A reverse (antibody capture) enzyme-linked immunosorbent assay for detection of antisperm antibodies in sera and genital tract secretions*

Transcription

1 FERTILITY AND STERILITY Copyright 1990 The American Fertility Society Vol. 54, No.5, November 1990 Printed on acid-free paper in U.S.A. A reverse (antibody capture) enzyme-linked immunosorbent assay for detection of antisperm antibodies in sera and genital tract secretions* Shafrira Shai, M.Sc. t Naomi Bar-Y oseph, t Eitan Peer, M.D.* Yehudith Naot, D.Sc.t Technion-Israel Institute of Technology, and Rambam Hospital, Haifa, Israel A reverse (antibody capture) enzyme-linked immunosorbent assay (ELISA) for detection of antisperm antibodies has been developed. The assay enables detection of immunoglobulin (Ig)M, IgG, IgA, or IgM, IgG, and IgA-antisperm antibodies in serum, cervical mucus, and seminal plasma samples. The reverse ELISA is more specific and sensitive than conventional ELISA in detecting human antisperm antibodies of different isotypes. Using this assay, statistically significant differences in levels of antibodies between infertile and fertile individuals were demonstrated in sera and in genital tract secretions. Studies with 143 infertile couples revealed that the presence of antibodies in sera was not necessarily reflected in individual's genital tract secretion and vice versa. These data emphasize the importance of detecting antisperm antibodies in sera as well as in genital tract secretions for correct evaluation of sperm immunity. Fertil Steril 54:894, 1990 Previous studies have demonstrated that spermatozoa are potential immunogens capable of eliciting antibody response in animals and causing adverse effects on their fertility.1 2 In humans, the incidence of antisperm antibodies has been reported to be higher in infertile than in fertile individuals. 3--B These antibodies, directed against antigens located at different sites of human spermatozoa, impair various in vitro spermal functions and biological activities that are crucial for reproduction. 7 8 Immunosuppressive treatment of infertile individuals exhibiting antisperm antibodies has been claimed to improve their reproductive perfor- Received January 15, 1990; revised and accepted July 12, * Supported by a grant from the Technion 2000 group, Los Angeles, California. t Department of Immunology, Faculty of Medicine, Technion-Israel Institute of Technology. :j: Department of Gynecology, Rambam Hospital. Reprint requests: Yehudith Naot, D.Sc., Department oflmmunology, Faculty of Medicine, Technion-Israel Institute of Technology, Bat-Galim, P.O. Box 9649, Haifa, Israel. mance, whereas a follow-up of untreated males revealed that fertility was inversely related to the presence of high titers of antibodies In view of this association between reproductive failure and antisperm antibodies detected in infertile females, males, and both partners, determination of these antibodies in sera and particularly in genital tract secretions is considered an essential step during clinical evaluation of infertile couples. Detection of antisperm antibodies has been performed using a variety of methods such as agglutination,3 12 complement-dependent immobilization, 13 immunofluorescence, 14 radio labeled antiglobulin, 9 and indirect immunobeads binding Past usage ofthese methods has given rise to a concern regarding the specificity, sensitivity, objectivity, and large-scale usefulness of these methods The dependence on availability of fresh, viable, high-quality motile spermatozoa, the inability to determine all antisperm isotypes immunoglobulin (Ig)G, IgM, and IgA, not only in sera but also in genital tract secretions, and the false-positive reactions reported in certain assays, prompted 894 Shai et al. Detection of antisperm antibodies Fertility and Sterility

2 the development of enzyme-linked immunosorbent assays (ELISA), which were generally shown to be more specific, sensitive, objective, and safer than previous methods. 19 ~ 21 This study describes the development of a specific and sensitive reverse (antibody capture) ELISA22 that uses solubilized sperm antigens prepared from pooled human semen samples. 