Varicocele and antisperm antibody: fact or fiction?

Objective: To study the level of antisperm antibodies (ASAB) in the serum and semen of infertile men with varicocele before and 6 months after varicocelectomy.

Materials and Methods: We performed a prospective study of 81 infertile men undergoing microsurgical inguinal varicocelectomy. Female factor was excluded. Semen analysis and seminal and serum ASAB (direct and indirect IgG and IgA), as measured by SpermMar test, were performed before and after surgery at 6 months. A control group consisting of idiopathic infertile patients was evaluated for ASAB.

Results: Eighty-one patients with a mean age of 28.7 years (23 to 42) completed our study. Mean sperm count, motility, and abnormal forms were improved after surgery and were statistically significant for sperm density and morphology (P < 0.05). Before surgery, 21 patients (26%) had a low positivity for ASAB (10 to 40%). Six months after surgery, the ASAB titer was reduced in 15 (A group), increased in 3 (B group), and reduced in another 3 (C group). The C group also had an increase in other titers. In the A group sperm count, motility and normal forms improved after surgery (P < 0.05). In the B group, motility was reduced after surgery. In the C group, motility and normal forms were reduced after surgery. Sixty patients were negative for ASAB before surgery. Of these, 48 showed an increase in at least one of the ASAB types to some degree that had no significant effect on semen parameters. In the control group, two patients (7%) were weakly positive for ASAB.

Conclusions: Varicocelectomy may reduce the ASAB level, and this reduction has a good effect on semen parameter quality. Also, it may raise the ASAB level in some patients. This positive conversion has no adverse effect on semen parameters.

Key Words: varicocele, infertility, antibody

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Varicocele is a common cause of infertility. Semen parameter aberrations, including oligospermia, impaired motility, and increased abnormal forms of sperm have been described by MacLeod. (1) Other detrimental effects of varicoceles are testicular growth failure, Leydig cell dysfunction, and histologic changes. (2) Despite data obtained from animal models as well as human studies, the pathophysiology remains unclear. (3) Higher intrascrotal temperature, (4) reflux of renal and adrenal metabolites from the renal vein, (5) decreased blood flow (6) and hypoxia, local hormonal imbalance, and intratesticular hyperperfusion injury (2) have all been proposed as the mechanisms by which varicoceles affect testicular function.

Varicocelectomy improves seminal parameters in approximately 70% of patients, most commonly in sperm motility, but also in regard to density and morphology. (7) Fertility may also be impaired as the result of the presence of antisperm antibodies (ASAB). Varicocele-related infertility with ASAB formation has been described in literature. (8,9) Knudson et al (10) demonstrated an increased incidence of sperm-bound antibodies in infertile varicocele patients and their adverse effect on motile sperm count compared with infertile patients without varicocele. However, the debate regarding the relationship of infertility, varicocele, and ASAB is ongoing.

A prospective investigation was done to assess the incidence of pre- and postoperative ASAB in varicocele-associated infertile patients.

Materials and Methods

Patients

We performed a prospective study of 81 varicocele-associated infertile men undergoing microsurgical inguinal varicocelectomy. Varicocele was determined by physical examination and Doppler ultrasound, categorized by a single examiner with the patient standing, as follows: grade 1, palpating an impulse in the scrotum during a Valsalva maneuver; grade 2, tortuous veins palpated without Valsalva maneuver; and grade 3, visible through skin.

All other probable causes of infertility, such as female factor, were excluded by a thorough evaluation comprising a history, physical examination, and laboratory tests including CBC, urea, electrolytes, and urinalysis. Hormonal studies (serum follicle-stimulating and luteinizing hormone and testosterone) were done if there was severe oligospermia (density<10,000,000/mL) or if there was any indication regarding history and examination. Semen analyses (WHO criteria) and ASAB were measured by the SpermMar (sMar) test performed before surgery and after surgery at 6 months. We also looked for the presence of risk factors, such as a history of trauma, torsion, cryptorchidism, vasectomy or vasectomy reversal, genitourinary infection, or previous inguinal surgery.

