Evaluation for antisperm antibodies after storage of sperm TEST-yolk buffer*

,---- FERTILITY AND STERILITY Vol. 66, No.3, September 1996 Copyright 'l" 1996 American Society for Reproductive Medicine Evaluation for antisperm antibodies after storage of sperm TEST-yolk buffer* Printed on acid-free paper in U. S. A.. In Hugh C. Hensleigh, Ph.D.H Patricia D. Javkin, M.D.t George E. Tagatz, M.D.t Jon L. Pryor, M.D.t ~ University of Minnesota, Minneapolis, Minnesota Objective: To determine if TEST-yolk buffer, consisting of TES (N-tris [hydroxymethyl] methyl-2-aminoethanesufonic acid), Tris (Tris[hydroxymethyl]aninomethane), and chicken egg yolk, affects the presence of antis perm antibodies on the sperm surface as detected by the Design: A prospective study of antisperm antibodies on sperm surface before and after incubation in TEST-yolk buffer. Direct immunobead test and indirect immunobead test were done the day of collection of the semen sample to detect the presence of human immunoglobulin class G (IgG) and immunoglobulin class A (IgA); immunobead tests were repeated on the same sperm samples after 24 hours of storage in TEST buffer. Setting: Academic tertiary institution. Participants: Patients undergoing evaluation for infertility. Results: There was no significant difference in the outcome of the direct immunobead test after extending semen samples with TEST-yolk buffer for 24 hours at 4 C. Eleven samples that were initially negative for IgG and 13 samples that were negative for IgA remained negative after 24-hour storage in TEST-yolk buffer. Eleven samples that were positive for IgG and nine samples that were positive for IgA by the direct immunobead test the first day remained positive the next day. Five extended sperm samples used in the indirect immunobead test with IgG positive serum gave positive results and four of five used with IgA positive serum gave positive results. Conclusions: These findings suggest that TEST-yolk buffer can be used to extend semen without affecting the presence of antibodies on the sperm surface as indicated by the direct The higher variability of the indirect immunobead tests indicates there may be some alteration of sperm antigens after storing in TEST -yolk buffer. These findings indicate that TEST-yolk buffer can be used to store semen for batched processing of samples or as a transport medium for delivery to a central laboratory for antibody testing. Fertil Steril 1996;66:454-8 Key Words: Antisperm antibodies, TEST-yolk buffer, immunobead test Antisperm antibodies have been shown to decrease fertility by numerous mechanisms, including decreasing motility (1), inhibiting sperm cervical Received December 23, 1995; revised and accepted April 11, 1996. * Supported by the Minnesota Medical Foundation, Minneapolis, Minnesota. t Department of Obstetrics and Gynecology. :j: Reprint requests: Hugh C. Hensleigh, Ph.D., Box 395 UMHC, Department of Obstetrics and Gynecology, University of Minnesota, Minneapolis, MN 55455 (FAX 612-626-0665). Department of Urologic Surgery. ~ Department of Cell Biology and Neuroanatomy. mucus penetration (2), reducing sperm binding to the human zona pellucida (3), decreasing zona penetration (4), decreasing sperm-oocyte fusion (5), and opsonizing sperm for phagocytosis (6). Further, antisperm antibodies may reduce zygote cleavage, implantation, and embryo survival (7). The direct immunobead test detects antisperm antibodies on the sperm surface whereas the indirect immunobead test detects antisperm antibodies in fluids such as seminal plasma, blood, or cervical mucus (8). The immunobeads are polyacrylamide beads coated with covalently bound rabbit antibodies directed against human immunoglobulin class G (IgG) and immuno- 454 Hensleigh et al. Antisperm antibodies after sperm storage Fertility and Sterility