21 This assay enables determination of IgG, IgM, and IgA antisperm antibodies in sera as well as in genital tract secretions. MATERIALS AND METHODS Patients and Samples Sera, cervical mucus (CM), and seminal plasma samples were obtained from 63 infertile females and 82 infertile males and another group of 143 couples with unexplained infertility of at least 2 years' duration with no indication for hormonal or physical causes for their infertility. Control samples were obtained from 90 healthy females and 55 healthy males with recent proven fertility of the same age group (mean± SD, 28.5 ± 3.2 years, range 20 to 42 years) as the infertile individuals. Sperm antibody-positive control serum and CM samples used in this study were obtained from an infertile female with unexplained infertility of 10 years' duration exhibiting high levels of antisperm antibodies in microagglutination 12 and immobilization tests.13 Sperm antibody-positive seminal plasma was obtained from an infertile male exhibiting high levels of antibodies in both blood and seminal plasma as tested in the above assays. Negative samples obtained from at least 10 healthy fertile volunteers were pooled to serve as negative control pools of sera, CM, and seminal plasma. After liquefaction period of 30 minutes, seminal plasma was separated from sperm cells by centrifugation of semen samples at 450 X g for 10 minutes. The supernatants (seminal plasma) were carefully collected and centrifuged again at 1,500 X g for 15 minutes. Separation of seminal plasma from sperm cells by two successive centrifugations was performed to avoid any residual contamination of seminal plasma by sperm cells. Because seminal fluids were subjected to bromelin and freezing and thawing, residual sperm cells might lyse and compete with labeled sperm antigens in the antibody d~tection assay. The supernatants collected were then incubated at 56 C for 30 minutes. It was our experience that most seminal plasma samples were soluble. However, in certain cases solubilization was needed. A standard procedure for all samples was, therefore, developed. Solubilization of seminal plasma samples diluted 1:2 with phosphate-buffered saline (PBS) was performed by incubating with bromelin (Cat. No. B-2252; Sigma, St. Louis, MO) at a concentration of 12.5 ~g/ml at 37oC for 30 minutes. Samples were then further incubated at 56oC for 1 hour. The solubilized seminal plasma samples were stored frozen until use. Samples of CM were collected 48 to 72 hours before ovulation after at least 48 hours of sexual abstinence. Collection of samples was performed using a 1-mL graduated syringe to which an extention of sterile plastic tubing was attached. The volume was recorded, and the sample immediately transfered to a plastic tube containing 0.5 ml sterile PBS. Solubilization of CM was achieved by subjecting samples diluted 1:10 with PBS to 25 ~g/ml bromelin for 1 hour at 37oC followed by additional incubation at 56oC for 1 hour. Particulate or insoluble materials were rarely visible after these procedures. If present, samples were clarified by centrifugation at 450 X g for 10 minutes. The solubilized samples of CM were stored frozen until tested. Under these experimental conditions, samples of CM and seminal plasma were solubilized without affecting the antibody titres.23 Enzyme-Linked Immunosorbent Assays Sperm Antigens Specimens were obtained from healthy volunteers with normal semen quality. The samples used for preparation of sperm antigens had at least 20 X 10 6 sperm cells/ml, 2 ml of volume, 40% progressive motility, and 50% normal morphology, and were found to be negative after cultures for the presence of Mycoplasma hominis and Ureaplasma urealyticum. On receipt, each specimen was centrifuged at 1,000 X g for 10 minutes and seminal plasma separated. Sperm cells were resuspended in Dulbecco's modified PBS (Cat. No. D-5773; Sigma) and washed two times. Washed cells from 7 to 10 individuals were pooled and suspended at 200 X 10 6 cells/ml in a volume of 3 to 10 ml and subjected to at least 20 cycles of freezing and thawing in liquid nitrogen. Microscopic examinations revealed that at least 80% of cells were lysed. To lyse the remain- Vol. 54, No.5, November 1990 Shai et al. Detection of antisperm antibodies 895

3 ing cells, the suspension was sonicated for 3 minutes at 4 oc at 12 ~-tm (MSE 150 watt ultrasonic disintegrator; Crawley, United Kingdom). Unbroken sperm cells, if present, were removed by centrifugation at 1,500 X g for 15 minutes. The supernatants collected contained 1.5 to 2 mg protein/ml. These sperm antigen preparations were then divided into 1-mL aliquots and stored at -700C. It was observed that such preparations could be stored for a period of 6 months without any significant effects on the quality of the sperm antigens to affect the performance of the sperm antibody assays. Soluble human sperm antigens were conjugated to alkaline-phosphatase (type VII; Sigma) according to the method of Engvall and Perlmann. 