The indirect mixed antiglobulin reaction test using a commercially available kit for the detection of antisperm antibodies in serum*

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1 FERTILITY AND STERILITY Copyright The American Fertility Society Printed in U. S.A. The indirect mixed antiglobulin reaction test using a commercially available kit for the detection of antisperm antibodies in serum* Aucky Hinting, M,D.t Lutgart Vermeulen, Ir.Biochem, Frank Comhaire, M.D., Ph,D,t Department of Internal Medicine, State University Hospital, Ghent, Belgium A simple procedure is described for the detection of antisperm antibodies of the IgG class in human serum using the indirect mixed antiglobulin reaction (MAR) test. The test uses only one dilution of serum (1/16) and no washing procedures. The test was performed in 361 sera. Comparative studies were conducted with the tray agglutination test (TAT) in 164 sera and with the direct MAR test on semen in 110 cases. In 24 sera with borderline test results, and in 13 sera with discordant findings, evidence of antisperm antibody activity was investigated by means of additional testing, namely, the adenosine triphosphate release cytotoxicity test (ARCT) and the indirect immunobead tests (IB) for IgA and IgM. Using the value of 40% reaction between motile spermatozoa and coated latex particles as the lower limit of significant activity, the indirect SpermMAR test has a sensitivity of 96% and specificity of 87%, The two false-negative indirect MAR results occurred in cases with IgM, the clinical significance of which is doubtful. Hence, the indirect MAR test appears to be an ideal screening procedure for sperm antibodies in serum since it is easy to perform and sensitive, Considering the false-positive rate of 13%, additional tests should, however, be performed on sera with a reaction of 40% or more in order to assess the relevance of the antibodies as a cause of infertility. Fertil Steril 49:1039, 1988 Detection of antisperm antibodies is an important step of the diagnosis of infertility.1 In 1978, Jager and co-workers have described the direct mixed antiglobulin reaction test (MAR test) as a simple method for the screening of antisperm antibodies on spermatozoa. 2 This test was modified by Vermeulen and Comhaire 3 by replacing the coated red blood cells by coated latex particles, and the Received December 9, 1987; revised and accepted February 26,1988. Supported by a grant from World Health Organization, Special Programme of Research, Development and Research Training in Human Reproduction. t On leave of absence from Department of Biomedics, FaCUlty of Medicine, Airlangga University, Surabaya, Indonesia. :j: Reprint requests: Frank Comhaire, M.D., Ph.D., State University Hospital, Department of Internal Medicine, Section of Endocrinology, De Pintelaan 185, B-9000 Ghent, Belgium. procedure was made commercially available (SpermMAR, Ortho Diagnostic Systems, Beerse, Belgium). Although antisperm activity can be detected simply and accurately in semen, serum still is an important and convenient source for the detection and titration of antibodies, particularly in female patients or in men with very low sperm count or motility precluding a direct MAR test. Among the methods for detection of sperm antibodies in serum, the tray agglutination test 4 is recommended for general use since it yields most information and requires only little donor spermatozoa. 5 However, the TAT is time consuming, reading of the test requires much experience, and reproducibility of results between different laboratories is poor. 6 Moreover, the method becomes very impractical when many sera have to be tested. Hinting et al. Indirect MAR test 1039

2 Elsewhere we have reported the preliminary results of the indirect SpermMAR test for IgG with coated latex particles. 7 In the present study, the diagnostic performance of the indirect MAR test was investigated further by comparing it with the tray agglutination test and the direct MAR test. In addition, the adenosine triphosphate release cytotoxicity test (ARCT)8 and indirect immunobead tests for IgA and IgM (Bio-Rad Laboratories, Richmond, CA) were performed in borderline cases and on sera with discordant results between the indirect MAR test and the tray agglutination test. Materials MATERIALS AND METHODS The study includes 361 consecutive sera from 242 male and 119 female patients who were investigated because of couple infertility. All sera were tested for antisperm antibodies by means of the indirect MAR test. In the early stage of the study, all sera were also tested in the TAT. After reference levels had been obtained, the TAT was performed only on sera with more than 25% positive indirect MAR activity. As a result, 164 samples were tested in both the indirect MAR test and the TAT test, 118 from male and 46 from female patients. The 95% confidence