J globulin class A (IgA); the beads will bind readily to antibodies on the surface of sperm cells. Because positive and negative controls must be done with each assay, a reliable method of storing sperm samples for at least a day to permit batching of assays greatly would increase laboratory efficiency. Further, this storage medium could be used to allow overnight transport of samples to a central laboratory for testing. TEST-yolk buffer consists oftes (N-tris [hydroxymethyl]methyl-2-aminoethanesufonic acid), Tris (Tris [hydroxymethyl]aminomethane), and chicken egg yolk. TES and Tris are zwitterionic buffers that are capable of stabilizing ph and CO 2 levels and of binding heavy metals. TEST-yolk buffer is a very effective extender for sperm used in artificial insemination and cryoprotectant when supplemented with glycerol (9). TEST-yolk buffer also has been used in the sperm penetration assay to store sperm overnight and may synchronize the acrosome reaction (10). However, the effect, if any, of TEST-yolk buffer on binding of antisperm antibodies to the sperm surface has not been studied. If TEST-yolk buffer stabilizes sperm without detectable changes in the binding of antisperm antibodies, batch processing of semen samples for the immunobead test would be possible. The purpose of this study is to establish if TEST -yolk buffer would affect the presence of sperm surface antibodies as detected by the direct or indirect Subjects MATERIALS AND METHODS The subjects were 22 patients undergoing evaluation for infertility. A semen sample was collected in the clinic by masturbation and delivered to the lab within 20 minutes. After liquefaction and within 1 one hour, a complete semen analysis was done as part of the evaluation. The specimens used in this study had the following minimal criteria: volume of 2:: 1.5 ml, concentration of20 to 200 X lo B /ml, motility 2:: 20%, and normal morphology 2:: 30% by World Health Organization criteria (11). A direct immunobead test was performed on half of the fresh sample and the other half of the sample was extended in TEST-yolk buffer, stored overnight at 4 C, and the direct immunobead test was repeated the next day. Five samples that were negative for antisperm antibodies by the direct immunobead test were used in an indirect The TEST Buffer In these experiments TEST-yolk buffer (Irvine Scientific, Irvine, CA) was used to extend half of the initial specimen for testing 24 hours later. Approximately half of each specimen was washed once in 10-mL aliquots of Ham's F-10 medium (GIBCO Laboratories, Grand Island, NY) supplemented with 0.3% human serum albumin (HF10A) at 600 X g for 10 minutes, and then resuspended in 0.5 ml HF10A and 0.5 ml of TEST buffer containing 0.5 M TES, 0.5 M Tris, 0.5 M sodium citrate, and 20% egg yolk. The extended sample was cooled slowly to 4 C by placing the 15-mL centrifuge tube containing the sample into a 100-mL beaker of water at room temperature that then was placed in a 4 C refrigerator for 24 hours. The next day the beaker of water containing the sample was removed from the refrigerator and was allowed to equilibrate to room temperature 1 hour. The direct and indirect immunobead tests then were performed as described below. Direct Immunobead Test A 100-ILL aliquot of the semen or 200-Ji,L aliquot of extended specimen was washed three times with 1 ml of Dulbecco's phosphate buffered saline (PBS; Sigma, St. Louis, MO) supplemented with 0.3% bovine serum albumin (BSA, fraction V; Sigma) and centrifuged for 10 minutes at 600 X g. After the final wash the sample was resuspended in PBS with 0.3% BSA to a concentration of approximately 10 X lob motile spermlml. Immunobeads prepared with rabbit anti-human IgA (Bio-Rad, Richmond, CA) and rabbit anti-human IgG (Bio-Rad) were used in this study. Five microliters of the immunobead preparation (IgG or IgA) and 5 ILL of 5% BSA in PBS were mixed with 10 ILL of the washed sperm on a microscope slide, a coverslip was applied, and the specimen was incubated for 10 minutes at room temperature in a humidified Petri dish. The slide was examined on a phase microscope at 40x magnification and the percent of motile sperm with beads attached was determined by examination of 200 