study was the evaluation of the fertility of vasovasostomized patients and the identification of those parameters that could best discriminate between potentially fertile and infertile patients.

MATERIALS AND METHODS

trol value. In each assay, positive and negative control sera and seminal plasma were tested. ZONA-FREE HAMSTER OVA ASSAY

Whenever motile spermatozoa were present in the ejaculate of a patient, the zona-free hamster ova assay was performed according to the technique of Rogers et aI.,14 with some modifications. 15 The sperm density was· adjusted to a final concentration of 30 x 106 cellslml before capacitation. After 6 hours of incubation, 20 to 50 zonafree ova were added to each aliquot of capacitated sperm. Three hours later the vitelli were removed, washed, and examined under a phase-contrast microscope. The percentage of zona-free ova fertilized was recorded; a fertilization rate higher than 15% was considered normal. In each assay, a semen sample from a donor of proven fertility served as a positive control sample.

PATIENTS

The vasovasostomies were performed in 47 men, 30 to 54 years of age (mean, 39), all of proven fertility prior to vasectomy. A single-layer macroscopic technique was employed in 7 cases and a two-layer microsurgical technique was used in 40. The presence of granulomas at the site of vasectomy and any other abnormalities observed during surgery were recorded. The duration of vasobstruction ranged from 1 to 11 years, with a mean of 5.6 years. Data on duct patency and pregnancy were available in all 47 cases. A subgroup composed of 32 men was further investigated. These men agreed to undergo an evaluation of their sperm antibody titer and their sperm fertilizing capacity. The postoperative follow-up period extended to more than 1 year in all cases. Of the other 15 men, 6 could not be contacted, 7 would not participate, and 2 had had a second vasectomy. SEMEN ANALYSIS AND SPERM ANTIBODY TESTS

Examination of the ejaculates included measurement of sperm count, motility, vitality, and morphologic characteristics. 12 Sperm antibodies were determined in sera and seminal plasma by the use of the gelatin agglutination test and the sperm immobilization test. 13 The latter test was considered positive when motility was reduced by more than 50% of the con198

Requeda et al. Fertilizing capacity after vasovasostomy

RESULTS OVERALL RESULTS OF THE VASOVASOSTOMIES

Spermatozoa reappeared in the ejaculate of 38 ofthe 47 vasovasostomized patients (duct patency rate, 80%). Nine patients remained azoospermic. One patient was oligospermic 4 months after surgery and azoospermic after 1 year. In four cases, surgical failure was associated with anatomic difficulties encountered during surgery: unilateral vas agenesis, extensive epididymal fibrosis, previous inguinal vasectomy, and one case of anastomosis under tension due to the removal of a long segment of the vas deferens at vasectomy. Two other patients experienced postoperative complications (hematoma and sepsis). When the characteristics (mean ± standard deviation [SDD of the azoospermic patients were compared with those of the patients with spermatozoa in the ejaculate, we found no significant difference in the ages of the subjects at vasectomy (31.9 ± 8.4 versus 32.9 ± 6.5; t = 0.425), duration of vasobstruction (7.0 ± 3.3 versus 5.1 ± 2.8; t = 1.876), and presence of granulomas (5/10 versus 14/37; X2 = 0.483). Thirty-nine of the 47 patients had requested vasovasostomy in an attempt to recover their fertility. The spouses of 18 patients became pregnant (pregnancy rate, 46%), and in all cases, pregnancy occurred within 1 to 15 months after Fertility and Sterility

Table 1. Characteristics of the Normospermic Patients in the Group With Pregnancy (Group D) and Without Pregnancy (GroupE) GroupD (n

Age at vasectomy" (yr) Duration of vasobstructiona (yr) Semen analysisa Volume (mD Sperm count (l06 sperm/mD Total sperm count (10 6 sperm/ejaculate) Motility at 2 hours (% motile) Vital staining (% viable) Morphology (% normal shape) aNot significant (P bMean ± SD.