19 Enzyme-conjugated sperm antigens were kept frozen at -20oC until use, and there was no change in the quality of the activity of enzyme-conjugated sperm antigens during a 3-month storage period. Conventional ELISA Conventional ELISA was performed as described by Wolff and Schill 20 with the following modifications. The wells of flat-bottomed microtiter plates (Cat. No ; Flow Laboratories, Inc., McLean, VA) were coated with 5 ~-tg/well sperm antigens. Wells were subsequently coated with 5% normal rabbit serum (Cat. No ; Flow Laboratories) in 0.15 M PBS containing 0.05% Tween 20 (Sigma). Serum samples were tested in triplicates at a 1:50 final dilution in PBS. Cervical mucus and seminal plasma samples were tested at 1:40 and 1:8 final dilutions, respectively. One hundred microliters of each sample were added to triplicate wells. Affinity-purified alkaline phosphatase-conjugated rabbit immunoglobulins to human IgM (Dako Patts No. D337, Copenhagen, Denmark) or human IgG (Dako Patts No. D336) or human IgA (Dako Patts No. D338) or human IgM, G, and A (Dako Patts No. D342) were used at 1:2,000; 1:2,500; 1:2,000; 1:1,000 predetermined optimal dilutions, respectively. One hundred microliters substrate for alkaline phosphatase, p-nitrophenylphosphate (PNPP, Cat. No ; Sigma) were added at 1 mg/ml to each well. After 1-hour incubation, the optical density ( OD) was determined using micro-elisa reader (Microplate auto reader EL309; Bio-Tek instruments, Bur lingtop, VT) at 405 nm. Results for each sample were determined as follows: mean absorbance for sample/mean absorbance for negative control pool (P/N). Test samples were considered positive when P/Nwas ~2. Reverse (Antibody Capture) ELISA The reverse ELISA was performed as previously described by Naot and Remington 22 with the following modifications. Wells of microtiter plates (PVC Cat. No. M-24; Dynatech, Kloten, Switzerland) were coated with the affinity-purified rabbit immunoglobulins to human IgG ( T chain specific, DAKO No. A 090) or to human IgM (~-t chain specific, DAKO No. A 091) or to human IgA (a chain specific, DAKO No. A 092) or to human IgM,G, and A (DAKO No. A 190). Wells were subsequently coated with 5% normal rabbit serum in 0.15 M PBS (ph 7.2) containing 0.05% Tween 20. One hundred microliters of human sera, CM, or seminal plasma samples diluted as described above were added into each of triplicate wells. One hundred microliters of alkaline phosphatase-conjugated soluble human sperm antigens were subsequently added to each well at a concentration of 2.5 ~-tg/ml. One hundred microliters of substrate for alkaline phosphatase, PNPP, Cat. No ; Sigma) were added at 1 mg/ml to each well. After 1-hour incubation at 37oC, the ODin wells was determined using micro ELISA reader at 405 nm. Results for each sample were determined as follows: (mean absorbance for test sample minus mean absorbance for negative pool)/(mean absorbance for a positive control sample minus mean absorbance for negative pool). 24 Test samples were considered positive when result was ~0.25. Statistical analysis of data was performed using Yate's corrected x 2 analysis for independence. RESULTS The Effect of Bromelin on Detection of Antisperm Antibodies in Genital Tract Secretions To quantitatively detect antisperm antibodies in genital tract secretions of different viscosities, solubilization of samples without affecting antibody levels is necessary. In a series of experiments, various concentrations of bromelin and different incubation periods were examined. To verify that under our experimental conditions, solubilization with bromelin does not affect the antisperm antibody levels initially present in clinical samples of CM or seminal plasma, control experiments were performed. We have prepared simulated samples of 896 Shai et al. Detection of antisperm antibodies Fertility and Sterility

4 Table 1 The Effect of Bromelin on Detection of Antisperm Antibodies in Genital Tract Secretions IgM IgG IgA Seminal plasma Cervical mucus Aliquots of positive and negative seminal plasma and cervical mucus samples were subjected to bromelin (treated samples). b Mean absorbance for untreated positive sample/mean absorbance for untreated negative control sample. c Mean absorbance for treated positive sample/mean absorbance for treated negative control samples. antisperm antibody-positive and antisperm antibody-negative CM or seminal plasma by adding positive or negative sera to either a soluble negative pool of CM or to a soluble negative pool of seminal plasma. These simulated preparations of antisperm antibody-positive or antisperm antibodynegative genital tract secretions were then subjected to additional treatment with bromelin. Antibody levels in the original preparations and in bromelin-treated preparations were compared in ELISA for IgM, IgG, or IgA. It can be seen in Table 1 that quantitative detection of antisperm antibodies of different isotypes is not affected by treatment with bromelin under optimal solubilization conditions. Comparison of