motile sperm and recorded as head, midpiece, or tail binding. The criteria for antisperm antibodies positive samples was 10% of the motile sperm with two or more beads bound to the surface. Samples with 50% binding to motile sperm are considered likely to have clinically significant levels of antisperm antibodies (12). Indirect Immunobead Test The indirect immunobead test was carried out using five positive and five negative sera previously tested. The sperm used were from five specimens that initially were antisperm antibody negative as shown by the direct Indirect immunobead tests were done using these samples initially and after 24 hours of storage in TEST-yolk Vol. 66, No.3, September 1996 Hensleigh et a!. Antisperm antibodies after sperm storage 455

Table 1 Immunoglobulin G Binding Initially and After 24 Hours Storage in TEST-Yolk Buffer* Oh Immunoglobulin G binding at 0 h 24 h Immunoglobulin G binding at 24 h Immunoglobulin G negative (n = 11) Immunoglobulin G low positive (n = 3) Immunoglobulin G high positive (n = 8) 46.8 ± 12.5 43.3 ± 11.5 41.9 ± 13.1 0.5 ± 0.8 13.0 ± 2.6 77.8 ± 16.6 31.1 ± 12.7 38.3 ± 5.8 31.9 ± 11.0 1.1 ± 2.4 16.3 ± 4.0 77.3 ± 17.6 * Values are means ± SD. Immunobead testing was done on washed samples initially (0 h) and after TEST-yolk buffer storage (24 h). buffer. A 250-Ji,L sample of heat-inactivated serum was mixed with 50 JLL of neat (unwashed) semen and incubated at 37 C for 1 hour. The sample then was washed by centrifuging three times in 10 ml of PBS with 0.3% BSA at 600 X g for 10 minutes. The immunobead test then was performed as for the direct immunobead test as described above. Five microliters ofthe immunobead preparation (IgG or IgA) and 5 JLL PBS with 5% BSA were mixed with 10 JLL of the washed sperm on a microscope slide, a coverslip was applied, and the specimen was incubated for 10 minutes at room temperature in a humidified Petri dish. The slide was examined on a phase microscope at 40x magnification and the percent of motile sperm with two or more beads attached was determined by examining 200 motile sperm and recorded as head, midpiece, or tail binding. The criteria for antisperm antibodies positive samples was identical to the criteria for the direct immunobead test (12). RESULTS There were no changes in the findings between the initial direct immunobead test and the delayed direct immunobead test performed after the samples were extended in TEST-yolk buffer for 24 hours. Eleven specimens were negative by the direct immunobead test for IgG antisperm antibodies the first day of the test and remained negative in the extended sample the following day. Thirteen specimens were negative by the direct immunobead test for IgA antisperm antibodies the first day of the test and remained negative in the extended sample. Eleven specimens that were positive for IgG and nine specimens that were positive for IgA by the direct immunobead test the first day of the test remained positive the following day. The binding pattern of beads on the sperm positive for IgG or IgA was recorded and statistically analyzed; no significant differences in head, midpiece, or tail binding were detectable and therefore total binding is reported. The IgG-positive specimens consisted ofthree lowpositive specimens with an average initial binding of 13.0% ± 2.6% (mean ± SD) and after 24-hour storage binding of 16.3% ± 4.0%; there were eight IgG high-positive samples with an average initial binding of 77.8% ± 16.6% with virtually no change after 24-hour storage at 77.3% ± 17.6% binding (Table 1). These differences were not statistically different (P < 0.05 by Student's t-test). The IgA-positive specimens consist of five low-positive samples and four high-positive samples (Table 2). None of the immunobead test tests for IgA changed from positive to negative or negative to positive. The average motility of the samples in each group decreased consistently by approximately 10% over the 24-hour storage period. The indirect immunobead test results were more variable