A control group comprising 27 patients with primary infertility (correlated with the study patients by age and infertility duration) who had no varicocele or any other abovementioned risk factors were evaluated for ASAB as well. All patients were completely informed and gave their consent.

Semen Analysis

Two consecutive semen samples were collected by masturbation into sterile, wide-mouthed containers after a 3- to 4-day period of abstinence. Samples were examined within 1 hour after ejaculation. After liquefaction, the sample was divided for semen analysis, performed with the cell soft system and ASAB assays.

Immunologic Evaluation

Seminal and serum ASAB (direct and indirect IgA and IgG) were measured by sMar test (document reference, FP09 102 RO1B.2 and FP09 I01 RO1A.3 produced by FertiPro N.V, Belgium). The direct sMar test detected sperm-coating antibodies on spermatozoa, which were isolated from the seminal plasma by one cycle of suspension, centrifuging, and resuspension in medium. The spermatozoa were then mixed with latex particles coated with antihuman anti-IgA and IgG. The formation of mixed agglutination of motile spermatozoa with latex particles indicated the presence of IgA, IgG antisperm antibodies on the spermatozoa. To perform a direct test, 10 [micro]L each of fresh semen and sMar latex particles were placed on a microslide with a capillary pipette. The sample was then mixed with the latex reagent 5 times with the edge of the cover slide. The mixture was observed under a light microscope using x400 to x600 magnification. The result was read after 3 minutes, looking for latex particles attached to the motile sperm. One hundred sperm were counted to determine the percentage of reactive sperm. The sample was read again after 10 minutes.

In the indirect sMar test, washed motile donor spermatozoa were incubated with diluted seminal plasma or decomplemented serum. If the tested fluid contained antisperm antibodies, these would cover the donor spermatozoa that would subsequently react positively to a sMar test. The reagent and seminal plasma or serum specimen were brought to room temperature. The specimen was then inactivated by heat (56[degrees]C for 30 minutes), washed, and diluted by one quarter using Earle medium, and the sperm concentration was adjusted to 20 million. One hundred microliters of the suspension was then incubated with 100 [micro]L of inactivated seminal plasma or serum, diluted by one quarter in oocyte culture medium for 1 hour at 37[degrees]C. Two milliliters of Earle medium was then added, mixed, and centrifuged for 10 minutes at 400 g. The supernatant was decanted or aspirated and resuspended in the pellet with 50 [micro]L of Earle medium. The sample was followed as in the direct test. The diagnosis of immunologic infertility was suspected (low probable positive) when 10 to 39% of the motile spermatozoa were attached to latex particles; if 40% or more of the spermatozoa were attached, immunologic infertility was determined to be highly probable.

Statistical Methods

Data were collected and analyzed using Wilcoxon signed ranks and analysis of variance test.

Results

Eighty-one patients completed our study. Their mean age was 28.7 years (23 to 42 years). Mean infertility duration was 4.29 years (1 to 11 years). All had left varicocele (grade 1: 6, grade 2: 51, grade 3: 24) and 7 had right varicocele and underwent a right varicocelectomy as well. Hormonal studies were performed in 18 patients; all had severe oligospermia and negative ASAB. Follicle-stimulating hormone was normal in 12 patients but was increased in 6. All of the patients were followed for 6 months, and none had recurrent varicocele on physical or Doppler examination. The average preand postoperative sperm density, motility, and morphology are summarized in Table 1.

Before surgery, none of the patients had high probable positivity for ASAB (>40%). Twenty-one (26%) had low probable positivity (10 to 40%). Results are as follows: 9 (11%) for direct IgA, 6 (7%) for serum indirect IgG, 3 (3%) for direct IgG, and 3 (3%) for direct IgA and seminal indirect IgA. The 21 patients who had low probable positivity were grouped according to postoperative ASAB changes. ASAB titer was reduced in 15 (A group) and increased in 3 (B group). The ASAB positivity was reduced, but other antibodies increased in another 3 patients (C group) 6 months after varicocelectomy. In the A group, sperm count, motility, and normal forms improved after surgery (P < 0.05). In the B group, motility was reduced, whereas in the C group, motility and normal forms were reduced after surgery.