Reverse and Conventional ELISA Results Positive- and negative-control sera were tested in parallel using either conventional or the reverse (antibody capture) ELISA. Results of IgM, IgG, and IgA ELISA for antisperm antibodies in sera are depicted in Figure 1. It can be seen that using the reverse method a negative pool composed of at least 10 sera from healthy fertile volunteers exhibits lower background activity especially at serum dilutions of 1:20 to 1:160, indicating the higher specificity of the reverse ELISA. The higher sensitivity of the reverse ELISA is apparent from the higher resolution between the positive and negative sera at each of the dilutions tested. Figure 1 also shows that using both ELISA methods, IgM, "E 2.0 lgm-c c: 0.2 II) ~ o.o c: Ia lgm-r.a ~.a 011( o.a lga-c !-.,..,...,..,..,..,...rt,,...,.,~nr,.-.t,...,..:'l=-! I ga-r '0 1d0 uo _ * *... o.o!-.--~4o~~1~&o~~84~0~2~5~8o~10~2~4~4~o~t8~o Reciprocal of Serum Dilution Figure 1 A positive control serum (e) and a negative control pool of sera(.") were tested at the specified dilutions for antisperm antibodies. IgM-C, IgG-C and IgA-C are results of the corresponding antisperm isotypes tested using the conventional ELISA. IgM-R, IgG-R and IgA-R are results of the corresponding antisperm isotypes tested using the reverse ELISA. Vol. 54, No.5, November 1990 Sbai et al. Detection of antisperm antibodies 897

5 Table 2 Reverse and Conventional IgM, lgg, IgA ELISA Results in Infertile and Fertile Individuals No. of positive/total Females Males Reverse ELISA Infertile Fertile Conventional ELISA Infertile Fertile Serum 21/63 (33.3)" 11/90 (12.2) 24/63 (38) 13/41 (31.7) CM Serum Seminal plasma 23/63 (36.5) 22/82 (26.8) 22/82 (26.8) 0/25 (0} 3/55 (5.4) 3/98 (3.1) 22/63 (34.9} 17/82 (20.0) 15/82 (18.2) 5/10 (50) 2/34 (5.8) 4/35 (11.4) Values in parentheses are percents. IgG, and IgA isotypes can be detected. These results justified further comparisons between conventional and reverse ELISA for simultaneous detection of all three isotypes (M,G, and A) in sera and genital tract secretions. Sera and CM samples from 63 infertile females as well as sera and seminal plasma samples from 82 infertile males were tested in both assays in parallel. Control groups of serum samples from 90 females and 55 males with a recently proven fertility were tested in the reverse assay. Sera from 41 of 90 fertile females and sera from 34 of 55 fertile males were also tested in the conventional assay. Cervical mucus samples were obtained from only 25 ofthese 90 fertile female volunteers. All of them were tested in the reverse assay, whereas only 10 CM samples with enough quantity were examined also in the conventional assay. Seminal plasma samples were obtained from 98 fertile males, 55 of whom also donated serum samples for the study. Thirty-five of these seminal plasma samples were tested in both ELISA methods, whereas the remaining 63 were tested only in the reverse assay. The results are summarized in Table 2. Statistical analysis of the results using the Yate's corrected x 2 test for independence showed significant differences between infertile and fertile individuals with regard to the presence of antisperm antibodies in female sera (P < 0.01), cervical mucus (P < 0.01), male sera (P < 0.01), and seminal plasma (P < 0.001) when tested using the reverse IgM, IgG, and IgA ELISA. On the other hand, no statistically significant differences were found between sera and genital secretions from infertile and fertile individuals when tested by conventional ELISA method. To further compare the two ELISA methods, we have analyzed the results obtained with the 145 infertile individuals (63 females and 82 males) who were tested independently in these two assays. Table 3 shows the distribution of positive and negative samples in both assays. Based on these data and considering the conventional ELISA as a standard, it was found that the reverse ELISA exhibits sensitivity of95.3% in sera and 100% in genital secretions; specificity of 98.0% in sera and 92.6% in genital secretions; diagnostic efficacy of 98.6% in sera and 94.5% in genital secretions, when compared with the conventional assay. Based on all these data, it can be concluded that the reverse (antibody capture) method is a significantly better assay than the conventional ELISA for detection of antisperm antibodies and differentiation between infertile and fertile individuals. Reverse ELISA Reproducibility and Specificity To determine the assay reproducibility, three serum samples exhibiting various levels of antisperm Table 3 Reverse and Conventional IgM, IgG, lga ELISA Results in 145 Infertile Individuals Serum samples Cervical mucus and seminal plasma samples Reverse ELISA Reverse ELISA Conventional ELISA Positive Negative Conventional ELISA Positive Negative Ppsitive (n = 41) Negative (n = 104) Total (n = 145) Positive (n = 37) 37 0 Negative (n = 108) Total (n = 145) Shai et al. Detection of antisperm antibodies Fertility and Sterility

