1 Department of Obstetrics and Gynecology, Niigata University School of Medicine, Niigata and 2 Department of Obstetrics and Gynecology, Nagaoka Chuo General Hospital, Nagaoka City, Japan
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, 1-757, Asahimachi-dori, Niigata, 951-8510, Japan. e-mail: obgy{at}med.niigata-u.ac.jp
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: genotype/HLA-DR/PCR-RFLP/unexplained recurrent abortion
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One group (Wegmann, 1987; Wegmann et al., 1993
) focused on the production of a diversity of cytokines by maternal immune-competent cells in decidual tissues, and proposed an immunotrophic theory, which was followed by the Th1/Th2 paradigms theory (Guilbert, 1996
; Raghupathy, 1997
; Raghupathy et al., 1999
; Chaouat et al., 2002
). Such immunological mechanisms for the maintenance of successful pregnancy suggest the implication of HLA antigen systems in the genesis of human abortions.
DNA analyses showed a lack of significant compatibility between patient couples compared with normal fertile couples (Christiansen et al., 1989; Takakuwa et al., 1992
; Ober et al., 1993
; Wagenknecht et al., 1997
), although one of these groups (Ober et al., 1993
) pointed out the possibility of significant compatibility of HLA-DQA1 and DQB1 alleles between patients and aborted fetuses using a PCR-sequence-specific oligonucleotides (SSO) method.
Recently, the association between HLA class II antigens and patients with unexplained recurrent abortion was elucidated by a large population study in a Caucasian population (Christiansen et al., 1994, 1996, 1999). The analyses, however, have been conducted in only a small population in Japan (Sasaki et al., 1997
; Takakuwa et al., 1999
). In this context, the frequency of HLA-DRB1 alleles were examined in a significant number of patients with unexplained recurrent abortion, using a PCR-restriction fragment length polymorphism (PCR-RFLP) method, in order to elucidate the association of HLA-DR antigens with unexplained recurrent abortion patients in the Japanese population.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Analyses of HLA-DRB1 genotypes
Analyses of HLA-DRB1 genotypes were performed using the PCR-RFLP method (Ota et al., 1992). The use of the method was validated by these authors.
Genomic DNAs, extracted by phenol extraction of sodium dodecyl sulphate (SDS)-lysed and proteinase K-treated peripheral lymphocytes from each individual, were amplified using the PCR procedure with 2.5 units of Taq DNA polymerase (Takara Co. Ltd, Kyoto, Japan). The reaction mixture, which contained 1 µmol/l each of the PCR 3' and 5' primers, 1 µg of genomic DNA, 10 µl of dNTP mixture (Takara Co. Ltd), a PCR reaction buffer (10 mmol/l TrisHCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2), and distilled water, to make a total volume of 100 µl in a 500 µl Eppendorf tube, was covered with 50 µl of mineral oil to prevent evaporation and subjected to 30 cycles of 1 min for denaturing (94°C), 1 min for annealing (60°C), and 2 min for extension (72°C) in an automated PCR thermal cycler (Thermal Cyclic Reactor, Toyobo Engineering Co., Tokyo, Japan).
For typing, seven group-specific primers, DR1, DR2, DR4, DR7, DR9, DR10 and DRw52-associated (DR3, -5, -6 and -8) antigen-specific primers, were used to obtain only the amplified product from the DRB1 gene. The DR7, -9, -10 alleles, which have no suballeles (DRB1*0701 and 0702 have the same nucleotide sequences in their 1 domain exons), were simply typed by the presence of amplified bands as DRB1*0701 or 0702, DRB1*0901 and DRB*1001 respectively.
After amplification, aliquots (6 µl) of the reaction mixture, together with an appropriate restriction buffer and restriction enzymes, were incubated for 13 h.