Sixty patients (74%) were negative for ASAB before surgery. Of these, 48 (80%) showed an increase in at least one of the ASAB types to some degree. This increase was significant for serum indirect IgG antibody (P < 0.01). It had no significant effect on semen parameters statistically.

Considering risk factors, antibodies revealed low probable positivity in six of nine patients with a history of significant scrotal trauma and three of six patients with a history of varicocelectomy. Antibody titers were reduced after surgery.

Antisperm antibody measurement in the control group revealed that none had high probability for ASAB. Two (7%) were weakly positive, both in direct IgA. Table 2 delineates pre- and postoperative semen parameters, based on the preoperative ASA status.

Discussion

Bouchot et al (11) showed that sperm motility and normal head spermatozoa density were significantly increased after varicocelectomy. In our study, sperm density improved in 62% of the cases (P = 0.018). Mean normal morphology and sperm motility improved in 66% (P = 0.001) and 37% (P = 0.2), respectively. In other reports, the most common improvement in semen parameters occurred in sperm motility after varicocelectomy (70%). (7)

The debate regarding the efficacy of varicocele ligation for the improvement of semen parameters is ongoing. Rumke (12) is credited with the novel idea of antisperm antibody and its relationship to varicocele. The causative role of ASAB in patients with varicocele has been proposed in the literature. (8,13) ASAB is present in 2% of normal fertile men and 10% of infertile men. (14) These antibodies may lead to premature acrosomal reaction, sperm-zona pellucida binding abnormality, and sperm capacitation inhibition. (15) The higher the antibody titer, the poorer the fertility issues.

Ozen et al (9) evaluated 65 patients with infertile varicocele. ASAB was detected by using the immunofluorescence method in 24.6% of patients. It had no relationship to the grade of the varicocele. Golomb et al (8) assessed ASAB (ELISA technique) in 32 infertile patients with varicocele, which revealed a 90% positivity, in contrast to 41% of the control group. We found an incidence of sperm-bound immunoglobulin in 26% of patients with infertile varicocele (weakly positive), and none of them had high probable positivity, whereas in the control group, only two patients (7%) were weakly positive for ASAB.

We did use the sMar test to evaluate ASAB. To the best of our knowledge, there is no study in the literature using the sMar test in varicocele patients before and after surgery. The sMar test has some advantages over the immunobead test (IBT). (16-18) It can be performed on less motile, unwashed sperm and is more sensitive. Unlike polyacrylamide particles in IBT, latex particles are the same size in sMar kits. sMar test results are reproducible and can be performed by any light microscope, even in the office.

Knudson et al (10) reported ASAB levels in 32 infertile patients with varicocele, using the immunobead test. In their study, 28% had a positive IBT, among whom IgG was bound to the surface of the sperm in 100% and IgA in 86%. Of the seven patients who were antibody positive initially, six (86%) remained so after varicocele ligation. One of 15 patients who were antibody negative before surgery became antibody-positive after surgery. Knudson et al were not able to show any significant difference between pre- and post-varicocelectomy ASAB level. There has been no significant change from the Knudson findings in the recent literature in regard to varicocele, ASAB, and infertility.

In this study, ASAB was weakly positive in 21 (26%) patients before surgery, and on follow-up it was reduced in 15 and increased in three, and in the last three patients, ASAB was decreased, but these patients had increased antibodies in another subtype. Considering the incidence of ASAB in the control group in our center (7%), infertile men with varicocele have a higher prevalence of sperm-bound immunoglobulins. According to the literature, there is some suggestion that men with varicoceles who also have sperm-bound immunoglobulins have more extensive damage to the seminiferous epithelia than men with varicoceles who lack this finding. (13) We demonstrated that ASAB does not have a harmful effect on semen parameters before surgery. In this regard, as none of our patients had high probable positivity for ASAB, the question remains as to whether the low levels of sperm-bound antibody found in our study would have a significant impact on fertility.

Antisperm antibody status did not affect semen parameter quality before surgery. Furthermore, after varicocele ligation, our patients exhibited the same improvement rate in semen parameters regardless of the preoperative antibody status. This improvement was only statistically significant on sperm density (P < 0.05) (Table 2).

