CELLULAR IMMUNITY TO LYMPHOCYTE ANTIGENS IN HUMAN INFERTILITY*

Transcription

1 FERTILITY AND STERILITY 1976 The American Fertility Society Vol. 27, No.4, April 1976 Printed in U.SA. CELLULAR IMMUNITY TO LYMPHOCYTE ANTIGENS IN HUMAN INFERTILITY* BRIAN D. TAIT, M.Sc.,t JEAN U. BARRIE, B.Sc., PH.D., Assoc. R.M.I.T.,* IAN JOHNSTON, M.D., M.G.O., F.R.C.O.G., AND PETER J. MORRIS, PH.D., F.R.C.S., F.R.A.C.S., F.A.C.S.~ Department of Surgery, University of Melbourne, and Department of Pathology, Royal Women's Hospital, Melbourne, Victoria, Australia The leukocyte migration test was used to detect cellular immunity to HL-A antigens present on the husbands' lymphocytes and spermatozoa in a group of fertile and infertile couples. Approximately 50% of the married female population appear to be sensitized. There was no difference between the fertile and infertile groups in the frequency of positive results, but a greater number of migration indices were below 0.7 and 0.6 in the infertile group. It is suggested that sperm cells are the most likely cause of immunization. The leukocyte migration test (LMT) has been shown to be an in vitro correlate of cell-mediated immunity. I. 2 In the present series the LMT was used to measure cellular immunity directed at antigens shared by both human lymphocytes and spermatozoa in a group of women attending an infertility clinic and in a control group of fertile and nulliparous women. Since HL-A antigens have been demonstrated on sperm cells,3 the use of the husband's lymphocytes in the LMT appeared to be a means of assessing cellular immunity to these antigens. Since non-hl-a antigens also may be common to both types of cells, the LMT may not assess cellular immunity to HL-A alone. Received July 16, *Supported in part by grants from the Australian Kidney Foundation and the National Health and Medical Research Council of Australia. trecipient of a research scholarship from the Royal Women's Hospital. *Serologist, Royal Women's Hospital. Honorary Consultant, Gynaecological Surgeon, Royal Women's Hospital. ~Present address: Nuffield Professor of Surgery, Nuftield Department of Surgery, University of Oxford, Radcliffe Infirmary, Oxford, United Kingdom. 389 The presence of immune antibodies directed at sperm antigens in the serum of some women is well-documented,4.5 although the significance of these antibodies in cases of unexplained infertility is debatable. 6 There are few reports of in vitro measurements of cellular immunity in normal and infertile subjects, and these studies describe the use of mixed leukocyte cultures and the lymphocyte response to sperm antigens in males MATERIALS AND METHODS Thirty-one couples who were attending the infertility clinic at the Royal Women's Hospital as outpatients were included as the test group. The period of infertility ranged from 12 months to 18 years. The control group consisted of 25 couples. Thirteen of these couples had produced children, while the fertility of the remaining twelve couples was unproven. Blood and Semen Samples Blood samples (10 ml heparinized and 10 ml allowed to clot) were obtained from each husband and wife at the infertility clinic. Semen samples were obtained from