=

8)

Group E (n

=

7)

36.1 ± 8.1b

31.9 ± 5.0

4.8 ± 3.4

6.0 ± 3.2

2.8 ± 1.7 90.1 ± 61.1

2.6 ± 0.9 38.4 ± 22.8

232.8 ± 221.4

105.3 ± 85.2

55.0 ± 10.3

50.0 ± 10.4

51.6 ± 11.8

46.9 ± 11.5

52.3 ± 10.3

53.4 ± 12.1

> 0.05 by t-test.

vasovasostomy. The duration of the period of vas obstruction in these fertile patients ranged from 1 to 11 years (mean, 4.6 years). A granuloma was found in at least one vasectomy site in 6 patients; it was absent in 12. RESULTS OF THE DETAILED STUDY

Thirty-two patients were further investigated. Based on the results that were obtained, they were classified into five groups. Group A: Azoospermic (n = 8) or necrospermic (n = 2) patients. Group B: Oligospermic (n = 4) patients (sperm count of less than 20 million/mI). Group C: Three patients requested vasovasostomy because of their fear of possible side effects from vasectomy. They had a normal semen analysis. Group D: Normospermic patients (sperm count > 20 x 106 /mI) whose wives became pregnant (n = 8). Group E: Normospermic patients (sperm count > 20 x 106 /mI) whose wives did not become pregnant (n = 7). In the normospermic patients (Table 1), the mean age at which vasectomy had been performed and the duration of vasobstruction were not statistically different for the group with pregnancy (group 0), compared with the group without pregnancy (group E). The volume, sperm count, total sperm count, percentage of motility, percentage of normal forms, and percentage of Vol. 39, No.2, February 1983

viable cells were comparable in both groups and were within the normal range in our laboratory. In the morphologic analysis of sperm, the same abnormalities were found in both groups and consisted mainly of microcephalic heads (8% and 7%) and bent, coiled, or absent tails (31% and 25%) for group D and group E, respectively. SPERM ANTIBODIES

Sperm antibodies were detected in 28 of the 32 patients. Serum agglutinins were the antibodies most frequently detected (87% of the cases); agglutinins in the seminal plasma (22%) and serum immobilizing antibodies (16%) were associated with high serum agglutinin titers. In group A, with one exception, serum agglutinins were present in all subjects, with titers ranging from 1:8 to 1:512 (Table 2). High titers above 1:256 were found in four subjects. One azoospermic man had seminal fluid agglutinins, and two had sperm-immobilizing antibodies in their sera. In group B (Table 3), the titer of serum agglutinins ranged from 1:8 to 1:1024; one patient had a titer of 1:16 in his seminal fluid. All four patients were free of immobilizing antibodies. In group C, serum agglutinins, at low titers of 1:4 and 1:8, were the only antibodies that could be detected in two of the three patients. In group D (Table 4), the titer of serum agglutinins never rose above 1:128; agglutinins were absent in the seminal fluid of all but one patient. None had immobilizing antibodies. In group E (Table 5), six of the seven subjects had serum agglutinins at a titer of1:256 or above, four had agglutinins in the seminal fluid, and three presented with sperm-immobilizing antibodies in the serum. Table 2. Sperm Antibody Titers in Azoospermic and Necrospermic Patients (Group A) Agglutinating antibodies Patient Serum

G. R. A.a

P. O. I.a D.A.V. D.E.M. G.A. G. G.L.A. M.A.T. V.E.Z. D. E. S. F. E. R.

Seminal plasma

1:128 1:8 1:512 1:16 1:256 1:32 1:256 1:64 1:256

Immobilizing antibodies (serum)

+

+ 1:4

aNecrospermic patients.

Requeda et al. Fertilizing capacity after vasovasostomy

199

Table 3. Fertilizing Capacity and Sperm Antibody Titers in Oligospermic Patients (Group B) Patient

L. A. R. C. O. S. B.E.N. S.T. L.

Fertilizing capacity (% fertilized ova) 0% 0% 0% 0%

(0/20)a (0/39) (0/40) (0/20)

Agglutinating antibodies Serum

Seminal plasma

1:8 1:16 1:256 1:1024

1:16

Immobilizing antibodies (serum)

aNumber of ova fertilized/number of ova recovered.