AvaII and PstI were used for digestion of the amplified DR1-DRB1, FokI, Cfr13I and HphI for DR2-DRB1, SacII, AvaII, HinfI, HaeII, HphI and MnlI for DR4-DRB1, AvaII, FokI, KpnI, HaeII, Cfr13I, SfaNI, SacII, BsaJI, ApaI and HphI for DR3, 5, 6 and 8-DRB1.
Samples of the amplified DNAs cleaved by restriction enzymes were subjected to electrophoresis using a 12% polyacrylamide horizontal gel in a minigel apparatus (AE-6450; Atto Corporation, Tokyo, Japan). Cleavage or non-cleavage of amplified fragments was detected by staining with ethidium bromide.
HLA-DRB1 genotypes were determined by comparing the restriction fragment patterns to those of the amplified DRB1 genes as reported previously (Ota et al., 1992).
Statistical analyses
A 2 analysis with Yates correction was used to analyse any significance in the difference between the rates of possession of each HLA-DRB1 allele in patients with recurrent abortion and those in the control population. A two-tailed Fishers exact probability test was used with small expected frequencies. P-values, corrected by multiplying by the number of tested alleles (n = 27) (Pc), were obtained using a previously published method (Svejgaard et al., 1974
). Odds ratios (OR) were calculated with a 95% confidence interval (CI), using the method of Woolf (Woolf et al., 1955
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
The rate of possession of the HLA-DRB1*1502 allele in the patients with secondary recurrent abortion was 57.1% (8/14 patients), while that in the normal fertile women group was 15.7% (18/115 patients). Thus, the rate of possession of the HLA-DRB1*1502 allele was significantly higher in the secondary recurrent abortion group compared with that in the normal fertile women group (OR, 7.19; 95% CI, 2.2323.2; P < 0.0001; Pc < 0.01) (Table III). The rate of possession of the HLA-DRB1*1502 allele in the patients with primary recurrent abortion was 27.8% (22/79 patients), which was higher compared with that in the normal fertile women group (OR, 2.08; 95% CI, 1.034.20; P < 0.05). However, after adjustment for the number of tested alleles (Pc), no significant difference was observed (Table III). In general, the rate of possession of the HLA-DRB1*1502 allele in patients with primary and secondary recurrent abortion was higher compared with that in normal fertile women (32.3 versus 15.7%, OR, 2.57; 95% CI, 1.324.99; P < 0.005; Pc = 0.081, not significant) (Table III).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It has been recognized for many years that the HLA system plays an important role in the aetiology of a number of diseases (Thomson, 1986, 1995; Nepom and Erlich, 1991
), and it is also possible that HLA systems are implicated in the genesis of human abortions. In particular, evidence that HLA antigens are markers for recurrent spontaneous abortion has gained increased attention. It has been reported (Christiansen et al., 1994
, 1996, 1999) that maternal HLA DR1, DR3 and DR10 were genetic markers for pregnancy loss in a large population study of Danish recurrent spontaneous abortion patients. With regard to the Japanese population, some investigators (including the present authors) reported a relationship between certain HLA class II genotypes and patient populations with unexplained recurrent abortion (Sasaki et al., 1997
; Takakuwa et al., 1999
). However, the size of the populations in these studies was very small compared with the present series. The increase in the rate of HLA-DRB1*1502 possession in the patient population in the present study was considered to indicate the possibility that HLA-DRB1*1502 is a risk allele for recurrent spontaneous abortion.
The appropriate selection of a control population in such a casecontrol study is crucial. In the present study, 115 normal fertile women were used as a control population. The frequency of each HLA-DRB1 genotype in this control group (data not shown) was not significantly different compared with that in 916 Japanese unrelated individuals reported previously (Hashimoto et al., 1994). Thus, the frequency of the HLA-DRB1 allele in the control group did not deviate from that of the total population.
In the present series, the population of unexplained recurrent aborters consisted of primary recurrent aborters and secondary recurrent aborters, and an increase in the rate of possession of HLA-DRB1*1502 was observed in both populations, though the immunological mechanisms responsible for the recurrent abortion in both populations may differ. The reason for such findings remains unknown.
