ANTISPERMANTIBODIES: THEIR EFFECT ON THE PROCESS OF FERTILIZATION. STUDIED IN VITRO*

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1 FERTILITY AND STERILITY Copyright 1981 The American Fertility Society Vol. 35, No.5, May 1981 Printed in U.8A. ANTISPERMANTIBODIES: THEIR EFFECT ON THE PROCESS OF FERTILIZATION. STUDIED IN VITRO* JEHOSHUA DOR, M.D.t EDWINA RUDAK, PH.D. R. JOHN AITKEN, M.Sc., PH.D.:j: Medical Research Council Reproductive Biology Unit, Centre for Reproductive Biology, Edinburgh EH3 9EW, Scotland The influence of antisperm antibodies on the fertilizing capacity of human sperm was investigated in a zona-free hamster egg system using heterologous and homologous antisera. The ability of these antisera to interfere with the fertilizing capacity of human spermatozoa correlated well with the antibody titers obtained using the Franklin-Dukes tube slide test. However, there was little correlation between the antifertility effects of these antisera and their activities in the gelatin and tray agglutination tests. The addition of complement to the incubation medium. amplified. the inhibitory activity exhibited by these antisera, although the extent of this inhibitory influence bore no relationship to the antibody titers obtained using the complement-dependent sperm-immobilization technique. The study demonstrates the importance of the hamster egg penetration test in evaluating the antifertility effect of antisperm antibodies. Fertil Steril35:535, 1981 The detection of antisperm antibodies in serum l - 3 and in the cervical secretions of infertile women 4 and their relationship to infertility has been investigated extensively, usingspermagglutination,5 immobilization,6 and indirect immunofluorescence 7 techniques. Unfortunately, these studies have been inconclusive, and no consistent relationship has been identified between fertility status and antibody titer.8 There is therefore a need to study the problem with more definitive methods. Received November 17, 1980; revised and accepted January 6, *Supported in part. by a grant from the Ruyadh AI-Kharj Hospital, Saudi Arabia, to ProfessorD. T. Baird, Department of Obstetrics and Gynaecology, Edinburgh. tpresent address: Department of Obstetrics and Gynaecology, The Chaim Sheba Medical Center, Tel-Hashomer, Israel. :j:reprint requests: Dr. R. J. Aitken, Medical Research Council Reproductive Biology Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, Scotland. 535 Several studies have been carried out in which the ability of antisperm antibodies to block the various sperm-egg interactions occurring during the fertilization process has been assessed in vitro.9 Tzartos,lO for example, has demonstrated that the fertilization of zona-free hamster eggs is inhibited by treating homologous spermatozoa with univalent antisperm antibodies. Menge and Black ll have also shown that, when human spermatozoa are treated with heterologous antisperm antibodies of unknown titer, there isa reduction of their ability to penetrate zona-free hamster eggs. In this study, we have used the zona-free hamster egg system 12 to examine the fertilizing capacity of human spermatozoa exposed to serial dilutions of heterologous and homologous antisperm antibodies of known titer. In this way a comparison has been made between the activity of the antisera according to conventional agglutination, immobilization, and immunofluorescence tests and their ability to interfere with the fertilizing capacity of human sperm.