6 Table 4 Reverse ELISA Reproducibility Optical density Incubation period with substrate Serum 1 Serum2 Serum3 1h Experiment (2.85)" (5.1) (6.5) Experiment (5.3) (5.2) (6.9) Experiment (3.1) (3.8) (3.5) Interassay variation (4) (5.8) (6.4) 2h Experiment (3) (4.9) (6.4) Experiment (5.5) (5.5) (8) Experiment (3.2) (4.0) (3.5) Interassay variation (4.5) (6.5) (6.9) a Coefficient of variation percents are in parentheses. antibodies were tested in triplicates for eight times on 3 separate days. Optical density in wells was determined after incubation with substrate for 1 hour and 2 hours. The IgM, IgG, and IgA ELISA results obtained are shown in Table 4. Evidently the intraassay and interassay variations were low, and the assay was highly reproducible regardless of incubation period with substrate. The specificity of the reverse ELISA method was further confirmed in a series of experiments in which two serum samples from two infertile females with high titers of antisperm antibodies were absorbed with washed sperm cells, washed human red blood cells, and washed human leukocytes. Incubation of sera with the respective cells was carried out at 37oC for 2 hours with constant mixing. After absorption, the cells were removed by centrifugation. Absorbed and unabsorbed sera were tested in the reverse assay for antisperm antibodies. Absorption of sera with either human red blood cells or leukocytes had no effect on the antisperm antibody activity of sera in ELISA. On the other hand absorption of sera with human sperm cells mark~ edly reduced their antibody levels in the ELISA, indicating that the antibodies detected in the ELISA were specific to sperm antigens. ELISA Results in Sera, CM, and Seminal Plasma From 143 Infertile Couples Sera, CM, and seminal plasma samples collected from 143 couples with unexplained infertility were tested for IgM, IgG, and IgA antisperm antibodies using the reverse ELISA. The results, as summarized in Table 5, clearly demonstrate the importance of testing sperm antibodies not only in sera but also in the genital tract secretions from infer- tile couples. In the group of 143 couples tested in this study, 13 (6.9%) females and 19 (13.3%) males exhibited antibodies only in genital secretions, being negative in their sera. These individuals may have been misevaluated and considered negative for sperm immunity if only their sera had been tested. Thirty-three (23%) females and 25 (17.5%) males showed antisperm antibodies only in sera, being negative in their respective genital secretions. This observation reflects the dichotomy between reproductive tract immunity and systemic immune status relative to the presence of antisperm antibodies. In general our data reveal that although sperm antibodies are detected more frequently in serum than in genital tract secretion of an individual, detection of antibodies in sera from either infertile females or males is not necessarily a true reflection of the sperm immunity in their CM or seminal plasma. DISCUSSION The results of these studies demonstrate the specificity, sensitivity, and reproducibility of areverse (antibody capture) ELISA developed in our laboratory for detection of IgM, IgG, IgA or IgM, IgG, and IgA antisperm antibodies in sera, CM, and seminal plasma. Based on results reported by others 21 on the diagnostic superiority of conventional ELISA methods using solubilized sperm antigens over whole sperm cells, our assay also utilized the soluble human sperm antigens that were prepared from freeze-thawed sperm pools and conjugated to alkaline phosphatase. A comparison between the reverse method with the conventional ELISA method for detection of sperm antibody isotypes related to infertility clearly demonstrates that the reverse ELISA is a more sensitive and specific assay and exhibits highly reproducible results. This study, employing the reverse ELISA, showed statistically significant differences between infertile individuals and fertile controls for sperm antibody levels not only in serum but also in their CM and seminal plasma specimens. Because such differences between infertile and fertile individuals were not found using the conventional ELISA method, it was concluded that the reverse ELISA should be the assay of choice when sperm immunity was suspected clinically as one of the causes of infertility. Higher sensitivity ofthe reverse method as compared with the conventional ELISA has been noted Vol. 54, No.5, November 1990 Shai et al. Detection of antisperm antibodies 899