Gilbert et al (13) and Golomb et al (8) suggested that the predictive value of serum ASAB measurement, that is, the percentage of time that sperm-bound immunoglobulins will be present when serum antibody is detected, is approximately 52% and 30%, respectively. We did not achieve similar results in this study. Inguinal surgery has been considered a possible risk factor for ASAB. In our study, 60 patients were negative for ASAB before surgery. Of these, 48 showed an increase in at least one of the ASAB types to some degree. This increase was significant statistically for serum indirect IgG antibody (P < 0.01).

Heidenreich et al (19) showed that only vasectomy and a history of epididymitis could be recognized as risk factors for ASAB. We found 15 patients with risk factors (9 with scrotal trauma, of which 6 were ASAB positive and 6 with previous varicocelectomy, of which 3 had positive ASAB). The ASAB level was reduced in all the positive cases after varicocelectomy. It should be mentioned that serum antibodies (like IgG) are nonspecific and can be falsely positive due to many circumstances, including fever, disease, surgery, and major stress. None of the above-mentioned states occurred in our patients during follow-up.

As reported by Ozen et al, (9) we did not find any correlation between varicocele grade and pre- and postoperative ASAB levels. Seven patients underwent bilateral varicocelectomy, which did not significantly change the semen parameters or ASAB titers.

Conclusion

Our data suggest that varicocele-associated injury to the seminiferous epithelium may induce ASAB formation and further impairment of fertility. Varicocelectomy may reduce the ASAB level, which has a positive effect on semen parameter quality. This procedure has also been found to raise the ASAB level in some patients albeit without significant adverse effect to semen parameters.

References

1. MacLeod J. Seminal cytology in the presence of varicocele. Fertil Steril 1965;16:735.

2. Francis X. Schneck: Abnormalities of the testes and scrotum and their surgical management. In Walsh PC, et al: Campbell's Urology, 8th edition, Philadelphia, W.B. Saunders Co, 2002, pp. 2353-2394.

3. Marmar JL. The pathophysiology of varicoceles in the light of current molecular and genetic information. Hum Reprod Update 2001;7:461-472.

4. Yamaguchi M, Sakatoku J, Takihara H. The application of intrascrotal deep body temperature measurement for the noninvasive diagnosis of varicoceles. Fertil Steril 1989;52:295-301.

5. Comhaire F, Vermeulen A. Varicocele sterility: Cortisol and catecholamines. Fertil Steril 1974;25:88-95.

6. Saypol DC, Howards SS, Turner TT, et al. Influence of surgically induced varicocele on testicular blood flow, temperature and histology in adult rats and dogs. J Clin Invest 1981;68:39-45.

7. Marmar JL, Kim Y. Subinguinal microsurgical varicocelectomy: A technical critique and statistical analysis of semen and pregnancy data. J Urol 1994;152:1127-1132.

8. Golomb J, Vardinon N, Homonnai Z, et al. Demonstration of antispermatozoal antibodies in varicocele-related infertility with an enzyme-linked immunosorbent assay (ELISA) Fertil Steril 1986;45:397.

9. Ozen H, Asgar G, Gungor S, et al. Varicocele and antisperm antibodies. Int Urol Nephrol 1985;17:97.

10. Knudson G, Ross L, Stuhldreher D, et al. Prevalence of sperm-bound antibodies in infertile men with varicocele: The effect of varicocele ligation on antibody levels and semen response. J Urol 1994;151:1260-1262.

11. Bouchot O, Prunet D, Gaschignard N, et al. Surgery of varicocele: results concerning sperm motility and morphology. Prog Urol 1999;9:703-706.

12. Rumke P, Hellinga G. Autoantibodies against spermatozoa in sterile men. Am J Clin Pathol 1959;31:357.

13. Gilbert B, Witkin SS, Goldstein M. Correlation of sperm-bound immunoglobulins with impaired semen analysis in infertile men with varicoceles. Fertil Steril 1989;52:469.