2 536 DORETAL. May 1981 MATERIALS AND METHODS Production and Assessment of Heterologous Antiserum Heterologous antibodies to human spermatozoa were raised in a rabbit by two intravenous injections, 3 weeks apart, of 400 x 106 and 150 x 106 human spermatozoa, respectively.10 Before injection, the spermatozoa were washed three times in 10 ml of Dulbecco's phosphate-buffered saline containing 1% polyvinylpyrrolidone. Serum was obtained from blood samples obtained on days 10 and 13 after the last injection. All serum samples were inactivated by heating to 56 C for 30 minutes before use. The activity of the antisperm antibodies in this heterologous antiserum was evaluated by three methods. Agglutination. The agglutinating activity of the serum was assessed by three methods. 1. The tube-slide agglutination test (TSAT), based on the method of Franklin and Dukes.5 The spermatozoa were washed in a Krebs-Ringer solution (BWW) modified to contain mm CaCl 2 instead of calcium lactate and 0.3% human serum albumin (HSA) (fraction V; Miles Laboratories, Elkhart, Ind., and Sigma Chemical Co., St. Louis, MO.),13 centrifuged at 600 x g for 5 minutes, and resuspended at a concentration of 40 x 106/ml, 0.1 ml of this sperm suspension was then added to 0.5 ml of serum and incubated for 1 hour at 37 C. A quantitative estimation of the agglutinating titer was made by using serially diluted serum samples in the range 1:10 to 1:1280. After incubation, the number of freely motile sperm, the number of motile agglutinated sperm, and the type of agglutination were recorded in 12 highpower ( x 400) fields. The test was considered positive when 10% or more of the motile sperm were agglutinated The gelatin agglutination test (GAT) was performed according to the method described by Kibrick et a1.14 Washed sperm at a concentration of 40 x 106/ml in BWW (see penetration test) were mixed with an equal volume of diluted serum and transferred to Kibrick tubes. The tubes were incubated at 37 C in 5% CO 2 in air and were assessed for agglutination after 2 hours. 3. The microtray agglutination test (TAT) was performed according to the method described by Friberg.15 The antiserum was serially diluted with 0.01 M phosphate-buffered saline (PBS) (ph 7.4),.and aliquots of each dilution were transferred to a microchamber tray under mineral oil; of semen, diluted to a concentration of 40 x 106/ml, was then added to each aliquot of antiserum and the mixture was incubated at 37 C for 2 hours prior to evaluation. Immobilization. The sperm immobilization test (MIT) was carried out according to the method described by Isojima et a1. 6 The sperm were washed in BWW and the pellet obtained after centrifugation at 600 x g for 5 minutes was resuspended in the same medium to give a concentration of 40 x 106 spermlm1. A portion ( ) of the sperm suspension was added to 250,",,1 of serum and of complement (WeUcome Laboratories, Research Triangle Park, N. C., diluted 1:10) and incubated at 37 C for 1 hour. Quantitative estimation of the immobilization titer was made by using serial dilutions of serum in the range 1:10 to 1:10,240. After incubation the numbers of motile and nonmotile sperm wer~ recorded for 12 high-power (x 400) fields. The sperm immobilization value (SlY) was then calculated: SIV = % motility in control serum/% motility in immune serum. Immunofluorescence. An indirect immunofluorescence technique was used, based on the method described by Hjort and Hansen. 7 Air-dried smears of washed spermatozoa at a concentration of 40 x 106/ml were prepared, fixed in absolute methanol for 30 minutes, then rehydrated in PBS. The rehydrated smears were incubated with serum in a humid chamber for 60 minutes, after which the slides were washed twice in PBS and incubated with fluorescein isothiocyanate-conjugated sheep antirabbit immunoglobulin (Wellcome Laboratories) diluted 1:5 in PBS. After repeated washing, the immunofluorescent staining patterns were observed under dark-field ultraviolet-blue illumination with a Leitz Ortholux II microscope equipped with a 50-watt mercury lamp, a KP 490-mm interference filter, a secondary K530 filter, and a BG-38 red suppressor filter. The test was considered positive when the characteristic staining was observed in the majority (>50%) of the spermatozoa.16 Source and Assessment of Homologous Antisera Both male and female human sera containing antibodies were obtained from the World Health Organization's Reference Bank for Reproductive Immunology, Aarhus, Denmark. The titer of antisperm antibodies in these sera had been assessed