7 Table 5 Results of ELISA in Sera, CM, and Seminal Plasma From 143 Infertile Couples No. of individuals positive for sperm antibodies Both serum and CM or seminal plasma Serum only CMor TotalinCM seminal plasma only Total in serum or seminal plasma Females (n = 143) Males (n = 143) Total (n = 286) 17 (11.9) a 16 (1) 33 (11.5) 33 (23.0) 25 (17.5) 58 (20.3) 13 (6.9) 50 (34.9) 30 (20.9) 19 (13.3) 41 (28.7) 35 (24.5) 32 (1) 91 (3) 65 (22.7) a Values in parentheses are percents. previously in assay for Toxoplasmagondii antibodies. 22 After the capture of human immunoglobulins to the ELISA plates coated with antibodies to human immunoglobulins, other components of serum or genital tract secretions are removed by washing in the reverse assay. Sperm antigens (conjugated to enzyme), which are subsequently added to these plates, react only with human antibodies without interference of other nonspecific components present in body fluids. Reports by other investigators 8 21 clearly suggest that usage of soluble extracts of sperm antigens is superior to whole sperm cells when employed in the conventional ELISA. Although crude preparation of soluble sperm antigens was used in the present study, it was possible that nonspecific reactions between the Fe portions of immunoglobulins and sperm cells were avoided in the reverse ELISA because of the initial capture of immunoglobulins to plates via their Fe portions. The need for further purification of soluble sperm antigens was thus not necessary. To further evaluate the diagnostic usefulness of the assay, we employed the reverse IgM, IgG, and IgA ELISA on sera, CM, and seminal plasma samples obtained from 143 infertile couples suffering from unexplained infertility. The results of these studies demonstrated that 11.9% of females and 1% of males had antisperm antibodies in both their sera and genital tract secretions. However, 23% of females and 17.5% of males exhibited antibodies only in their sera but not in their respective genital tract secretions. On the other hand, 6.9% of females and 13.3% of males were positive for antibodies only in CM or seminal plasma, whereas exhibiting negative IgM, IgG and IgA ELISA results in their sera. Our data illustrate the importance of detecting sperm antibodies not only in sera but also in genital tract secretions of infertile individuals d}lring evaluation of the sperm autoimmunity The reverse ELISA developed in this study obviates the dependence on availability of fresh, viable, motile spermatozoa from high-quality semen specimens and enables specific and sensitive large scale determinations of different isotypes. This assay is highly reproducible, and the results are interpreted objectively. We therefore consider this assay to be one of the best ways to reliably determine the levels of antisperm antibodies in sera and genital tract secretions and for further studies on their physiological role in infertility. REFERENCES 1. Edwards RG: Immunological control of fertility in female mice. Nature 203:50, Beer ARE, Neaves WB: Antigenic status of semen from the viewpoints of the female and male. Fertil Steril29:3, Shulman S: Sperm antigens and autoantibodies: effect on fertility. Am J Reprod Immunol Microbiol10:82, Ingerslev HJ: Antibodies against spermatozoal surfacemembrane antigens in female infertility. Acta Obstet Gynecol Scand Suppl100:1, Menge AC, Medley NE, Mangione CM, Dietrich JW: The incidence and influence of antisperm antibodies in infertile human couples on sperm-cervical mucus interactions and subsequent fertility. Fertil Steril 38:439, Mathur S, Williamson HO, Petrina V, Genco PV, Rust PF, Fundenberg HH: Female's isoimmunity to sperm is associated with sperm autoimmunity in their husbands. J Clin Immunol5:166, Bronson R, Cooper G, Rosenfeld D: Sperm antibodies: their role in infertility. Fertil Steril42:171, Alexander NJ, Anderson DJ: Immunology of semen. Fertil Steril4 7:192, Haas GG, Jr, Cines DB, Schreiber AD: Immunologic infertility: identification of patients with antisperm antibody. N Engl J Med 303:722, Rumke P, Van Amstel N, Messer EN, Bezemer PD: Prognosis of fertility of men with sperm agglutinins in serum. Fertil Steril25:393, Rumke P: The origin of immunoglobulins in semen. Clin Exp lmmunol12:287, Friberg J: A simple and sensitive micro-method for demonstration of sperm-agglutinating antibodies in serum from infertile men and women. Acta Obstet Gynecol Scand Suppl36:21, Isojima S, LI TS, Ashitaka Y: Immunologic analysis of sperm immobilizing factor found in sera of women with unexplained sterility. Am J Obstet Gynecol101:677, Shai et al. Detection of antisperm antibodies Fertility and Sterility

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