2 390 TAITETAL. April 1976 each husband after 72 hours of sexual abstinence by either masturbation or coitus interruptus. Heparinized blood samples (10 ml) were obtained from each husband and wife in the control group of couples. Leukocyte Migration Test The method is based on the original leukocyte migration test described by Soborg and Bendixen. 2 The technique for both positive and negative control groups has been described previously.lo Briefly, leukocytes from the wife's heparinized specimen, separated from erythrocytes by dextran sedimentation, were used as the migrating cells. Thrice frozenthawed husband's lymphocytes, isolated from the heparinized specimen by an Isopaque-Ficoll method,l1 were used as the antigen. Capillary tubes (20-p,1 volume) containing the migrating cells (5 x 10 7 /ml) were centrifuged for 7 minutes at 600 x g, cut at the cell fluid interface, and placed horizontally in wells of 0.5-ml volume (Sterilin plates) containing either HEPES (N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid) buffered medium 199 alone or medium containing the appropriate antigen. The open end of the tube was approximately at the center of the well. The tubes were held in place by a small pellet of high-vacuum grease (Dow Corning Corporation, Midland, Mich.), which was also lightly smeared around the rim of the well so that a cover slip formed an airtight seal. After incubation at 31' C for 16 hours, areas of cell migration were obtained by light projection onto a screen. The areas were traced onto paper, cut out, and weighed on a Paul Bunge balance to the nearest milligram. The areas of cell migration in the presence of antigen were expressed as a decimal fraction of the area obtained in medium alone (migration index). Each test was performed in triplicate. Migration indices (MIs) under 0.8 were considered positive and indicative of sensitization. HL-A Typing and Cytotoxic Cross-Match Each infertile couple was typed for the following 23 HL-A antigens by the Terasaki microlymphocytotoxicity method. 12,13 First locus: HL-A1, 2, 3, 9, 10, 11, W28, W19; second locus: HL-A5, 7, 8, 12, 13, W5, W10, W14, W15, W16, W17, W18, W22, W27, M3 (W21). A cytotoxic cross-match was also performed in each case between the wife's serum and the husband's lymphocytes. The sera were further screened against a panel of selected lymphocyte donors possessing 90% of the known HL-A antigens. Semen Analysis and Sperm Antibody Tests As a routine procedure the fresh semen was examined macroscopically and microscopically and note was made of the following factors: volume and viscosity of the ejaculate, sperm motility, sperm count, abnormal sperm forms, and cell survival at room temperature over a period of several hours. The sperm cells were also examined for spontaneous aggregation. When the sperm count approached normal values 14 and the motility was greater than 50%, sera from both husband and wife were examined for spermagglutinins and immobilisins by the following methods.. Immobilisins. A portion (0.25 ml) of the wife's serum sample was mixed in a Wasserman tube with ml of semen and 0.05 ml of guinea pig complement (sodium azide-free) and incubated for 1 hour at 31' C. The motility was again determined microscopically. In each case a similar test was performed with the husband's serum and

3 Vol. 27, No.4 ANTIGEN IMMUNITY IN HUMAN INFERTILITY 391 a control in the form of fresh human serum which was blood group-compatible with the husband's cells. The criterion for significant sperm immobilization was regarded as a 50% or greater reduction in the motility, compared with control values. Agglutinins. A 0.25-ml sample of heat-inactivated serum from the wife was mixed in a Wasserman tube with ml of semen and incubated at 31' C for 4 hours. In each case a similar test was performed using the husband's serum or fresh human serum group-compatible with the husband's cells. Spermagglutination was considered to have occurred if groups of two or more sperm cells were joined by the heads or tails and maintained forward progressive move- mep.t. Doubling dilutions of the test sera in serum group-compatible with the husband's cells were prepared to determine the titer of antibody present. Patient Classification On the basis of clinical studies and seminal analyses, the most likely cause of infertility was ascertained by one of us (I. J.) without prior knowledge of the LMT result. RESULTS Leukocyte Migration Test. The results of the LMT performed on both infertile and control groups are depicted in Figures 1 and 2 and summarized in Table 1. Although some apparent differences were 1'2 NULLlPAROUS PAROUS 1,' ',0 )( III Q Z z 0 ;: c III: " 0 ' o a a aa u '..... A 0'5 0 4 FIG.!. Results of leukocyte migration tests obtained in nonpregnant control females, using the husbands' lymphocytes as the antigen. Cell migration in the presence of antigen is expressed as a decimal fraction of that obtained in medium alone (migration index). MI < 0.8 was considered positive.