3 Vol. 35, No.5 ANTISPERM ANTIBODIES: EFFECT ON FERTILIZATION IN VITRO 537 by the World Health Organization (WHO), using tray,15 gelatin,14 and immobilization 6 tests. In addition, the concentration of agglutinating antibodies was determined in the authors' laboratory using the tube-slide test.5 Control human serum samples were obtained from healthy fertile males, and all serum samples were heat-inactivated (56 C for 30 minutes) before use. 0 Assessment of Fertilizing Capacity The fertilizing capacity of human spermatozoa was assessed by using a zona-free hamster egg technique based on the method described by Yanagimachi et al.12 The medium used for washing the sperm samples and recovering the hamster ova was BWW12 containing 0.3% HSA, whereas the spermatozoa were capacitated by a 6-hour incubation in BWW containing 1.8% HSA (high-albumin BWW). Semen samples were obtained from healthy donors of either proven or presumed fertility, who were known to exhibit a high penetration rate in the zona-free hamster egg system. The semen was washed three times in BWW and centrifuged after each wash at 600 x g for 5 minutes. After the last wash, the spermatozoa was resuspended in high-albumin BWW at concentrations of 10 x 10 6 spermlml for the control and antiserum-alone incubations and 12 x 10 6 spermlml for the antiserum plus complement cultures. To assess the effect of agglutinating antibodies on the fertilizing capacity of human spermatozoa, serial dilutions were prepared in the range 1:40 to 1:1280 for the heterologous antiserum and in the range 1:10 to 1:160 for the homologous antisera. For the last hour of the 6-hour preincubation period, 25-J..LI aliquots of appropriately diluted antiserum were added to the sperm suspension. To assess the effect of complement on the ability of antisperm antibodies to inhibit the fertilizing capacity of human spermatozoa, serial dilutions from 1:160 to 1:10,240 and 1:10 to 1:160 were prepared from the heterologous and homologous antisera, respectively, as well as appropriate control sera. For the last hour of the 6-hour preincubation period, 25 J..LI of diluted serum and 100 J..LI of complement were added to the sperm suspension. In order to minimize the nonspecific effects of complement on sperm motility, we used a complement concentration of 10% for the penetration assay, which we had found to be the lowest concentration sufficient to reveal the immobilizing activity of antisperm antibodies. After 1 hour, the serum-treated sperm suspensions were centrifuged at 600 x g for 5 minutes and the sperm pellet was resuspended in 1 ml of high-albumin BWW to give a final concentration of approximately 10 x 10 6 /ml. Aliquots (20 J..Ll) of the preincubated sperm suspension were then transferred to a Petri dish (Falcon Plastics, Los Angeles, Calif.) and covered with paraffin oil. Eggs were recovered from superovulated adult female golden hamsters (Mesocricetus auratus), 8 to 12 weeks old. Ovulation was induced with an intraperitoneal injection of 30 IU of pregnant mare serum gonadotropin on day 1 of the estrous cycle followed by an intraperitoneal injection of 40 IU of human chorionic gonadotropin on day 3. The animals were killed 17 to 18 hours after the injection of human chorionic gonadotropin and the cumulus masses were recovered from the swollen ampullary region of the excised fallopian tubes. The ova were freed from the cumulus cells by incubation with 0.1 % hyaluronidase in BWW, washed twice with BWW, and the zonae pellucidae were then digested off with 0.1% trypsin in BWW. The zona-free eggs were washed twice in BWW and transferred to the pre incubated sperm droplets, 5 to 12 eggs/20-j..li drop. Eggs and sperm were incubated together at 37 0 C in 5% CO2 in air for 3 hours. After incubation with the spermatozoa, the eggs were rinsed in BWW and then transferred with a small amount of accompanying medium to the center of a microscope slide. The ova were then gradually compressed under a cover slip to a depth of about 30 J..Lm and viewed at x 400 magnification using phase-contrast optics. Ova containing one or more swollen sperm heads with associated tails were considered to be penetrated. Heterologous Serum RESULTS Agglutination, Immobilization, and Immunofluorescence Titers. The agglutinating and immobilizing activity exhibited by serial dilutions of the heterologous rabbit antiserum are shown in Table 1. The TSAT gave positive results at serum dilutions of 1:10 to and was negative at higher dilutions. The dominant patterns of sperm association were the tail-to-tail variety, although a small proportion of spermatozoa exhibited headto-tail or head-to-head agglutination. The control serum showed some agglutinating activity at a

4 538 DORETAL. TABLE 1. Antisperm Antibody Titers in Heterologous Rabbit Antiserum as Determined by Five Separate Techniques Test used to a"""ss titer Tube-slide agglutination test (TSAT) Gelatin agglutination test (GAT) Tray agglutination test (TAT) Immobilization test (MIT) Indirect immunofluorescence (IF) Titer 1:10,240 1:256 1:5,120 dilution of 1:10 (12.8% agglutination) but was negative at higher dilutions. In the gelatin and tray agglutination tests this antiserum gave positive results up to a dilution of 1:10,240 and 1:256, respectively, whereas in the immobilization assay a titer of 1:5120 was observed. The immunofluorescent staining was positive in the immune serum at dilutions of 1:2 to but was negative when control serum was used. The staining patterns observed consisted of acrosome and tail fluorescence in 87% of spermatozoa, postacrosomal cap fluorescence in 3.5%, equatorial segment fluorescence in 2.8%, midpiece fluorescence in 2.1%, and whole-cell staining in 4.5%. Penetration Assay Using Zona-Free Hamster Eggs. The influence of serially diluted heterologous antiserum on the ability of human spermatozoa to penetrate zona-free hamster ova in the absence of complement is shown in Table 2. Total inhibition of penetration was observed when the sperm were incubated in immune serum dilutions of 1:10 and 1:40; there was a significant decrease at dilutions of (X 2 = 31.0; P < 0.001) and 1:160 (X 2 = 6.84; P < 0.01); inhibition at a dilution of 1:320 was not significant. The influence of the serially diluted heterologous antiserum on egg penetration rates in the presence of complement is shown in Table 3. A total inhibition of penetration was observed at a May 1981 dilution of 1:160, and the penetration rate observed at a dilution of 1:320 was significantly decreased (X 2 = 6.5; P < 0.01) with respect to the appropriate controls. Some inhibition of egg penetration was observed with higher immune serum dilutions, but the differences were not found to be statistically significant. Homologous Sera Agglutination and Immobilization Titers. The agglutinating and immobilizing activities exhibited by the homologous antisera (two female, three male) are depicted in Table 4. The TSAT was positive in the two female sera tested but was negative in the three male serum samples. The MIT was positive in one of the female sera and in two of the male samples, while the TAT and GAT were positive for all of the serum samples tested. No agglutinating or immobilizing activity was observed in any of four control serum samples subsequently used in the hamster egg penetration assay. Penetration Assay Using Zona-Free Hamster Eggs. The influence of serially diluted homologous antisera on the ability