Clin. exp. Immunol. (1976) 25, 73-79.

Human sperm antigens and antisperm antibodies II. AGE-RELATED INCIDENCE OF ANTISPERM ANTIBODIES K. S. K. TUNG, W. D. COOKE, JR, TERESITA A. McCARTY & P. ROBITAILLE Department of Pathology, School of Medicine, University of New Mexico, Albuquerque, New Mexico, U.S.A., and Department of Pediatrics, University ofMontreal, Montreal, Canada

(Received 25 November 1975) SUMMARY

Naturally occurring human antisperm antibodies (ASA) detectable by immunofluorescence, were found to have a peak incidence of 90°/, in both sexes before puberty. Thereafter, the incidence declined to about 60%/o and persisted through life. This age-related incidence resembled that for foreign and not self antigens. The natural incidence of the six antibodies under investigation varied: the most frequent were antibodies to acrosomal antigen Acl and equatorial antigen; the least frequent were antibodies to acrosomal antigen Ac2 and sperm nuclear protamine; and between these were antibodies to the mainpiece of tail and post-acrosomal region. Irrespective of their natural incidence, these antibodies increased at a comparable rate in men following vasectomy indicating these six sperm antigens have comparable immunogenicity. These results provide evidence for the lack of immunologic tolerance in man toward many sperm antigens. The variation in the natural incidence of individual ASA is explanable by differences in prevalence of crossreaction between each sperm antigen with exogenous antigens. Thus, antibodies to acrosomal antigen Acl and equatorial antigen, which occurred most frequently, appeared to crossreact with ubiquitous microorganisms. INTRODUCTION The question of whether immunological tolerance exists for sperm remains unanswered. Several lines of evidence suggest that sperm may deviate immunologically from other autoantigens: (1) sperm antigens develop at puberty, when the immune system is fully mature; (2) sperm is normally separated from the immune system by an effective blood-testis barrier (Fawcett, Leak & Heidger, 1970; Johnson, 1970); and (3) immune response to sperm as well as an unusually vigorous granulomatous tissue reaction follow spillage of sperm outside its normal residence (Glassy & Mostofi, 1956). Whether a given antigen is self or foreign is indicated by the age-related incidence of its homologous antibody. Thus, the incidence of antinuclear (Cannat & Seligmann, 1965), antithyroid (Goodman et al., 1963), and antigastric parietal cell (Doniach & Roitt, 1964) antibodies, which is negligible in childhood and young adults, rises gradually after middle age. Such finding has been interpreted as demonstrating the existence of immunological tolerance to the different self antigens, and that tolerance becomes gradually terminated as the immune system ages (Walford, 1969). On the other hand, antibodies to foreign antigens tend to have an early onset, reach a peak before puberty and gradually decline in incidence (Friedberger, Bock & Furstenheim, 1929; Thomsen & Kettel, 1929; Lewi, 1968). Until recently, antisperm antibodies (ASA) were detected by their reaction with sperm surface antigens resulting in sperm immobilization or agglutination. In the past several years, some investigators (Hjort & Hansen, 1971; Tung, 1975) have applied immunofluorescence (IF) to the detection and semiquantitation of ASA. When we used this method to examine the types and incidence of ASA in men Correspondence: Dr Kenneth S. K. Tung, Department of Pathology, School of Medicine, University of New Mexico Albuquerque, New Mexico 87131, U.S.A.