IN VITRO FERTILIZING CAPACITY

Spermatozoa from men of proven fertility (semen donors for artificial insemination) were used as controls with each assay for fertilizing capacity. The percentage of zona-free ova fertilized by the spermatozoa ofthese men ranged from 50% to 100%. In group B patients, the fertilizing capacity of the spermatozoa was abnormal in all four cases (Table 3), even though the sperm concentration in the assay was adjusted to correct for their oligospermia. The spermatozoa of one patient in group Chad normal fertilizing capacity. In the other two patients, the spermatozoa fertilized only 5% and 6% of the zona-free ova. The results for the patients whose partners became pregnant (group D) are presented in Table 4. Six of the eight patients gave normal fertilization rates, ranging from 24% to 48%. In the other two cases (patients L. E. D. and C. O. M.), where the partners became pregnant, the percentage of ova fertilized was 0% (measured on two occasions at different dates). The seven patients in group E had abnormal fertilization rates (Table 5). DISCUSSION

The patency rate of 80% observed in our series of 47 vasovasostomized patients is similar to the rates of surgical success reported in recent studies. 1 Higher success rates, of the order of 90%,16 have been attained where no anatomic difficulties were encountered and where proper alignment of the two ends of the sectioned vas deferens could be established. The pregnancy rate of 46% observed in our series was calculated relative to the number of men who desired to recover their fertility. Others have reported pregnancy rates ranging from 12%3 to 60%.16 200

Requeda et al. Fertilizing capacity after vasovasostomy

All of the studies concerning vasovasostomy, including ours, clearly demonstrate a discrepancy between patency and pregnancy rates, reaffirming the consensus that the reappearance of spermatozoa in the semen is not a reliable index of the return of fertility. In some patients, poor semen quality alone can account for the low fertility prognosis after vasovasostomy. Such cases include severe oligospermia, teratospermia, asthenospermia, and necrospermia. The underlying mechanisms responsible for these conditions have yet to be elucidated; high sperm antibody titers have not been consistently found in this category of patients, even though the fertilizing capacity is abnormal. The group of patients that most attracted our attention were those who became normospermic after surgery and of whom a detailed investigation could be done. Of the 15 couples in this category, 8 achieved a pregnancy within 1 year (group D), while the other 7 couples could not achieve the desired pregnancy (group E). When the characteristics of the patients in the two groups were compared (Table 1), we found no significant differences between their ages at the time of vasectomy, the time interval between vasectomy and vasovasostomy, and the presence of granulomas at the sites of vasectomy. The mean sperm count and the mean total sperm count were higher in the group with pregnancy, compared with the group without pregnancy; however, these differences were not statistically significant. Sperm motility and viability as well as the percentage of normal forms were the same in both groups and comparable to the normal values in our laboratory. The assessment of the sperm fertilizing capacity by the technique of zona-free hamster ova yielded more meaningful results. The percentage of ova fertilized was abnormal in all of the patients in the group of couples without pregnancy. On the other hand, the percentage of ova fertilFertility and Sterility

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&

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Table 4. Fertilizing Capacity and Sperm Antibody Titers in Normospermic Patients Uiith Pregnancy (Group D) Patient

Fertilizing capac· ity (% fer· tilized ova)

Time from conception"

Agglutinating antibodies Serum

Seminal plasma

1:2 1:16 1:16 1:32 1:128 1:128

1:8

Immobilizing antibodies (serum)

nw

D. U.B. M. A. R. P. A. R. D. U.M. R. O. B. V.A.L. L.E.D. C.O.M.

+ + + +

3 2 1 2 - 5, - 4 + 12 - 1, + 1 + 4, + 8

48% (l0/21)b 31 % (8/26) 24% (7/29)

46% (6/13) 40%, 45% (4110, 13/29) 31% (6/19) 0%, 0% (0/26, 0120) 0%, 0% (0/14, 0/15)

aTime interval between estimated date of conception and date on which the tests were performed. bNumber of ova fertilized/number of ova recovered.