limits were calculated for the percentage of positive indirect MAR reaction in relation to each titer in the tray agglutination test, with exclusion of the discordant cases. The result of the indirect MAR test and TAT was considered discordant when more than 70% of motile spermatozoa had particles attached in the former but the agglutinin titer was lower than 1132 in the latter, or when less than 40% of the spermatozoa had particles attached but the serum agglutinin titer was more than 1/32 in the TAT. Since the TAT measures only the agglutinating capacity of the serum but not the immobilization or any other effect on sperm function, additional procedures were performed in discordant and borderline cases. These included the ARCT and indirect immunobead tests for IgA and IgM. A direct MAR test was performed on the semen of 110 patients. Methods Direct Mixed Antiglobulin Reaction Test The direct MAR test was performed by mixing on a microscope slide one drop (about 10 l in vol Hinting et a1. Indirect MAR test ume) of fresh semen, one drop of latex particles coated with IgG, and one drop of antiserum from rabbit against human IgG (SpermMAR kit). Reading was performed after 2 to 3 minutes under the microscope. The percentage of motile spermatozoa with particles attached was calculated. Indirect Mixed Antiglobulin Reaction Test The indirect MAR test was performed by adding 25 l of a suspension of washed motile spermatozoa (concentration 20 million/ml) from a donor (known to be free of antibodies) to 25 l of complement-inactivated patient serum diluted 1116 with Earle's medium. After incubation at 37 C for 1 hour, one drop (about 10 l in volume) of the mixture was processed in a regular MAR test by mixing it with coated latex particles and anti -IgG. The percentage of motile spermatozoa with particles attached was calculated after 2 to 3 minutes. Tray Agglutination Test The TAT was performed as described by Friberg 4 and included dilutions from 118 to of complement-inactivated patient serum. Aliquots of 5 l of each dilution were transferred to a microchamber tray under mineral oil, and 1 l of a suspension of washed donor spermatozoa (concentration 40 million/ml) was added. After incubation at 37 C during 2 hours, the presence of agglutination was evaluated in an inversion microscope. Adenosine Triphosphate Release Cytotoxicity Test (ARCT) The ARCT was performed according to the procedure described by Suominen and co-workers8 and included dilutions from 112 to of complement-inactivated patient serum. Fifty l of diluted serum, 20 l of guinea pig complement, and 50 l of washed donor sperm suspension (concentration 20 million/ml) were incubated at 37 C for 90 minutes. In parallel, a mixture was incubated of diluted complement-inactivated patient serum and sperm suspension, but with inactivated guinea pig complement. After incubation, 100 l of the suspension was then mixed with 100 l nucleotide-releasing substance (NRS), and 300 l of Tris-acetate, EDTA buffer. Finally, 100 l of adenosine triphosphate (ATP) monitoring reagent (LKB Wallac, Wallac Oy, Turku, Finland) was added, and light emission was measured in a luminometer (LKB Wallac, 1250 Luminometer). Fertility and Sterility

3 A sample was considered positive at a particular dilution titer if the A TP content was reduced by 50% or more as compared with the parallel sample containing inactivated guinea pig complement. An ARCT titer of 114 or more was considered evidence of cytotoxic antibody activity.3. Indirect Immunabead Test far IgA and IgM If' The indirect IBT was performed with slight modification of the method described by Clarke and co-workers. 9 Fifty l of washed motile spermatozoa of a donor (concentration 20 million/ml) were added to 400 l of 114 diluted, complementinactivated patient serum. After 1 hour incubation at 37 C, the spermatozoa were washed three times, and the final pellet was resuspended in 100 l of Earle's solution containing 3% bovine serum albumin. One drop (about 10 l in volume) of resuspended spermatozoa was mixed with one drop of bead suspension (Bio-Rad Laboratories, Richmond, CA; Anti-IgA, cat no ; anti-igm, cat no ) on a microscope slide and covered with a coverslip. A sample was considered positive if 20% or more of the motile spermatozoa were bound to one or more beads. RESULTS Among 361 sera tested with the indirect MAR test, 232 (64.3%) had particles attached to less than 20% of motile spermatozoa, 61 (16.9%) to between 20% and 39%, 25 (6.9%) to between 40% and 79%, and 43 (11.9%) to between 80% and 100% of motile spermatozoa (Fig. 1). Concordant Results in Both Tests A comparative study of the indirect MAR test and the tray agglutination test was performed on 40 ;; ii '" i! 20 Z! I '0 r Figure 1 Frequency distribution of serum samples with var ious levels of percentage of reaction in the indirect MAR tests (% motile spermatozoa with particles