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before and after vasectomy (Tung, 1975), we were surprised to find that ASA existed in over 60% of men before vasectomy. In order to gain more insight on these 'natural' antibodies, and further evaluate the question of immunological tolerance to sperm, we have studied the sera from a number of children, adults and elderly individuals of both sexes for the incidence of different types of ASA. Unlike other autoantibodies, ASA reached their peak incidence of 90%/0 between the ages of 1 and 10 years, were more equally distributed in both sexes, and that the most common types of ASA appeared to react with several ubiquitous micro-organisms. MATERIALS AND METHODS Subjects. The sera for this study came from 373 individuals (Table 1). The sera from 174 children and teenagers came from Albuquerque, New Mexico, Montreal, Canada, and Denver, Colorado, and were obtained from patients attending the respective pediatric clinics. Healthy adult males between the ages of 22 and 51 years were candidates for the bilateral vasoligation operation; and of these 114 men, who were studied before their operation, 112 were also studied 2 months after vasectomy and seventy-one between 7 and 9 months after vasectomy. The thirty-eight adult females included twenty-four Catholic nuns and fourteen laboratory personnel, and their ages ranged from 22 to 55 years. Forty-seven elderly residents of a geriatric home, aged between 70 and 85, were also studied. Sera were shipped to Albuquerque on dry ice and kept at - 70'C until study. Detection of antisperm, antinuclear, antimitochondrial, and antiadrenal antibodies by indirect IF. This method has been described in detail elsewhere (Tung, 1975). Human IgG and monoclonal human IgM were prepared by chromatographic methods. Rabbits were immunized with these antigens in Freund's complete adjuvant. From the rabbit serum IgG was isolated and the antihuman IgG and IgM antibodies were made y and p specific respectively by appropriate immunoabsorptions. Following conjugation to fluorescein isothiocyanate (FITC), the labelled IgG fractions were further chromatographed on DEAE with step-wise increase in ionic strength. The fractions with a fluorescein: protein ratio of 2 were used. The specificity of the FITC-conjugated antisera was determined by (1) their reactivity, or lack of it, with tissue bound IgG or IgM in human renal biopsies, (2) a reduction of fluorescence following absorption of the FITC antisera with its specific antigen, and (3) by their lack of reaction with human sperm and sections of mouse kidney or human adrenal gland. Sperm for IF study came from two healthy donors with 0 blood group. Liquescent semen samples were washed twice by centrifugation at 200 g for 10 min at 4VC in phosphate-buffered saline, 0-15 M, pH 7-2 (PBS). For detection of the different ASA, four types of sperm smears were prepared. (1) Regular sperm smears: sperm were smeared on clean glass slides, air dried, fixed in absolute methanol for 30 min, again dried, and stored at - 20'C for use within 1 week. (2) Swollen sperm smears: sperm smeared on glass slides and fixed by absolute methanol for 5 min were incubated briefly in a solution of 50 mm TrisHCl, 0-4 mm dithiothreitol (DTT) and 10 pgg/ml of trypsin, pH 9 (Sigma Chemical Company, St Louis, Missouri) until swelling of the sperm head was apparent under phase microscopy, rinsed in distilled water, air dried and stored at - 20°C (Kolk, Samuel & Rumke, 1974; T. Samuel, personal communication 1975); (3) Acid-treated sperm smears: sperm smeared on glass slides were fixed for 10 min in absolute methanol, incubated in citrate buffer, 0-15 M, pH 3, for 30 min, washed in PBS (Tung, 1976). (4) Controlfor acid-treated sperm smears: as in (3), except that citrate buffer was replaced by PBS. Frozen sections of testis and epididymis of healthy young men obtained within 4 hr of accidental death, fixed in absolute methanol for 30 min, were also used for detection of ASA. To detect antinuclear antibodies, frozen sections of mouse kidney were fixed for 30 min in acetone, air dried and stored at - 20°C. Antimitochondrial antibodies were detected by unfixed, frozen sections of mouse kidney. The procedure for the indirect IF staining was carried out as described previously (Tung, 1975). The slides were examined with a Leitz Ortholux II fluorescent microscope using a 150 W xenon light source, To eliminate nonspecific staining, all studies were carried out with sera diluted at 1: lOin PBS. To semiquantify ASA by IF, the sperm smears were incubated with serial dilutions of the patients' serum (in PBS); and the titre was the highest dilution that gave a positive reaction. In order to minimize the variations inherent in the IF test, the study always included serial dilutions of a positive control. The experimental results were accepted only when the titre of the positive control fell within one tube dilution of its expected titre. Each serum sample was studied twice using sperm smears from different donors. When ASA titre was 40 or higher, the results were reproducible. With sera having ASA of 10, the results between the two studied differed in about 10%/ of cases. These equivocal sera were then studied for the third time, and those that exhibited antibody activity in two of three studies were also considered positive. The percentage of sperm with fluorescence in a positive slide varied according to the antibody titre. Thus, strong antibody stained literally all of the sperm, and sera with antibody of 10 stained around 50%. of the sperm on the slide. Staining of suspension of viable sperm by immunofluorescence was carried out as described previously (Tung, Unanue & Dixon, 1971) Absorption ofantibodies by micro-organisms and tissue extracts. Pure cultures of Staphylococcus aureus, S. albus, ,B-haemolytic Streptococcus, Streptococcus pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Klebisella pneumoniae and Candida albicans isolated from patients were washed and lyophilized in PBS. Tissues were obtained at autopsy from five young individuals within 4 hr of their accidental death, and they included brain, testis, liver, kidney, adrenal gland, heart, spleen and ovary. The