tween sperm antibodies and male infertility.2o, 2t Several authors have emphasized the importance of the sperm antibody titer: the higher the titer, the more remote are the chances of pregnancy. This conclusion followed studies of the naturally occurring sperm antibodies in infertile men and can be applied to those cases where the antibodies are found following vasovasostomy. These antibodies are produced as a consequence of vasectomy and can be found in the serum and less frequently in the seminal fluid of vasectomized men. 22 The persistence of sperm antibodies in the serum 10 years after vasectomy has been documented. 23 Vasovasostomy does not result in the disappearance of circulating sperm antibodies; this has been shown in the rhesus monkey2 and in man. 4, 24 There is only one case in the literature where a man became free of sperm antibodies after vasovasostomy; most patients show unchanged or slightly modified levels. 6 In our study, 87% of the vasovasostomized patients had detectable serum agglutinins. This percentage is substantially higher than the usual incidence reported for these antibodies in vasectomized men; however, it compared well with the results obtained by others.3, 4, 25 In six of our vasovasostomized patients, a blood sample was also obtained

ized was normal in six of the eight men of the group with pregnancy. The occurrence of pregnancy in two couples in spite of an abnormal fertilizing capacity is difficult to explain. Certain authors have raised the possibility that fertility in the male may not remain constant with the passage of time. 17 It would not appear, at least in one patient (L. E. D.), that we can rule out a change in fertility due to time, since the tests were performed 1 month before and 1 month after the approximate date of conception. This possibility cannot be ruled out in the other patient (C. O. M.), in whom the analyses were done 4 and 8 months after conception. A more likely explanation was suggested in two other studies, where it was found that the spermatozoa from some donors of proven fertility can fertilize human ova in vitro or in vivo, whereas they fail to fertilize zona-free hamster ova. lB , 19 In our two patients (L. E. D. and C. O. M.) we tend to exclude this possibility, mainly because of the high sperm antibody titer in the serum and also in the seminal plasma in one case (C. O. M.); definite proof would involve unethical tests to confirm paternity. Evidence is accumulating in the literature in support of a cause-and-effect relationship be-

Table 5. Fertilizing Capacity and Sperm Antibody Titers in Normospermic Patients Without Pregnancy (Group E) Patient J.E.A. L.O.N. T. R. E. D.E. S. B. E. A. M.U.R. D.E.N.

Fertilizing capac· i!r (% fertilized ova) 4% (1!26)a 11% (3/26)

0% (0/13) 0% (0/14) 0% (0/19) 0% (0/50) 0% (0/17)

Agglutinating antibodies Serum 1:16 1:512 1:256 1:512 1:1024 1:1024 1:2048

Seminal plasma

1:32 1:4 1:8 1:16

Immobilizing antibodies (serum)

+ + +

aNumber of ova fertilized/number of ova recovered. Vol. 39, No.2, February 1983

Requeda et aI. Fertilizing capacity after vasovasostomy

201

prior to surgery; in some cases a dramatic increase of the sperm antibody titer was observed after the vasovasostomy (in patient B. E. A., the titer of serum agglutinins rose from 1:2 to 1:1024). This increase probably explains the high incidence of sperm antibodies observed in our study and might result from antigen withdrawal 2 or booster effects due to sperm antigens at the time of vasovasostomy. 24 It has been previously shown that sperm antibodies can be absent in the semen before vasovasostomy and appear after surgery4; even though these antibodies were not measured before vasovasostomy in our study, the high incidence in the seminal plasma of 22% of our patients supports this concept. While the presence of sperm antibodies in the serum of most men after reversal of vasectomy is well established, their immunologic consequences on fertility are still under discussion. Thomas et al. 25 failed to find any significant difference in the level of antibody titers, measured in the preoperative period, between fertile and infertile patients. In contrast, several studies suggest that sperm antibodies can exert deleterious effects on fertility in vasovasostomized rhesus monkeys2 and in vasovasostomized men. 3 - 6 Linnet et al. 4 found a close association between high serum titers before vasovasostomy and the ability to cause pregnancy. Our study supports this conclusion, since the highest agglutinin titers (> 1:128) were found exclusively in those patients who failed to impregnate their wives. Furthermore, in the six fertile patients who produced spermatozoa with a normal fertilizing capacity, the agglutinating antibodies were either absent or detected at low titers of 1:32 or less. The presence of sperm antibodies in the serum at titers of up to 1:32 is frequently encountered in vasovasostomized patients whose wives have conceived3 ,5 demonstrating that low titers are compatible with fertility. Pregnancies were observed in two couples . where the men had sperm antibody titers of 1:128 for the agglutinins and had spermatozoa that failed to penetrate zona-free hamster ova. This finding could imply that the in vitro fertilization assay is more sensitive to the effects of antibodies than is in vivo fertilization and that pregnancy can occur even when such a high titer is encountered. The presence of sperm agglutinating antibodies in the seminal plasma after vasovasostomy has been closely associated with failure to cause 202