4 392 TAITETAL. April 1976 '-41 NULLIPAROUS I I, I,., I PAROUS 0 I> I> I> I> ',0 I> 0 I> o g )( I> 0 0 Q o I>! I> z I> ~ 0 7 l- e 0:: ~ :E >. I> 0 5 0'4.. I> 0 FIG. 2. Results of leukocyte migration tests obtained in primary and secondary infertility patients, using the husbands' lymphocytes as the antigen. Cell migration in the presence of antigen is expressed as a decimal fraction of that obtained in medium alone (migration index). MI < 0.8 was considered positive. a, Sperm antibody tests not done; 0, sperm antibody tests negative; e, sperm antibody tests positive;., sperm autoantibody detected in husband's serum. seen, namely that there were more subjects in the infertile group with low MIs, these differences were not significant. The MIs of 12 of the 31 infertile women were below 0.7, compared with 5 of 25 in the control group; the MIs of 5 of the 31 infertile women were below 0.6, compared with only 1 of the 25 control subjects (a nulliparous woman who had been married for 3 years). There was no difference in the frequency of positive LMT results between nulliparous and parous women in both the control and infertile groups. Seminal Antibody Studies. Sperm immobilisin and agglutinin studies were performed in 15 cases. Among the infertile group, sperm antibodies were detected on at least one occasion in three of these patients. In a fourth case autoagglutinins and autoimmobilisins were detected in: the husband's serum. Three of these cases were included in the group of five with an MI under 0.6. Two of the.three patients with sperm antibodies were advised to practice occlusive methods of contraception (condoms) in an attempt to lower the titer of serum antibody present. In one case, occlusive therapy was begun 2 weeks and in the other case 3 months before the LMT was performed. On retesting, the LMT eventually became negative in both cases (Fig~ 3). In one female patient (HOP) the im-

5 Vol. 27, No.4 ANTIGEN IMMUNITY IN HUMAN INFERTILITY 393 Patient classification TABLE 1. Summary of Leukocyte Migration Tests in Fertile and Infertile Couples No. of patients MI< 0.8 Control group Nulliparous 12 7 Parous 13 8 Infertile group Nulliparous Parous 6 2 ami < 0.8 was considered positive. No. positive u MI < 0.7 MI< 0.6 MeanMI SD mobilizing antibody disappeared and the agglutinating titer decreased from 1:128 to 1:16. In the second patient (LAD) occlusive contraception had no effect on a strong immobilizing antibody. HL-A Typing and Cytotoxic Cross Match. A positive cytotoxic cross-match between wife's serum and husband's lymphocytes was not found among the infertile couples, nor were cytotoxic antibodies detected in the sera of the wives on testing against a panel of lymphocytes. There was a lowered frequency of antigen HL-A2 among males whose partners were nonimmunized (2 of 17) when compared with the immunized group (9 of 14). This difference was significant by the X 2 test with Yates correction... x c z 1'0 0 9,///0"0 ". HOP,/ 0'8+---~' ,~ ,,,,,,,,,,,.' 0'5 '--_...,...-_ ,.. -.-_--, 4 10 TIME IN MONTHS SINCE COMMENCEMENT OF OCCLUSIVE THERAPY FIG. 3. Results of leukocyte migration tests obtained on two patients after a period of occlusive contraception. In both cases the LMT reverted to negative values. (P < 0.02). However, the significance was lost when a correction was applied for the number of comparisons made (0.02 x 23 = 0.46). Possible Causes of Infertility. After extensive clinical investigation and seminal analyses a likely cause of infertility could no-t be found in four cases. The MI results and HL-A typings of these couples are shown in Table 2. TABLE 2. Results of HL-A Typings and Migration Indices of Patients with Infertility of Unknown Etiology Patient GRIF HUD PAT UGA HL A type" W:2,5,12,4a,4b H: 2, 5, 4a, 4b W: 1,2, W5, 4a,4b H: 10, W28, W5, 4a W: 1,2,8, W27,4b H: 1, 3, 7, 8, 4b W:2,3, WI0,4a,4b H: W28, 5, W27,4a,4b aw, Wife; H, husband. bmi < 0.8 was considered positive. DISCUSSION MI' The present study was designed to test the hypothesis that within an infertile group of the population there may exist cases in which cellular or humoral immunity in the female, directed at HL-A antigens of the male, may be responsible for this condition, since HL-A antigens are known to be expressed in haploid form on human spermatozoa. 3 The LMT was used to determine cellular immunity in the wife against HL-A on the husband's lymphocytes, although it is possible that