14. Sinsi AA, Difinizo B, Pasquali D. Prevalence of antisperm antibody by spermMar test in subjects undergoing a routine sperm analysis for infertility. Int J Androl 1993b;16:311-314.

15. Mahony MC, Blackmore PF, Bronson RA, et al. Inhibition of human sperm-zona pellucida tight binding in the presence of antisperm antibody positive polyclonal patient sera. J Reprod Immunol 1991;19:287-301.

16. Maclure RD. Sperm check: A simplified screening assessment for immunologic infertility. Fertil Steril 1989;52:650.

17. Kay DJ, Boettcher B. Comparison of the sperm Mar test with currently accepted procedures for detection of human sperm antibody. Reprod Fertil 1992;4:175.

18. Rajah SV, Parslow JM, Howell RJ, et al. Comparison of mixed antiglobulin reaction and direct immunobead test for detection of spermbound antibody in subfertile males. Fertil Steril 1992;57:1300.

19. Heidenreich A, Bonfig R, Wilbert DM, et al. Risk factors for antisperm antibodies in infertile men. Am J Reprod Immunol 1994;31:69-76.

Hooman Djaladat, MD, Abdolrasol Mehrsai, MD, Modjtaba Rezazade, PHD, Yasaman Djaladat, MD, and Gholamreza Pourmand, MD

From the Department of Urology, Mohammadi Hospital, Hormozgan University of Medical Sciences; the Department of Urology, Urology and Transplant Research Center, Sina Hospital, Tehran University of Medical Sciences, Tehran, Iran; and the Department of Embryology, Rooyan Research Institute.

Reprint requests to Dr. Hooman Djaladat, Urology and Transplant Research Center, Sina Hospital, Department of Urology, Tehran University of Medical Sciences, Tehran, Iran. E-mail: [e-mail]

Accepted July 29, 2005.

RELATED ARTICLE: Key Points

* Varicocelectomy may reduce antisperm antibody level.

* Varicocelectomy may induce antibody formation in those with preoperative negative antibody level.

* Reduction of antisperm antibodies after varicocelectomy is associated with improving count, motility, and normal forms of sperm.

Table 1. Pre- and postoperative sperm density, motility, and abnormal
forms in 81 infertile patients with varicocele (using Wilcoxon signed
ranks test)

                      Preoperative         Postoperative       P (a)

Sperm density         22.6 [+ or -] 1.9    32.2 [+ or -] 2.8   0.018
  (mean [+ or -] SD)    x [10.sup.6]         x [10.sup.6]
Sperm motility        30.8 [+ or -] 16.8%  34.8 [+ or -] 19%   0.2
  (mean [+ or -] SD)
Sperm abnormal form   74.03 [+ or -] 18%   53.07 [+ or -] 12%  0.001
  (mean [+ or -] SD)

(a) P < 0.05 is considered statistically significant.

Table 2. Semen parameters and antibody status

Ig status                    No.  Mean age (yr)  Count (million)

Infertile non-varicocele     27   26.7           28.0 [+ or -] 6.4
  control patients
Infertile varicocele
  patients before surgery
  Weak positive              21   26.4           19.6 [+ or -] 1.4
  Negative                   60   29.5           23.6 [+ or -] 2.2
Infertile varicocele
  patients after surgery
  Weak positive              21    --            21.4 [+ or -] 2.5
  Negative                   60    --            36 [+ or -] 2.9 (a)

Ig status                    Motility (%)        Abnormal form (%)

Infertile non-varicocele     30.7 [+ or -] 12    64 [+ or -] 6.8
  control patients
Infertile varicocele
  patients before surgery
  Weak positive              27.2 [+ or -] 13.4  73 [+ or -] 17.4
  Negative                   32 [+ or -] 17.6    74.4 [+ or -] 18.4
Infertile varicocele
  patients after surgery
  Weak positive              30.2 [+ or -] 17.8  48.4 [+ or -] 16
  Negative                   36.4 [+ or -] 19.4  54.7 [+ or -] 10.6

(a) P < 0.05, 2-tailed t test. All parameters are reported as mean
[+ or -] SD.