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tissue was homogenized in PBS (w/v = 1/1) and lyophilized. Seminal plasma from vasectomized men was collected and lyophilized. To absorb, 1 mg of antigen was incubated with 100 Jul of serum for 1 hr at 370C, then overnight at 4VC. This procedure was repeated twice. As controls, the same serum was incubated (1) without the micro-organism or tissue, and (2) with human sperm. The serum for absorption contained antisperm antibodies of well-defined patterns with titres above 1: 40. Before absorption, the serum was diluted to two tube-dilutions below the antibody titre and absorption was considered positive when the absorbed serum failed to stain the sperm by IF.

RESULTS Human ASA detected by IF IF revealed antibodies to at least eight sperm antigens. Regular sperm smears detected antibodies to two antigens in the acrosome: Acl and Ac2 (Tung, 1976), one antigen each in the equatorial region (E), the post-acrosomal region (PA), the mainpiece of tail (T) (Hjort & Hansen, 1971), and the tip of tail (Johnson & Menge, 1975). Sperm smears pretreated with DTT and trypsin detected antibodies to two additional sperm antigens: the midpiece of the tail (Tung, 1975) and sperm nuclear antigen which was shown to be protamine (N) (Kolk & Samuel, 1975) Since Ac2 was found to be more resistant to acid pH than was Acl (Tung, 1976), their identification was based on their reaction with acid-treated sperm smears. In this study, we confined our attention to six of the antibodies: anti-Acl, anti-Ac2, anti-E, anti-PA, anti-T and anti-N. ASA detected by IF were sperm-specific since the reaction was readily absorbed by sperm and testicular extracts; and except for anti-Acl which cross-reacts with human adrenal extracts (Hjort & Hansen, 1971; Tung, 1976), was not absorbed by other tissues. These were not antibodies to blood-group or sperm-coating antigens (Weil & Rodenburg, 1962) since they stained group 0 sperm, were not absorbed by seminal plasma, and reacted with sperm in frozen sections of human testis and epididymis. Since the staining pattern of several hundreds of the ASA studied remained consistent regardless of sperm donors, the antibodies were not likely to be alloantibodies. 100 _ E

80

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0 A

B

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Age groups*

FIG. 1. Age-related incidence ofantisperm and antinuclear antibodies detected by indirect immunofluorescence in the males (o or 0) and the females (a or *). Of the antisperm antibodies, circle denote incidence of antibodies at titre 10, squares denote incidence of antibodies with titres of 40 or higher. Age groups: A, < 1 year; B, 1-10 years; C, 11-20 years; D, 22-55 years; E, 70-85 years.

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ASA detected by IF were directed to intracellular antigens of sperm because the study was made on dried sperm smears, and ASA did not stain the surface of viable sperm in suspension. The age-related incidence of antisperm and antinuclear antibodies In contrast to antinuclear antibodies, ASA occurred most frequently early in life, and the incidence did not differ significantly according to sex. As shown in Fig. 2, almost 90% of the children between the ages of 1 and 10 years had ASA of titre 1:10 or higher. The incidence was reduced to about 60%/o in the next decade; and then persisted at this level through life. Since the scoring of ASA of low titre (1:10) tended to vary in about 10% of the sera between different studies, the incidence of those antibodies with titres of 1:40 or over was also analysed. The results (Fig. 1) showed that the incidence of the high titred antibodies likewise paralleled the overall incidence, again with a peak in the age group of 1-10 years. TABLE 1. Incidence of human antisperm antibodies according to age and sex