6 394 TAITETAL. April 1976 other minor antigen systems are shared by both lymphocytes and spermatozoa. There appear to be two major problems in the investigation of the role of histoincompatibility in infertility. The first, which is common to previous studies of sperm antibodies in infertility, is that a percentage of proven fertile women appear to be immunized. 4,15 The second is that infertility is often a multifactorial problem. For example, immunologic reasons for infertility may be demonstrated in an infertile couple in the presence of clinical conditions capable independently of preventing conception. In this series more than one possible cause for infertility was found in 12 couples after clinical and seminal evaluation. These findings make it difficult to define the role of cellular immunity to histocompatibility antigens in infertility. The secretor status of the female may be the determining factor in the extent of the clinical effect of humoral antibody. Studies of sperm immobilization in vitro, using cervical mucus in preference to serum, appear to correlate better with infertility and probably represent a truer refelction of the in vivo situation.16 The presence of antibody in serum does not necessarily indicate that this antibody is free to combine with antigen within the reproductive tract. In cellular immunity the degree of sensitization as measured by the leukocyte migration test may be important, provided the test can be applied quantitatively.17 The second problem can only be overcome by studying large numbers of patients and selecting a small group in which the infertile condition could not possibly be a result of any other known factor. Despite the fact that humoral antibody to HL-A antigens was not detected in any of the cases studied, results obtained in both the nulliparous group and the group of infertile couples practicing occlusive contraception strongly suggest that cellular immunity to antigens shared by spermatozoa and peripheral blood lymphocytes was being measured. This dichotomy between humoral and cellular immunity in the HL-A system has been observed in other situations and is possibly related to the mode of immunization. 10 Beer et au 8 have observed that intrauterine injections of spermatozoa incite a strong transplantation immunity in rats, but intravenous injections do not. In human pregnancy, HL-A antibodies are usually formed as a result of the entrance of either fetal leukocytes or (possibly) trophoblast cells to the maternal circulation.19,20 The elicitation of cellular immunity directed at antigens on sperm cells may depend on an intravaginal or intrauterine method of immunization, as well as the number of cells involved and possibly the amount of antigen present on each type of cell. Two of the three patients in whom sperm antibodies were designated as the prime cause of female infertility were included in the group of five patients with MIs under 0.6. This lends support to the theory that the group with low MIs may represent the group to which immunologic causes of infertility may be attributed. Although it is not certain that the LMT is quantitative in measuring degrees of sensitization, it has been shown that subjects who have been sensitized by skin grafting react with increasing levels of inhibition when confronted with one, two, three, or four HL-A antigens in common with the sensitizing donor.17 Other factors, such as variation in "strength" among HL-A antigens and individual differences in immune responsiveness, may be important in determining the degree of inhibition in the LMT. The possibility also arises that the sperm antibodies detected in these three patients are directed at the same antigens operative in the LMT. The fact that lymphocytotoxic antibodies were not detected may be explained on the basis of either lack of sensitivity of this test or