of human spermatozoa to penetrate zona-free hamster ova in the absence of complement is shown in Table 5. Serum 106 exhibited a significant inhibition of penetration at dilutions up to 1:40 (X2 = 13.18; P < 0.001), but not at higher dilutions. Serum 38 was also capable of inhibiting sperm penetration at serum dilutions of 1:20 (X2 = 7.1; P < 0.001) and 1:40 (X2 = 29.4; P < 0.001), although no significant inhibition was observed at. Serum samples 55, 95, and 118 did not exhibit any inhibitory activity at a nominal dilution of 1:10; sperm penetration rates were even slightly increased in the presence of two of the antisera (95 and 110). TABLE 2. Penetration of Zona-Free Hamster Eggs by Human Spermatozoa Following Incubation in Serially Diluted Heterologous (Rabbit) Antisperm Antiserum Without Complement Control (serum free) 1:10 12/29 (41.4) 1:40 17/34 (50.0) 24/31 (77.4) 1:160 17/34 (50.0) 1:320 40/43 (93.0) 1:640 40/43 (93.0) 1:1,280 40/43 (93.0) almmune serum versus control serum. bp < cp < Penetrated eggs/total eggs (%) Immune serum 0/37 (0) 0/38 (0) 4/45 (8.9) 1128 (3.6) 18/31 (58.1) 26/35 (74.2) 40/43 (93.0) Control serum 10/35 (28.6) Total 12/31 (38.7) Total (62.0) 85 b 7/29 (24.1) 85 c 23/32 (71.9) (72.4) None 13/15 (86.6) None

5 Vol. 35, No.5 ANTISPERM ANTIBODIES: EFFECT ON FERTILIZATION IN VITRO 539 TABLE 3. Penetration of Zona-Free Hamster Eggs by Human Spermatozoa Following Incubation in Serially Diluted Heterologous (Rabbit) Antisperm Antiserum and Complement Penetrated eggs/total eggs (%) Control (serum-free) Immune serum Control serum 1:10 12/29 (41.4) 0/37 (0) 7/25 (28.0) Total 1:160 24/31 (77.4) 0/35 (0) (67.7) Total 1:320 24/31 (77.4) 8/42 (19.0) 16/37 (43.2) 56.0 b 1:640 19/26 (73.1) 9/24 (37.5) 10/23 (43.5) :1,280 23/34 (67.6) 13/49 (26.5) 29/69 (42.0) :2,560 37/59 (57.6) 25/98 (25.5) 19/61 (31.1) :10,240 37/59 (57.6) 37/107 (28.9) 46/105 (43.8) 34.0 acontrol serum versus immune serum. bp < The ability of three of the WHO antisera to inhibit the fertilizing capacity of human sperm in the presence of complement is shown in Table 6. Serum 106 showed a highly significant inhibition of penetration at dilutions of 1:40 (X 2 = 35.4; P < 0.001), (X 2 = 38.3; P < 0.001), and 1:160 (X 2 = 18.5; P < 0.001), although no inhibition was observed at 1:320. Serum samples 55 and 95 at dilutions of 1:10 exhibited no ability to inhibit the fertilizing capacity of human spermatozoa in the presence of complement. DISCUSSION Our study has demonstrated the importance of the hamster egg penetration test in evaluating the antifertility activity of antisperm antibodies. The ability of the various antisera tested to inhibit the fertilizing capacity of human sperm in the absence of complement correlated best with the results of the Franklin-Dukes tube-slide (TSAT) test. In three of the homologous antisera tested (WHO 55, 95, and 118), a negative TSAT was associated with an inability to inhibit the fertilizing capacity of human sperm despite the presence of significant titers according to the gelatin and tray agglutination tests. WHO serum 106 inhibited the fertilizing capacity of human sperm at a dilution of1:40 and gave an equivalent titer in the tube-slide test, whereas the gelatin and tray Antiserum no. agglutination tests gave titers of 1:64 and 1:256, respectively. Both WHO serum 38 and the heterologous serum inhibited the fertilizing capacity of human sperm at a slightly higher dilution than that which produced significant agglutination in the tube-slide test; even so, the titer produced by this test showed a better correlation with the results of the penetration assay than any of the other agglutination tests employed'in the study. The correlation observed between the results obtained in the tube-slide test and the dilution at which the fertilizing capacity of human sperm is inhibited is possibly related to the fact that this agglutination test is sensitive for antibodies directed against the sperm head. 17 In view of the results obtained in