attached). '00 -:-- ":" I 90,.0 II: 70 i 10 so! 40 J I Iii.. 20! f 30.. WI I F... n.'" - II. II "" III '2,"g. AGGLUTlN'N TITER CTAT I Figure 2 Comparison between the result of the indirect MAR test (% motile spermatozoa with particles attached) and the agglutinin titer as assessed with the tray agglutination test. 164 sera (Fig. 2). Among 94 sera with agglutinin titers of <1132 in the TAT, less than 50% of the motile spermatozoa had particles attached in the indirect MAR test. Thirty-three sera with agglutinin titers of> 1/32 had more than 60% of the motile spermatozoa with particles attached, and 28 of these presented a reaction with more than 90% of the motile spermatozoa. In 24 sera with agglutinin titer equal to 1132, particles were bound to between 15% and 99% of the motile spermatozoa. Figure 3 shows the 95% confidence interval of the percentage particles attached to the motile spermatozoa in the indirect MAR test, related to the agglutinin titer in the TAT, excluding the 13 discordant cases. It appears that the criterion level for negativity in the indirect MAR test is situated at 50% reaction. too II: 70 Z is!: 50 ;:: 40 iii f 30 ;: '---L...- Mlan D_t2SD... nog. til tl AGGlUTININ TITER I TAT 1 Figure 3 The 95% confidence limit for the percentage ofpositive indirect MAR reaction in relation to each agglutinin titer in the tray agglutination test. Hinting et al. Indirect MAR test 1041

4 Table 1 The Results of Antisperm Antibodies Tests in Borderline Cases (TAT Agglutination Titer 1132) Indirect Direct Indirect IBT MAR MAR ARCT IgG IgG (cytotoxic IgA IgM No. (%) (%) titer) (%) (%) 1 15 F' 112 NOb NOb t e 3 16 F' t e F' NOb NOb NOb 7 32 F' t e 8 40 F' t e 4 t e t" 68 t e NF'" 0 3 t e 8 t e NOb NOb NOb NOb NOb t e F' t e F' t e 4 t e NOb t e 40 t e NOb t e 2 t e F' Ttd.e NOb NOb NOb NOb t e NF'" 0 8Td t e 6 t e F' t e 3 t e NOb t e 2 t e o NF = not feasible. b NO = not done. e F = female patient. d T = tail (principal piece). e t = tip-tail. Sera with Borderline TAT Results In the sera with agglutinin titer equal to 1132, the 95th confidence limit of the indirect MAR varies from 0% to 100%. In order to provide more infor- mation about the antisperm activity of those sera, additional testing for evidence of antibody activity was performed using the ARCT test and the indirect IBT test for IgA and IgM. The direct MAR results were also compared whenever both serum and semen were available. Table 1 shows that the discriminating level of the indirect MAR was situated at 40% reaction, since all sera with <40% reaction displayed no significant antibody activity in the other test. Discordant Results Thirteen sera gave discordant results between the indirect MAR test and the TAT. Eleven sera had an agglutinin titer <1132, but between 79% and 100% of the spermatozoa reacted with particles. Two sera with agglutinin titer >1/32 had particles attached to only 19% and 36% of the motile spermatozoa. The sera from the discordant cases were tested for other evidence of antibody activity with ARCT and the indirect IBT for IgA and IgM, as well as with the direct MAR test. Table 2 shows that eight TAT results should be considered false-negative (Table 2, nos. 1 to 6, 10, and 11), three were falsepositive indirect MAR tests (nos. 7,8, and 9), and two were false-negative indirect MAR tests (nos. 12 and 13). However, the latter sera displayed a significant antibody activity for IgM only. Comparison Between the Direct and Indirect MAR Test Comparison was made between results of the indirect MAR test and the direct MAR test in 110 Table 2 The Results of Antisperm Antibodies in Discordant Cases Indirect IBT TAT Indirect MAR OirectMAR ARCT (Agglut. IgG IgG (cytotoxic IgA IgM No. titer) (%) (%) titer) (%) (%) NOb t' 2t' NF" HTtd,e., It' t' 3 t' F' thdj NOb t' NF" F' NOb 0 It' 4t' NOb 114 It' 58 t' t' Hd F' t o NF = not feasible. b NO = not done. e F = female patient. dh = head. e T = tail (principal piece). 't = tip-tail Hinting et al. Indirect MAR test Fertility and Sterility

5 100..-, 10 I< ao n C Z i 50!I! 40 ;: f 30 =" :. o I-ll ,. POSITNE DIRECT MAR Figure 4 Comparison between the result of the indirect MAR test and the direct MAR test. patients. The direct MAR test was not feasible in 16 semen samples because the ejaculate contained too few motile spermatozoa. Taking 40% or more motile spermatozoa reaction with particles in both the direct and the indirect MAR test as criterion level, there was an excellent agreement between the two procedures (Fig. 4). Three indirect MAR tests were positive, but there was no significant direct reaction in semen. Two of these sera had an agglutinin titer of 1132 (Table 1, nos. 9 and 14) and 1 had an agglutinin titer of DISCUSSION The indirect MAR test is easy to perform because it employes only one dilution of serum and there is no need for washing procedures of the serum-sperm suspension. Moreover, reading