7 Vol. 27, No.4 ANTIGEN IMMUNITY IN HUMAN INFERTILITY 395 lack of cytotoxicity of these immobilisin antibodies. If some sperm antibodies are reactive toward antigens present on lymphocytes they may be effective in blocking in vitro the positive LMT reaction. This is being investigated at present. In a previous study of pregnant women,10 positive LMTs directed at antigens present on cord lymphocytes were found to be associated with reduced placental weight. It was suggested that the mother's cellular immunity to fetal antigens could inhibit trophoblastic invasion. All of the positive LMT values in the pregnant group were between 0.6 and 0.8. Values under 0.6 may indicate a situation in which trophoblastic invasion is inhibited to the point where pregnancy is nonviable. On the other hand, the immune reaction may occur before fertilization. Specific immunity to lymphocyte antigens present on spermatozoa may result in elimination of these cells before fertilization occurs. Subsequent to the work reported here one of the patients in the strongly positive LMT group became pregnant but aborted at approximately 8 weeks' gestation. This is the only evidence available from our studies which suggests that fertilization may occur in the face of strong preexisting cellular immunity. Lindblom et a1. 21 have reported that in an infertile group of 569 couples, they were able to select 8 couples whose infertility could not possibly be explained by any known cause. There appeared to be a low incidence in this small group of HL A haplotypes known to show linkage disequilibrium in Caucasians (e.g., HL AI, 8; HL-A2, 12; HL-A3, 7). They suggested that certain haplotypes may favor fertilization and that this may explain the excess of these haplotypes in a population. In the group of four patients in this series with unexplained infertility, positive haplotype associations were seen in two females and one male (GRIF, PAT). The diminished frequency of HL-A2 antigen in the male members of the nonimmunized group is an interesting observation, although the numbers are too small to draw any firm conclusions. It is possible that HL-A2 may be associated with a factor responsible for an increased penetrative ability of sperm cells. This ability may result in spermatozoal penetration of the epithelial cell lining of the uterus and increase the possibility of contact with lymphoid cells and the chance of sensitization. Although the present investigation failed to demonstrate conclusively that sensitization to lymphocyte antigens can be a cause of unexplained infertility, it did demonstrate that cellular immunity does exist in certain members of the married female population. It is also possible that humoral factors or "blocking" type antibodies modify the in vivo action of cellular immunity during the reproductive process. Acknowledgments. The authors are indebted to Mrs. Kaye Turner, Mrs. Barbara Jacobe, Mr. David Williams, and Mr. Alan White, technical staff in the Department of Pathology, Royal Women's Hospital, who performed the seminal analyses and antibody tests. 1. George M, Vaughan JH: In vitro cell migration as a model for delayed hypersensitivity. Proc Soc Exp BioI Med 111:514, Soborg M, Bendixen G: Human lymphocyte migration as a parameter of hypersensitivity. Acta Med Scand 181:247, Fellous M, Dausset J: Probable haploid expression of HL-A antigens on human spermatozoa. Nature 225:191, Dukes CD, Franklin RR: Sperm agglutinins and human infertility: female. Fertil Steril 19:263, Kolodny RC, Koehler BC, Toro G, Masters WH: Sperm agglutinating antibodies and infertility. Obstet Gynecol 38:576, Isojima S: The nature of antibodies against spermatozoa found in women with unexplained sterility. Proceedings of the Seventh World Congress on Fertility and Sterility. In Excerpta Medica Int Congr Ser 278, Amsterdam, 1972, p 105

8 396 TAIT ET AL. April Ohama K, Kadotani T: Lymphocyte reaction in mixed wife-husband leukocyte cultures in relation to infertility. Am J Obstet Gynecol 109:477, EI Alfi OS, Bassili F: Blastoid transformation of lymphocytes in response to seminal antigen in cases of nonobstructive azoospermia. J Reprod Fertil 21:23, Mumford DM, Barsales PB, Ball KD, Gordon HL: Technique and patterns of responsiveness to autologous and allogeneic semen from normal and infertile male subjects. J Urol 105:858, Tait BD, d'apice AJF, Morris PJ: Maternal cell mediated immunity to foetal transplantation antigens. Tissue Antigens 4:586, Boyum A: Separation of leukocytes from blood and bone marrow. Scand J Clin Lab Invest [Suppl] 97:31, Teresaki PI, Vredevoe DL, Mickey MR: Serotyping for homotransplantation. X. Survival of 196 grafted kidneys subsequent to typing. Transplantation 5:1057, Mittal KK, Mickey MR, Singal DP, Teresaki PI: Serotyping for homotransplantation. XVIII. Refinement of microdroplet lymphocyte cytotoxicity test. Transplantation 6:913, Scientific Tables, Edited by K Diem, C Lentner. Summit NJ, Ciba-Geigy Corporation, 1970, p Waldman RH, Cruz JM, Rowe DS: Sperm migration-inhibiting antibody in human cervicovaginal secretions. Clin Exp Immunol 12:49, Parish WE, Ward A: Studies of cervical mucus and serum from infertile women. J Obstet Gynaecol Br Commonw 75:1089, Falk RE, Thorsby E, Moller E, Moller G: In vitro assay of cell mediated immunity: the inhibition of migration of sensitised human lymphocytes by HL-A antigens. Clin Exp Immunol 6:445, Beer AE, Billingham RE, Hoerr RA: Elicitation and expression of transplantation immunity in the uterus. Transplant Proc 3:609, Billington WD, James DA, Kirby DRS: Some effects of genetic dissimilarity between mother and foetus. J Reprod Fertil [Suppl] 3:1, Walknowska J, Conte FA, Grumbach MM: Practical and theoretical implications of foetal maternallymphocyte transfer. Lancet 1:1119, Lindblom JB, Friberg J, Hogman CF, Gemzell C: HL-A haplotypes and unexplained infertility. Tissue Antigens 2:352, 1972