this study the ability of antisperm antibodies to inhibit the fertilizing capacity of sperm is clearly not a simple consequence of their ability to agglutinate spermatozoa. Similar conclusions have been reached by other workers 10, 11 from animal experiments involving the use of Fab antibody fragments, which retain their sperm binding but not their agglutinating and immobilizing activity. Antibodies directed against the sperm head might achieve their antifertility by interfering with the acrosome reaction or possibly by occluding the receptors on the outer plasma membrane for the sperm binding sites on the zona pellucida or oolemma. In the presence of complement, the antifertility activity of all of the antisera tested became ampli- TABLE 4. Agglutination and Immobilization Titers of Homologous Antisperm Antisera Sex Test used to assess titer" Franklin Dukes Friberg Kibrick TSAT TAT GAT WHO F 1:40 1:256 1:64 WHO 38 F 1:20 1:64 1:4 WHO 55 M 0 1:32 1:32 WHO 95 M 0 1:256 1:256 WHO 118 M 0 1:1,024 1:256 a TAT, GAT, and MIT performed by WHO; TSAT was carried out in the author's laboratory. bt_t, tail-to-tail; H-H, head-to-head; TT-TT, tail tip-tail tip. I~~Ta Mode of agglutination b 1:512 T-T, TT-TT, H-H 0 H-H 1:4 T-T 1:32 T-T 0 TT-TT

6 540 DORETAL. May 1981 TABLE 5. Fertilizing Capacity of Human Spermatozoa After Incubation in Homologous Antisera Without Complement Antiserum no. WHO 106 1:40 1:160 WHO 38 1:20 1:40 WHO 55 1:10 WHO 95 1:10 WHO 118 1:10 aimmune serum versus control serum. bp < Control (serum-free) 27/41 (65.9) 27/41 (65.9) 24/34 (70.6) 27/41 (65.9) 4/19 (21.1) 17/35 (48.6) (57.9) 17/35 (48.6) 12/25 (48.0) 8/22 (36.4) Penetrated eggs/total eggs (%) Immune serum Control serum 6/34 (17.6) 18/31 (58.1) 69.7 b 12/29 (41.4) 10/37 (27.0) None 17/30 (56.7) 15/32 (46.9) None 9/21 (42.9) 7/20 (35.0) None 8/50 (16.0) 12/30 (40.0) 60.0 b 5/45 (11.1) 19/27 (70.4) 84.2b 17/23 (73.9) 19/23 (82.6) /44 (25.0) 12/35 (34.3) /19 (52.6) 6/18 (33.3) None (51.2) 7/19 (36.8) None fied, presumably because the addition of complement introduces an immobilization effect into the activity spectrum of the antiserum and further disrupts the structural integrity of the sperm plasma membranes. Since complement is present in the female reproductive tract, these results emphasize the importance of including complement in any assessment of the antifertility activity of anti-sperm antibodies. The results ofthe Isojima immobilization test were poorly related to the capacity of the respective antisera to inhibit the fertilizing capacity of human sperm; the titers of those samples which exhibited positive activity in the immobilization test (the heterologous antiserum and WHO sera 106 and 95) were invariably higher than the dilutions at which fertilization was inhibited. A possible explanation for this lack of correlation became apparent when examination of the culture dishes at the end of the penetration test consistently revealed a higher percentage of live sperm in the incubation mixtures than would have been predicted from the results of the immobilization test. We therefore investigated the possibility that the difference in sperm concentration used in the immobilization test (3.1 x 106/ml) and the egg penetration assay (10 x 106/ml) was responsible for this discrepancy. We performed the immobilization test at the sperm concentration recommended for routine clinical analysis, 3.1 x 106/ml and at 9.3 x 106/ml for serum dilutions of 1:320 to 1:1280. The SIV for the low sperm concentration was always 5 to 6 times greater than that at the high concentration. From these results it is clear that the amount of antigen, represented as number of spermatozoa, present in the assay is highly critical in producing a positive or negative response. A high sperm concentration can seriously dimini~h the effect of the complement-fixing antibodies, and those spermatozoa which remain motile have the ability to penetrate eggs. These findings may help to explain the clinical observation that