Requeda et al. Fertilizing capacity after vasovasostomy

pregnancy.4 Our study confirms this finding, since these antibodies were often found in infertile patients but in only one patient whose wife became pregnant. Sperm-immobilizing antibodies were found in the serum of two azoospermic patients and of three normospermic infertile patients; however, they were not detected in the fertile patients. Similar observations have been made in vasovasostomized men 3 and in couples with natural infertility. How these antibodies exert their deleterious effects on fertility is not well understood. Spontaneous agglutination or immobilization of freshly ejaculated spermatozoa was not observed in the group of infertile normospermic patients. In summary, the reappearance of spermatozoa in the ejaculate and even a normal semen analysis were not reliable indices of the restoration of fertility in the vasovasostomized patients investigated in this study. On the other hand, the zonafree hamster ova assay was quite reliable in evaluating the likelihood of pregnancy. In view of the fact that a severe autoimmune reaction toward the spermatozoa is associated with infertility, it is evident that the measurement of sperm antibodies should be done prior to, as well as after, vasovasostomy.

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547, 1979 2. Alexander NJ: Vasectomy and vasovasostomy in rhesus monkeys: the effect of circulating antisperm antibodies on fertility. Fertil Steril 28:562, 1977 3. Gupta I, Dhawan S, Goel GD, Saha K: Low fertility rate in vasovasostomized males and its possible immunologic mechanism. Int J Fertil 20:183, 1975 4. Linnet L, Hjort T, Fogh-Andersen P: Association between failure to impregnate after vasovasostomy and sperm agglutinins in semen. Lancet 1:117, 1981 5. Friedman S: Immunologic aspects of vasovasostomy. Andrologia 10:251, 1978 6. Royle MG, Parslow JM, Kingscott MMB, Wallace DMA, Hendry WF: Reversal of vasectomy: the effects of sperm antibodies on subsequent fertility. Br J Urol 53:654, 1981 7. Samuel T, Rose NR: The lessons of vasectomy: a review. J Clin Lab Immunol 3:77, 1980 8. Yanagimachi R, Yanagimachi H, Rogers BJ: The use of zona-free animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa. BioI Reprod 15:471, 1976 9. Haas GG, Sokoloski J, Wolf DP: The interfering effect of human IgG antisperm antibodies on human sperm penetration of zona-free hamster eggs. Am J Reprod Immunol 1:40,1980

Fertility and Sterility

10. Huang TTF, Tung KSK, Yanagimachi R: Autoantibodies from vasectomized guinea pigs inhibit fertilization in vitro. Science 213:1267, 1981 11. Dor J, Rudak E, Aitken RJ: Antisperm antibodies: their effect on the process of fertilization studied in vitro. Fertil Steril 35:535, 1981 12. Zaneveld LID, Polakoski KL: Collection and physical examination of the ejaculate. In Techniques of Human Andrology, Edited by ESE Hafez. Amsterdam, ElsevierNorth Holland Publishing Co., 1977, p 147 13. Menge AC: Immunoandrology. In Techniques of Human Andrology, Edited by ESE Hafez. Amsterdam, ElsevierNorth Holland Publishing Co., 1977, p 225 14. Rogers BJ, Van Campen H, Ueno M, Lambert H, Bronson R, Hale R: Analysis of human spermatozoal fertilizing ability using zona-free ova. Fertil Steril 32:664, 1979 15. Bousquet D, St-Jacques S, Sullivan R, Chapdelaine A, Roberts KD, Bleau G: Evaluation of fertilizing potential of human spermatozoa using a technique of in vitro fertilization. Union Med Can 109:840, 1980 16. Silber SJ: Perfect anatomical reconstruction of vas deferens with a new microscopic surgical technique. Fertil Steril 28:72, 1977 17. Karp LE, Williamson RA, Moore DE, Shy KK, Plymate SR, Smith WD: Sperm penetration assay: useful test in evaluation of male fertility. Obstet Gynecol 57:620, 1981 18. Overstreet JW, Yanagimachi R, Katz DF, Hayashi K, Hanson FW: Penetration of human spermatozoa into the

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