Incidence of antibodies* No. of Subjects with subjects antibodies (Y.) Acl

Subjects Children, total (under 21 years) Children, less than 1 year Children, 1-10 years Children, 11-20 years Children, all males Children, all females Adults, both sexes (22-25 years) Adults, males Adults, females Aged adults, total (70-85 years) Aged adults, male Aged adults, female

174 29 87 58 88 86 152 114 38 47 17 30

124 (71) 10 (34) 75 (86) 39 (67) 60 (68) 64 (74) 83 (55) 64 (56) 19 (50) 32 (68) 14 (82) 18 (60)

53 10 75 43 52 55 32 31 34 43 47 40

Ac2

E

PA

T

N

2 0 1 3 2 1 5 6 0 11 18 7

31 31 31 29 32 29 20 25 3 21 29 17

9 0 8 14 5 13 16 11 32 11 12 10

12 5 15 9 7 16 4 4 5 2 12 0

0 0 0 0 0 0 0 0 0 0 0 0

* Percentage of total number of patients.

When the incidence of the six ASA were analysed individually, their age-related incidence was found to vary considerably (Table 1; Fig. 2) Most frequently observed were anti-Acd and anti-E, which accounted for the overall age-related incidence of ASA shown in Fig. 1. Antibodies that were rarely found were anti-N and anti-Ac2. Anti-N was not found until after vasectomy (see later), and anti-Ac2 was detected rarely except in old men. Finally, anti-PA and anti-T tended to occur with an intermediate frequency between the above groups. Significant differences were noted between adult men and adult women with respect to anti-PA and anti-E antibodies, but the basis for this finding is obscure. Incidence of ASA, antinuclear and antimitochondrial antibodies after vasectomy ASA increased in incidence following vasectomy (Fig. 3). The slopes of the curves showing increment in incidence are comparable between the different antibodies irrespective of their incidence before vasectomy, thus indicating that all of the sperm antigens elicited autoantibodies to a similar extent. None of the patients developed antinuclear or antimitochondrial antibodies at 6-9 months following vasectomy.

Immunoglobulin classes of ASA One hundred and six antisperm antibodies from children, eighty-three from adults between 25 and 55 years, and thirty-two from elderly people were studied for immunoglobulin classes by IF using FITCconjugated H chain-specific antisera. Differences were not found between the different age groups; and

Age incidence ofhuman antisperm antibodies

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D

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D

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FIG. 2. Age-related incidence of six antisperm antibodies in the males (o) and females (X). Age groups: sce legend to Fig. 1.

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FIG. 3. Incidence of six individual antisperm antibodies before, at 2 and at 6-9 months after vasectomy. The increase in incidence of antibodies with a low natural incidence (- -) is comparable to that of antibodies with ). a higher natural incidence (

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Tung et A

in each, about 60%/o of the antibodies were of IgG and IgM classes, 30%/O were restricted to IgG, and 10% to IgM. Crossreaction between ASA and antigens of micro-organisms When sera with anti-Acl antibodies were absorbed with lyophilized Staphylococcus aureus, S. albus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans, they no longer reacted with sperm by indirect immunofluorescence. This finding suggests that anti-Acl antibodies crossreact with certain antigen(s) in these microorganisms. Similarly, anti-E antibody was found to cross-react with Pseudomonas aeruginosa. In contrast, the reactivity of anti-Ac2, anti-PA, anti-T and anti-N antibodies with sperm was not absorbed by these micro-organisms.