the test is unequivocal. The serum dilution of 1/16 was found to be optimal after trial and error in serial dilutions. At the dilution <1116 the sensitivity of the test was reduced, possibly because higher serum concentrations contain such an important amount of IgG that this blocks the mixed antiglobulin reaction. Using serum dilutions >1/16 did not improve the sensitivity of the test. The present study shows a good agreement between the indirect MAR test and the TAT, as well as with the direct MAR test. All patients with more than 40% particles attached to the motile sperm in the direct MAR test also had a positive result of the indirect MAR test in their serum. Since it is the sperm-bound antibodies that interact with the female reproductive tract, this finding is of clinical importance. Serum testing having some practical advantages, the indirect MAR test seems to be valuable for daily practice. Furthermore, the pres- ence or absence of other evidence of sperm antibody activity (either cytotoxic or IgA or IgM) has clarified the borderline and discordant cases. The finding of 40% or more motile spermatozoa reacting with particles in the indirect MAR test should be considered as evidence of a significant antisperm activity of the serum. Although the 95% confidence limit for negativity in the indirect MAR test is situated at 50% reaction, there is evidence of antisperm activity in borderline case (TAT = 1132) starting at 40% reaction. Since a screening test should aim at sensitivity rather than specificity, the 40% reaction was accepted as limit of positive reaction. At this level the diagnostic performance of the indirect MAR test was indeed highly sensitive (96%) and reasonably specific (87%). Interestingly, eight cases were found to present a false-negative result in the tray agglutination test. This may be due to nonagglutinating activity of the antibodies. The two cases considered as false-negative indirect MAR tests resulted from the presence of IgM antibodies in serum. From a clinical point of view, these antibodies are probably of little importance since they do not transudate into the seminal fluid. 10 Although the indirect MAR test is an excellent procedure for detection of antisperm antibodies in terms of sensitivity and specificity, its usefulness for antibodies titration is limited. Considering the false-positive rate of 13%, additional tests should be performed on sera with 40% or more reaction in the indirect MAR test. For these reasons titration with the tray agglutination test is still needed in sera with 40% or more of reaction in the indirect MAR test. When this level of reaction was used as criterion value, no more than 20% of sera of infertile couples needed additional testing in the TAT. REFERENCES 1. World Health Organization (prepared by Comhaire FH, de Kretser DM, Farley TT, Rowe PJ): Towards more objectivity in diagnosis and management of male infertility. Int J Androl Supplement 7:1, Jager S, Kremer J, Van Slochteren-Draaisma T: A simple method of screening for antisperm antibodies in the human male: detection of spermatozoal surface IgG with the direct mixed antiglobulin reaction carried out on untreated fresh human semen. Int J Fertil 23:12, Vermeulen L, Comhaire F: Le test "MAR" aux particles de Latex, et Ie test spermatoxique selon Suominen: simplification et nouveaute dans l'arsenal du diagnostique immunologique. Contraception-Fertilite-Sexualite 11(suppl):381, 1983 Hinting et al. Indirect MAR test 1043

6 4. Friberg J: Immunoglobulin reaction in serum and seminal fluid from men with and without sperm-agglutinating antibodies. Am J Obstet Gynecol 136:671, Linnet L, Suominen JJO: A comparison of eight techniques for the evaluation of the auto-immune response to sperm after vasectomy. J Reprod Immunol 4:133, Boettcher B, Hjort T, Rumke P, Shulman S, Vyazov OE (eds): Auto- and iso-antibodies to antigens of the human reproductive system. 1. Results of an international comparative study on antibodies to spermatozoa and other antigens detected in sera from infertile patients deposited in the WHO Reference Bank for Reproductive Immunology. Acta Pathol Microbiol Scand 258(suppl):1, Comhaire FH, Hinting A, Vermeulen L, Schoonjans F, Goethals I: Evaluation of the direct and indirect mixed antiglobulin reaction with latex particles for the diagnosis of immunological infertility. Int J Androlll:37, Suominen JJO, Multamaki S, Djupsund BM: A new method for measurement of cytotoxic antibodies to human spermatozoa. Arch Androl 4:257, Clarke GN, Stojanoff A, Cauchi MN, Johnston WIH: The immunoglobulin class of antispermatozoal antibodies in serum. Am J Reprod Immunol 7:143, Haas GG Jr, Beer AE: Immunologic influences on reproductive biology: sperm gametogenesis and maturation in the male and female genital tracts. Fertil Steril 46:753, Hinting et al. Indirect MAR test Fertility and Sterility