than the direct immunobead test results (Table 3). All the samples used in the IgG tests remained positive after the 24-hour incubation in TEST-yolk buffer. However, one of five extended specimens tested with IgA-positive serum shifted from positive to negative. All the negative indirect immunobead tests remained negative after extending the sperm samples. DISCUSSION These findings suggest that TEST -yolk buffer can be used to extend semen without affecting the pres- Table 2 Immunoglobulin A Binding Initially and Mter 24 Hours Storage in TEST-Yolk Buffer* Oh Immunoglobulin A binding at 0 h 24 h Immunoglobulin A binding at 24 h Immunoglobulin A negative (n = 13) Immunoglobulin A low positive Immunoglobulin A high positive (n = 4) 46.5 ± 13.0 39.0 ± 10.2 45.0 ± 12.9 1.7 ± 2.2 16.6 ± 5.3 82.8 ± 23.6 33.2 ± 12.7 30.0 ± 12.7 33.8 ± 2.5 0.5 ± 1.2 31.2 ± 32.9 73.5 ± 23.9 * Values are means ± SD. Immunobead testing was done on washed samples initially (0 h) and after TEST-yolk buffer storage (24 h). 456 Hensleigh et al. Antisperm antibodies after sperm storage Fertility and Sterility

Table 3 Indirect Immunobead Test With Fresh and TEST-Yolk Stored Spermatozoa* Fresh sample Test buffer stored Fresh sample Test buffer stored Immunobead test Binding (%) IgG 56.4 ± 16.3 IgG 46.6 ± 30.1 IgA 60.8 ± 13.4 IgA 51.2 ± 33.7 * Values are means ± SD. Indirect immunobead test was done using fresh washed samples initially (0 h) and after TEST-yolk buffer storage (24 h). ence of antibodies on the sperm surface as indicated by the direct immunobead test and without affecting binding of antisperm antibodies as indicated by the indirect Use of TEST-yolk buffer will allow the storage of semen samples for batching of immunobead tests. Shipping of samples from physician's offices to reference laboratories also may be possible if the samples can be maintained at 4 C. In these experiments, sperm was stored successfully for 24 hours before immunobead testing; the presence of IgG or IgA antisperm antibodies on the sperm did not affect sperm motility over the 24-hour storage in TEST-yolk buffer. The percent binding of immunobead tests for IgA were a bit more variable than that for IgG (Tables 1 and 2) and may be due to the small sample size after positive results were divided into low positives and high positives. However, none ofthe specimens tested changed from positive to negative or, conversely, from negative to positive. The indirect immunobead test involves de novo binding of antisperm antibodies to sperm. The higher variability observed may indicate that there are changes in the surface antigens present on the sperm surface over time when sperm are stored in TEST-yolk buffer. None of the samples used for the IgG indirect immunobead test gave results that changed from positive to negative. However, one of five samples used for the IgA indirect immunobead test changed from positive (65% binding) to negative (4% binding) and had reduced but positive binding for IgG. In this particular sample, the binding was nearly all head binding. We speculate that this sample was subjected to temperature shock during processing, with possible shedding of antigens from the sperm head, however, the acrosomal status of this sample was not determined. These findings suggest that a fresh donor sample be used for the indirect immunobead test to minimize variability of the test. It was reported previously that sperm incubated with antisperm antibodies and stored in TEST-yolk buffer had subsequent reduced antisperm antibodies as indicated by the immunobead test, which seems to contradict the present work (Lam TC, Chan PJ, Tredway DR, abstract). However, there are important differences between these studies. Lam and associates used frozen sperm and labeled with posi- tive serum before an incubation with TEST-yolk buffer. If high titers of antisera were used, the observed decrease after incubation in TEST-yolk buffer may be due to initial nonspecific antisperm antibodies binding. In contrast, the present work was designed