there is no significant difference in the pregnancy rates between infertile couples showing positive or negative results with the immobilization test. 8 It would therefore appear that the immobilization test, like the gelatin and tray agglutination tests, produces titers which exceed the ability of an antiserum to inhibit the fertilizing capacity of human sperm. TABLE 6. Fertilizing Capacity of Human Spermatozoa After Incubation in Homologous Antisera in the Presence of Complement Antiserum no. WHO 106 WHO 55 WHO 95 1:40 1:160 1:320 1:640 1:10 1:10 "Control serum versus immune serum. bp < Control (serum-free) 14/20 (70.0) 14/20 (70.0) 8/22 (36.4) 3/10 (30) 3/10 (30) 17/35 (48.6) 12/25 (48.0) Penetrated eggs/total eggs ('li-) Immune serum Control serum 1123 (4.3) 18/20 (90.0) 95.2 b 1130 (3.3) (80.0) 95.9 b 1130 (3.3) 12/24 (50.0) 95.3 b 10/22 (45.5) 10/20 (50.0) 4.8 6/16 (37.5) 5/14 (35.7) 0 9/33 (27.3) 16/42 (38.1) /25 (36.0) 7/25 (28.0) 0

7 Vol. 35, No.5 ANTISPERM ANTIBODIES: EFFECT ON FERTILIZATION IN VITRO 541 Acknowledgments. We are extremely grateful to Miss Fiona Newman, B.Sc., and Mr. David Richardson, M;Sc., of the Medical Research Council Unit of Reproductive Biology, Edinburgh; O. Djahanbakhch, M.D., F.R.C.O.G., Department ofobstetries and Gynaecology, Edinburgh; and T. Hargreave, M.B., F.R.C.S., Department of Surgery /Urology,Edinburgh, for their help in the study. R. V. Short, Ph.D., D.Sc., F.R.S., gave a valuable criticism of the manuscript. REFERENCES 1. Isojima S, Kunio K, Koyama K, Tanaka C, Naka 0, Adachi H: Further studies on sperm-immobilizing antibody found in sera of unexplained causes of sterility in women. Am J Obstet GynecoI112:199, Jones WR, Ing RMY, Kaye MD: A comparison of screening tests for anti-sperm activity in the serum of infertile women. J Reprod Fertil 32:357, Shulman S, Jackson H, Stone M: Antibodies to spermatozoa. VI. Comparative studies in sperm-agglutinating activity in groups of infertile and fertile women. Am J Obstet Gynecol 123:139, Dor J, Nebel L, Soffer Y, Mashiach S, Serr DM: Cell mediated and local immunity to spermatozoa in infertility. Int J Fertil 24:94, Franklin RR, Dukes CD: Antispermatozoal antibody and unexplained infertility. Am J Obstet Gynecol 89:6, 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, Hjort T, Hansen KB: Immunofluorescent studies on human spermatozoa. I. The detection of different spermatozoal antibodies and their occurrence in normal and infertile women. Clin Exp Immunol 8:9, Jones WR: The use of antibodies developed by infertile women to identify relevant antigens. In Karolinska Symposia on Research Methods in Reproductive Endocrinology, Seventh Symposium: Immunological Approaches to Fertility Control. Copenhagen, Scriptor, 1974, p Russo I, Metz CB: Inhibition of fertilization in vitro by treatment of rabbit spermatozoa with univalent antibody. J Reprod Fertil 38:211, Tzartos SJ: Inhibition of in vitro fertilization ofintact and denuded hamster eggs by univalent anti-sperm antibodies. J Reprod Fertil 55:447, Menge AC, Black CS: Effects of antisera on human sperm penetration of zona-free hamster ova. Fertil SteriI32:214, Yanagimachi R, Yanagimachi H, Rogers BJ: The use of zona-free animal ova as a test system for the assessment of. fertilizing capacity of human spermatozoa. BioI Reprod 15:471, Rudak E, Jacobs PA, Yanagimachi R: Direct analysis of the chromosome constitution of human spermatozoa. Nature 274:911, Kibrick S, BeldingDL, Merrill B: Methods for detection of antibodies against mammalian spermatozoa. II. A gelatin agglutination test. Fertil Steril 3:430, Friberg J: Clinical and immunological studies on spermagglutinating antibodies in serum and seminal fluid. Acta Obstet Gynecol Scand [Suppl] 36:3, Johnson WL, Menge AC: Evaluation of human sera for antibodies against sperm by immunofluorescence. Fertil Steril 26:721, World Health Organization: Auto- and iso-antibodies to antigens of the human reproductive system. Acta Pathol Microbiol Scand Suppl 258, 1977