Human leukocyte antigens group A in couples with unexplained infertility*

Transcription

1 FERTILITY AND STERILITY Copyright 98 The American Fertility Society Vol. 0, No., July 98 Printed in U.SA. Human leukocyte antigens group A in couples with unexplained infertility* Claes Nordlander, M.D.t Tommy Fuchs, M.D., Ph.D.t:!: Lennart Hammarstrom, M.D., Ph.D. C. I. Edvard Smith, M.D., Ph.D. Karolinska Institute, Danderyd and Huddinge University Hospitals, Stockholm, Sweden The human leukocyte antigen (HLA) frequencies and antigen sharing were studied in couples with unexplained infertility. There were no significant differences in the frequency of HLA-A, B, C, or DR antigens, as compared with normal control subjects. Furthermore, the degree of HLA sharing did not deviate from that theoretically expected. An increased frequency of HLA-DR sharing, as compared with the expected frequency, was observed in a control group consisting of parents of children with leukemia. When infertile spouses were tested for mixed lymphocyte culture reactivity, there were no indications of an altered reactivity, as compared with control subjects. Fertil Steril 0:60, 98 About 0% of all couples trying to conceive are found to be infertile.! In a small number of these couples, the cause of infertility is unknown despite thorough examination. The possibility that a human leukocyte antigen (HLA)-associated cause might contribute to infertility has been studied by several investigators. A significantly higher incidence of circulating anti-hla antibodies with primary or secondary unexplained infertility was reported by Stolp et a. Other studies have failed to provide evidence of gametic selection based on HLA or of HLA compatibility be- Received December 7, 98; revised and accepted March 7,98. *Supported by the Swedish Medical Research Council, the Torsten and Ragnar Soderberg Foundation, and the Claes Groschinsky Foundation. tdepartment of Obstetrics and Gynecology, Karolinska Institute, Danderyd Hospital. q*reprint requests: Tommy Fuchs, M.D., Ph.D., Department of Obstetrics and Gynecology, Danderyd Hospital, Danderyd, Stockholm, Sweden. Department of Clinical Immunology, Huddinge University Hospital, and Department of Immunobiology, Karolinska Institute. 60 Nordlander et ai. HLA and fertility tween reproductive partners with long-term unexplained infertility. The latter study, however, indicated a negative association between certain haplotype combinations and infertility. A high frequency of shared HLA-A and B antigens has been reported in couples with repeated abortions. 5,6 Three recent studies on HLA-A, B, C, and DR matching and habitual abortion all claim an increased HLA compatibility between the spouses. 7-9 In this study we report the HLA-A, B, C, and DR frequencies and the degree of HLA sharing as well as the mixed lymphocyte culture (MLC) reactivity of infertile couples. MATERIALS AND METHODS CRITERIA FOR PARTICIPATION IN THE STUDY Fourteen couples with unexplained infertility were examined. In order to participate in the study, the following requirements had to be fulfilled: () primary sterility of more than years' duration; () two normal spermiograms according to standardized procedures recommended by the Fertility and Sterility