to correspond to routine clinical testing of fresh sperm samples that were negative or positive for antisperm antibodies when collected. The present study indicates that immunobead testing for antisperm antibodies can be performed routinely after incubation in TEST-yolk buffer. TEST-yolk buffer conserves sperm motility (10), which is essential to demonstrate clearly binding of the beads in this assay. TEST-yolk buffer, with its high concentration of cholesterol, may allow sperm to undergo some of the physiological changes of capacitation (13) but it also may stabilize the acrosomal membrane and reduce spontaneous acrosome reactions (14). An important requisite to using TEST-yolk buffer as a storage medium for the immunobead test may be slow rates of initial cooling before storage and warming after storage. Indeed, a rapid warming (temperature shock) of sperm after storage in TEST-yolk buffer as part of a sperm penetration assay has been reported to increase the rate of the acrosome reaction (13, 15). However, an acrosomereacted sperm would be expected to alter the plasma membrane over the head of the sperm, with possible shedding of head-bound antibody. No statistical difference in the percent binding on sperm heads observed in the direct immunobead tests of this study suggests preservation of acrosomal membranes. Slow cooling and warming rates used in this research may facilitate the conservation of viable, motile, capacitated, but acrosome- intact sperm for the REFERENCES 1. Isojima SL, Li TS, Ashitaka Y. Immunologic analysis of sperm-immobilizing factor in sera of women with unexplained sterility. Am J Obstet Gynecol 1968; 101:677-83. 2. Jager S, Kremer J, Kuiken J, Mulder 1. The significance of the Fc part of antispermatozoal antibodies for the shaking phenomenon in the sperm-cervical mucus contact test. Fertil Steril 1981;36:792-7. 3. Bronson RA, Cooper GW, Rosenfeld DL. Sperm-specific isoantibodies and autoantibodies inhibit the binding of human Vol. 66, No.3, September 1996 Hensleigh et al. Antisperm antibodies after sperm storage 457

spenn to the human zona pellucida. Fertil Steril 1982; 38:724-9. 4. Tsukui S, Noda Y, Yano J, Fukuda A, Mori T. Inhibition of sperm penetration through human zona pellucida by antispenn antibodies. Fertil Steril 1986;46:92-6. 5. Aitkin RJ, Parslow JM, Hargreave TB, Hendry WF. Influence of antispenn antibodies on human sperm function. Br J Urol 1988; 62:367-73. 6. Bronson RA, Cooper GW, Rosenfeld DL. Complement-mediated effects of sperm head-directed human antibodies on the ability of human spermatozoa to penetrate zona-free hamster eggs. Fertil Steril 1983;40:91-5. 7. Naz RK. Effects of antibodies on early cleavage of fertilized ova. BioI Reprod 1992;46:130-9. 8. Clarke GN, Clarke FM, Wilson S. Actin in human spennatozoa. BioI Reprod 1982;26:319-27. 9. Graham E. Fundamentals of the preservation of spenn. In: The integrity of frozen spermatozoa. Washington, DC: National Academy of Sciences, 1978:4-44. 10. Rogers BJ. The sperm penetration assay: its usefulness reevaluated. Fertil Steril 1985;43:821-40. 11. World Health Organization. Laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 3rd ed. New York: Cambridge University Press, 1993. 12. Clarke GN. Detection of antispenn antibodies using immunobeads. In: Keel BA, Webster BW, editors. Handbook of the laboratory diagnosis and treatment of infertility. Boca Raton (FL): CRC Press, 1990:177-92. 13. Johnson AR, Syms AJ, Lipshultz LI, Smith RG. Conditions influencing human sperm capacitation and penetration of zona-free hamster ova. Fertil Steril 1984;41:603-8. 14. Falk RM, Silverberg KM, Fetterolf PM, Kirchner FK, Roger BJ. Establishment of TEST-yolk buffer enhanced spenn penetration assay limits for fertile males. Fertil Steril 1990; 54:121-6. 15. Bielfeld P, Jeyendran RS, Holmgren WJ, Zaneveld WD. Effect of egg yolk medium on the acrosome reaction of human spennatozoa. J Androl 1990; 11:260-9. 458 Hensleigh et al. Antisperm antibodies after sperm storage Fertility and Sterility