DISCUSSION This study demonstrates clearly that ASA detectable by IF were found most frequently in prepubertal children of either sex. The incidence of antibodies then gradually declines with age. Since sperm antigens are not present at this early age, the ASA almost certainly represent the result of immune response in early childhood to exogenous antigens that crossreact with human sperm. The children in this study were patients attending pediatric clinics, therefore, they might be considered a select group and not representative of the normal population. However, when the clinical histories of fifty children were reviewed, it was found that over 90% of them had only minor physical trauma or common colds; and their incidence of ASA was comparable to those of the entire group of children. The IF studies on human antisperm antibodies by Hjort & Hansen (1971) and of Husted & Hjort (1974) in fact provided data that was somewhat comparable to that reported here. For example, 58% of prepubertal children were found to have antisperm antibodies, albeit of low titre, as compared with an overall incidence in adults of around 20%. However, the results of these studies were not discussed in the context of agerelated incidence of antisperm antibodies as they differ from those of other autoantibodies. The age-related incidence of ASA is akin to that found for antibodies to exogenous antigens. For example, the incidence and titre of 'natural' antibodies to sheep erythrocytes (Friedburger et al., 1929) and isoagglutinins to A or B blood group antigen in blood group 0 individuals (Thomsen & Kettel, 1929) have been found to appear early, peak at the age of 10 years and then decline. In contrast, antibodies to self antigens, such as antinuclear antibodies (Cannat & Seligmann, 1965), antithyroid antibodies (Goodman et al., 1963), antiparietal cell antibodies (Doniach & Roitt, 1964) and rheumatoid factor (Heimer, Levin & Rudd, 1963) are found late in life. This study evaluated antibodies to six sperm antigens, and their age-related incidence was found to differ markedly. Three possible explanations should be considered: (1) immunological tolerance exists only to some but not to all of the six sperm antigens; (2) these sperm antigens or their cross-reactive exogenous antigens differ in their immunogenicity; and (3) different sperm antigens crossreact with exogenous antigens to different extents. The first possibility is not likely since following vasectomy, when men presumably are stimulated by sperm, they develop an increased incidence of antibodies to all six sperm antigens, but not to those antigens (such as DNA and mitochondria) in sperm for which immunologic tolerance exists. Evidence against the second possibility is the finding that vasectomy leads to a comparable increment in incidence of the different ASA. The third possibility is considered most likely since anti-Ac 1 and anti-E, which are the most common of all ASA, are found to cross-react with many ubiquitous micro-organisms. Cross-reaction between human antisperm antibodies and microbial antigen has been previously demonstrated by Sarkar (1974). Based on results of the present study, several tentative conclusions concerning the immunological status of some of the sperm-specific antigens may be formulated. (1) Sperm antigens, such as Acl, Ac2, E, PA, T and N, are essentially foreign antigens, and to them, we do not develop immunological tolerance. (2) These antigens in sperm are immunogenic and will elicit antibodies when men are exposed to sperm as in vasectomy. (3) Exogenous antigens, such as micro-organisms that cross-react with sperm antigens Acl and E, can elicit similar antibodies which account for the age-related ASA in both

Age incidence of human antisperm antibodies

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sexes. (4) Finally, the frequency of the various ASA are related to the extent of cross-reaction that exists between each individual antigen and exogenous antigens. It is possible that the origin of natural ASA that has been described in rabbits (Symons & Herbert, 1971), guinea-pigs (Johnson, 1968), and mice is explanable on a similar basis. Sperm antigens detectable by IF are in all likelihood different from those detected by spermagglutination or spermimmobilization (Husted & Hjort, 1974; Kolk et al., 1974). Whether any of the conclusions that we have reached in this study also apply to antigens related to spermagglutination or spermimmobilization remains, of course, open to question. The authors are extremely grateful to Dr Blaise E. Favara, Denver, Colorado, for providing the children's sera. The sera from geriatric patients was a generous gift of Dr Ralph Williams. This research was supported by National Institutes of Health contract number HD-3-2717; and, in part, by NIH grant number HD-0-7591.