2 World Health Organization 0; () one normal postcoital test (at least 0 motile sperm per highpower field); () normal hysterosalpingography; (5) indirect evidence of ovulation defined by at least a + b: (a) biphasic basal body temperature curve, (b) serum progesterone, and (c) endometrial curettage; and (6) laparoscopy or laparotomy showing normal genital organs. CONTROL SUBJECTS Forty fertile couples who had been tissue-typed in order to find a donor for kidney (n = 7) or bone marrow transplantation (n = ) served as control subjects. suspended in Eagle's medium. Half the cells were given irradiation of 0 Grey;.5 x 0 5 untreated (responder) cells were mixed with.5 x 0 5 irradiated (stimulator) cells in quadruplicate in microtiter plates. After 5 days of incubation (7 0 C, 5% CO, humidified air),!lci of H-thymidine was added to each well. On the sixth day, the microplates were harvested using a Skatron harvesting machine (A. S. Flow Laboratories, Lierbyen, Norway). Radioactivity was measured in a scintillation spectrophotometer. Relative response (RR) was calculated as follows: RR % = (Resp cells + Stirn cellsirrad) - (Resp cells + Resp cellsirrad) x loo/(resp cells + Control cellsirrad) - (Resp cells + Resp cellsirrad)' TISSUE TYPING HLA-A, B, and C antigens were detected by the lymphocytotoxic microtechnique as described by Kissmeyer-Nielsen and KjaerbyeY HLA-DR antigens were analyzed according to methods described by Bodmer et al. The following HLA specificities were determined: HLA-A, A, A, A9, A0 (5,6), A, A8, and Aw 9; HLA-B5, B7, B8, B, B, B, B5, B7, B8, B7, B7, B0, Bw6, Bw, Bw, Bw5, and Bw; HLA Cw, Cw, Cw, Cw, and Cw6; HLA-DR, DR, DR, DR, DR5, DR7, DRw8, and DRw0. Certain antisera have been demonstrated to react with lymphocyte surface antigens in a fashion indicating the presence of "supertypic" HLA specificities. The following HLA-DR combinations were used for study of the possible association with "supertypic" specificities: HLA-DR, DR (MB, MT, DC, DRPub); DR, DR7 (MB, DC, BR); DR, DR5 (MB, BR); DR, DR5, DR8 (MT, BR, DRPub); and DR, DR7, DRwl0 (MT, BR). The possible corresponding supertypic antigen is shown in parentheses. Since we were not able to test for DRw6 or DRw9, the influence of these antigens could not be studied. MIXED LYMPHOCYTE CULTURE One-way mixed lymphocyte cult~res between cells from the infertile spouses were performed. A pool of frozen lymphocytes from healthy blood donors served as controls. Thirty milliliters ofheparinized blood was diluted : with saline, ph 7., and thereafter separated by Ficoll-Isopaque gradient centrifugation. Lymphocytes were collected, washed three times with saline, and thereafter ESTIMATION OF THE THEORETIC FREQUENCY OF HLA SHARING The theoretic frequency of HLA sharing between spouses was calculated by the following method. The frequency of sharing =! (phenotype frequency) - x (genotype frequency) x! (genotype frequency of antigens already used in the calculation). Example: The phenotypel genotype frequency of HLA-Al, A, and A in a Swedish control population is 0.90/0.576, Table. HLA A, B, C, and DR Phenotype Frequencies in Couples with Unexplained Infertility Control HLAa Women (n = ) Men (n = ) Total (n = 8) subjects (n = 7 6 ) Al A A A Aw B B B B B B Bw Cw DRI DR DR DR DR aonly HLA antigens with a frequency in the control material of > 0.5 are presented. bin the DR group, n = 07. Vol. 0, No., July 98 Nordlander et al. HLA and fertility 6

3 Table. Tissue Types and MLC Reactivity Against Partner in Couples with Unexplained Infertility Couple A,,8 HLA antigens B C 7,5, 7,5 DR Sharing more than one antigen A B C D o o o MLC RR a 7 5,9, 7, 7 o o o 79 56,5 5,7 5 o o o o 5 66,8 8, 7,5,7 o o o o 8 60,0 (6), 5,7 5,7,8 8 o 96 66, 5, 7,8, o o 7 50,,9,0,7 o o 9 8,9, 7,0 5,7 o o o 68 55,,7 5,7,8 o o 8 0 9, 5,7 8,5 5 7 o o o,,7 8,0,8 o o o -5,8, 7,0 8,,, o o o 7 78,9,8 7,0 7,0,, o 8 0 (6), 8,5,,8 o o 8 5 Total arelative response. btechnical failure , and 0.99/0.75, respectively. Thus, the frequency of individuals sharing Al is 0.90 = 0.08; A (but not AI) X 0.99 X = 0.5; A (but not Al or A) X 0.75 X ( ) = Similar calculations were made from control materials for all HLA-A, B, or DR 5 antigens. The theoretic frequency of HLA sharing calculated as described above was 0.98, 0., and 0. for HLA-A, B, and DR, respectively. Only antigens which could be detected were included in the calculation. Thus, DR "blanks" were not included. 6 Nordlander et al. HLA and fertility RESULTS HLA FREQUENCIES IN COUPLES WITH UNEXPLAINED INFERTILITY Fourteen couples with unexplained infertility were examined for HLA-A, B, C, and DR antigens. The phenotype frequencies for antigens present in more than 5% of a control population are presented in Table. As can be seen, there were no significant differences, as compared with a control population for any of the antigens tested (P > 0.05). Furthermore, there were no signifi- Fertility and Sterility