REFERENCES sera for antibodies against sperm by immunofluorescence. Fertil. Steril. 26, 721. KOLK, A.H.J. & SAMUEL, T. (1975) Isolation, chemical and immunological characterization of two strongly basic nuclear proteins from human spermatozoa. Biochim. biophys. Acta (Amst.), 393, 307. KOLK, A.H.J., SAMUEL, T. & ROMKE, P. (1974) Autoantigens of human spermatozoa. I. Solubilization of a new auto-antigen detected on swollen spermheads. Clin. exp. Immunol. 16, 63. LEWI, S. (1968) Observations sur les iso-anticorps du supplement 10, 105. systeme ABO et sur les effects de la stimulation par les FRIEDBERGER, E., BoCK, G. & FURSTENHEIM, A. (1929) Zur substances de groupe. Gerontologia, 14, 160. Normalantikorperkurve des Menschen durch die verschiedenen Lebensalter und ihre Bedeutung fur die SARK"R, S. (1974) Carbohydrate antigens of human sperm and auto-immune induction of infertility. J. Reprod. Med. Erklirung der Hautteste (Schick, Dick). Z. Immunitats13, 93. forsch. 64, 294. GLASSY, F.J. & MOSTOFI, K.F. (1956) Spermatic granuloma SYMoNs,D.B.A. & HERBERT, J. (1971) Incidence of Immunoglobulins in the secretion of rabbit genital tracts and the of the epididymis. Amer. ]. clin. Path. 26, 1303. distribution of IgG-globulin in the tissues of the female GOODMAN, M., ROSENBLATT, M., GOTTLIEB, J.S., MIUER, tract. 3. Reprod. Fertil. 24, 55. J. & CHEN, C.H. (1963) Effect of age, sex, and schizophrenia on thyroid autoantibody production. Arch. gen. THOMSEN, 0. & KETTEL, K. (1929) Die Starke der menschlichen iso-agglutinine und entsprechenden BlutkorperPsychiat. 8, 518. chenrezeptoren in verschiedende Lebensaltern. Z. HEIMER, R., LEvIN, M. & RUDD, E. (1963) Globulins Immunitatsforsch. 63, 67. resembling rheumatoid factor in serum of the aged. TUNG, K.S.K. (1975) Human sperm antigens and antisperm Amer. ]. Med. 35, 175. antibodies. I. Studies on vasectomy patients. Clin. exp. HJORT, T. & HANSEN, K.B. (1971) Immunofluorescent Immunol. 20, 93. studies on human spermatozoa. I. The detection of different spermatozoal antibodies and their occurrence TUNG, K.S.K. (1976) Human sperm antigens and antisperm antibodies. III. Studies on acrosomal antigens. Clin. exp. in normal and infertile women. Clin. exp. Immunol. 8, 9. Immunol. (In press). HUSTED, S. & HJORT, T. (1974) Comparison of the occurrence of spermatozoal antibodies in male and female TUNG, K.S.K., UNANUE, E.R. & DIXON, F.J. (1971) Pathogenesis of experimental allergic orchitis. II. The blood donors. Clin. exp. Immunol. 17, 61. role of antibody 3. Immunol. 106, 1463. JOHNSON, M.H. (1968) Characterization of a natural antibody in normal guinea-pig serum reacting with homo- WALORD, R.R. (1969) The Immunologic Theory of Aging. Munksgaard, Copenhagen. logous spermatozoa. ]. Reprod. Fertil. 16, 503. JOHNSON, M.H. (1970) An immunological barrier in the WEIL, A.J. & RODENBURG, J.M. (1962) The seminal vesical as the source of spermatozoal-coating antigen of seminal guinea-pig testis. ]. Path. 101, 129. plasma. Proc. Soc. exp. Biol. (N.Y.), 109, 567. JOHNSON, W.L. & MENGE, A.C. (1975) Evaluation of human

CANNAT, A. & SELIGMANN, M. (1965) Les anticorps antinucleaircs de celes par immunofluorescence et par d'autres methodes immunologiques. Semin. Hopit. 41, 1090. DONIACH, D. & Roirr, I.M. (1964) An evaluation of gastric and thyroid auto-immunity in relation to hematologic disorders. Semin. Hemat. 1, 313. FAWCETT, D.W., LEAK, L.V. & HEIDGER, P.M. (1970) Electron microscopic observations on the structural components of the blood-testis barrier. J. Reprod. Fertil.

Human sperm antigens and antisperm antibodies. II. Age-related incidence of antisperm antibodies.

Clin. exp. Immunol. (1976) 25, 73-79. Human sperm antigens and antisperm antibodies II. AGE-RELATED INCIDENCE OF ANTISPERM ANTIBODIES K. S. K. TUNG,...
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