4 Table. HLA Sharing in Infertile Couples and Control Subjects Infertile couples (n = ) Fertile couples (n = 0) Leukemia (n = 8)a Kidney disease (n = 7)a Miscellaneous (n = 5)a Expectedb Percentage of couples sharing more than one antigen HLA A HLA B HLA DR c adiagnosis of child investigated for transplantation. bexpected values; calculation based on HLA antigen frequencies in normal control subjects (see Materials and Methods). CO.Ol < P < 0.05, when compared with the expected value, using the x test without Yates' correction and not corrected for the number of comparisons being made. cant differences in the frequency of the less com mon antigens (data not shown). HLA SHARING IN COUPLES WITH UNEXPLAINED INFERTILITY As can be seen from Table, the sharing of more than one HLA-A, B, or DR antigen was evident in 6 of (%), 6 of (%), and 5 of (6%) sterile couples, respectively. The compatibility frequency in infertile couples, as compared with control subjects, is presented in Table. Since it was not possible to DR-type a control subject because of a lack of antisera, we made a comparison with parents of patients whose cases were being investigated for transplantation. Three groups were formed, based on the diagnosis: () leukemia; () kidney disease; and () miscellaneous (all patients in this group were candidates for a bone marrow transplant). This might suggest the introduction of familial bias. Therefore, the expected compatibility frequencies in subjects without any known disease were also calculated (see Materials and Methods). As can be seen, the two separate control groups did not differ significantly with regard to compatibility frequency for HLA-A, B, or DR loci (Table ). However, in the leukemia group a significant increase of HLA-DR compatibility was noted, compared with what was expected. The infertile spouses, as compared with control subjects, did not show any increased sharing of A, B, or DR antigens. Thus, unexplained infertility does not seem to be associated with an increased DR compatibility between husband and wife. In Table, the relationship between common HLA-DR antigens and MLC (RR%) in infertile couples, as compared with control subjects, is demonstrated. No significant difference in response between infertile and fertile women could be noted (Student's t-test). The results support that infertility, as defined here, was not correlated with any overrepresent at ion of compatibility between the spouses. Since supertypic antigens related to the HLA DR system have been described, we found it relevant to study the possible influence of these. However, none of the nine couples not sharing a DR antigen shared a supertypic antigen. DISCUSSION Infertility is found in about 0% of couples trying to conceive. Fourteen couples participated in our study. This study group might seem small, but our couples' infertility is representative of so-called unexplained infertility, where there is no known cause, like tubal occlusion, endometriosis, anovulation, or abnormal sperm. In this study, we have investigated the HLA-A, B, C, and DR antigen frequencies in couples with unexplained infertility. There was no indication of an increased HLA compatibility when our couples' frequencies were compared with the expected frequency of sharing in a control population or when compared with those of fertile couples investigated for HLA types in connection with bone marrow or kidney transplantation. Furthermore, there were no changes in the HLA phenotype fre- Table. Relationship Between Common HLA-DR Antigens and MLC (RR%) in Infertile Couples and Control Subjects Infertile couples (n = ) Control subjects (n = 0) Common DR loci No. of couples Mean ofrr (%) in MLC Vol. 0, No., July 98 Nordlander et al. HLA and fertility 6

5 quencies, as compared with those of a control population. However, one of the control groups, parents of children with leukemia, demonstrated an increased sharing of HLA-DR antigens. The reason for this increased sharing is not known. However, it is possible that tumor viruses originating from the father more easily could infect a DR-compatible mother and then later the fetus. An increased compatibility of A and B antigens in parents of children with leukemia or aplastic anemia has previously been reported.6 The fact that we could not confirm the results on HLA-A and B sharing could simply be due to the size of the patient sample. This could also explain the observation that the infertile couples did not differ significantly in their degree of sharing, as compared with the leukemia group. Couples in which the woman has habitual abortions have been reported by several groups to have an increased sharing of HLA-A, B, and DR antigens, as compared with control couples.7-9 The reason for this increased sharing of HLA antigens is unknown. However, disease-predisposing genes coding for HLA antigens7 or antigens in linkage disequilibrium with HLA is one possibility. Thus, a trait, often associated with some type of disease, inherited by the fetus from both parents, could be anticipated to result in abortion with high frequency. Another possibility is that an increased sharing merely results in a lower degree of mismatch and that the important issue is the number of paternal antigens foreign to the mother. Thus, in the case of habitual abortions, the idea that reactivity against certain trophoblastllymphocyte cross-reactive antigens would result in the formation of protecting or blocking factors has been advanced.8 We also investigated the frequency of HLA-A, B, or DR antigen mismatch between the spouses. However, there were no indications of a changed frequency in this group as compared with our control group. However, one important issue that has not been carefully dealt with is whether there is an increased compatibility in couples with habitual abortions or whether the control population demonstrates decreased compatibility. If the expected degree of HLA sharing is calculated as described in this report, using the data of Gerencer et ai.,8 it rather seems as if the control couples have a decreased degree of sharing. The fact that major histocompatibility locus-associated mating can take place has been elegantly demonstrated in the mouse system. Yamazaki et ai.9 have report- ed a mating preference between major histocompatibility complex congeneic mouse strains, possibly due to chemosensory discrimination. It is possible that a similar mechanism could be of importance in humans, although the reinterpretation of the findings of Gerencer et ai. 8 would argue in favor of a negative rather than a positive association. If a decreased HLA compatibility in the normal human population is found, this could certainly alter the interpretation of our findings in infertile couples. To our knowledge, this is the first study where couples with infertility of unknown origin were HLA-DR-typed. In contrast to the condition in spouses with habitual abortions, no similar increase in compatibility frequency could be found, possibly arguing in favor of the idea that unexplained sterility is not due to early spontaneous abortions. REFERENCES. Southam AL: What to do with the "normal" infertile couple. Fertil Steril :5, 960. 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