The precise mechanisms underlying the association of most diseases with particular MHC haplotypes are not yet fully understood, though several possibilities exist. One of the first such reports was of the so-called immunotrophic theory, whereby some cytokines produced by maternal cells that recognize fetal antigens promote the proliferation of trophoblastic cells and sustain pregnancy continuation (Wegmann, 1987; Wegmann et al., 1993
). Moreover, some investigators demonstrated the importance of a T helper 2 (Th2) bias for normal pregnancy, indicating the crucial role of the activation of maternal humoral immunity following recognition of fetal antigens during pregnancy (Guilbert et al., 1996
; Raghupathy, 1997
; Raghupathy et al., 1999
; Chaouat et al., 2002
). Fetal tissue expresses HLA-DR antigens as early as the ninth week of gestation, and these may elicit maternal immune responses (Trebichavsky and Nyklicek, 1992
). MHC class II molecules, especially HLA-DR molecules, bind peptides derived from the degradation of proteins ingested by MHC class II-expressing antigen-presenting cells (APC), and display them at the cell surface for recognition by CD4-positive T lymphocytes (Margulies, 1999
). In the decidua, CD4-positive T lymphocytes expressing HLA-DR antigens are reported to be activated during early pregnancy (Chao et al., 1999
).
Thus, it is possible that the increased rate of possession of the HLA-DRB1*1502 allele in patients with unexplained recurrent spontaneous abortion may have implications for the lack of recognition of fetal antigens by the maternal immune system.
It is also possible that some genes, which have linkage disequilibrium with certain HLA class II haplotypes, are responsible for the genesis of recurrent spontaneous abortion. Although placental cells, which are in contact with maternal blood or tissue, are devoid of HLA class II antigens, HLA class I antigens, such as HLA-C or HLA-G antigens are expressed on the cells (King et al., 1996; Hammer et al., 1997
). It was reported that the expression of HLA class I antigens is controlled by the transporter associated with antigen processing (TAP) genes and proteosome genes in the human placenta, and these genes have strong linkage disequilibrium with HLA-DR regions (van Endert et al., 1992
; Roby et al., 1994
). Thus, HLA-DR antigens may be implicated in the genesis of recurrent spontaneous abortions. All of these hypotheses, however, require further investigation.
The association between HLA-DRB1*1502 and unexplained recurrent spontaneous abortions in the present study was not very strong, and a new independent study must be conducted in order to provide conclusive proof of any such association.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chaouat, G., Zourbas, S., Ostogic, S., Lappree-Delage, G., Dubanchet, S., Ledee, N. and Martal, J. (2002) A brief review of recent data on some cytokine expression at the materno-fetal interface which might challenge the classical Th1/Th2 dichotomy. J. Reprod. Immunol., 53, 241256.[CrossRef][ISI][Medline]
Christiansen, O.B., Riisom, K., Lauritsen, J.G. and Grunett, N. (1989) No increased histocompatibility antigen sharing in couples with idiopathic habitual abortion. Hum. Reprod., 4, 160162.[Abstract]
Christiansen, O.B., Rasmussen, K.L., Jersild, C. and Grunett, N. (1994) HLA class II alleles confer susceptibility to recurrent fetal losses in Danish women. Tissue Antigens, 44, 225233.[ISI][Medline]
Christiansen, O.B., Pedersen, B., Mathiesen, O., Husth, M. and Grunett, N. (1996) Maternal HLA class II alleles predispose to pregnancy losses in Danish women with recurrent spontaneous abortions and their female relatives. Am. J. Reprod. Immunol., 35, 239244.[ISI][Medline]
Christiansen, O.B., Ring, M., Rosgaard, A., Grunnet, N. and Gluud, C. (1999) Association between HLA-DR1 and -DR3 antigens and unexplained repeated miscarriage. Hum. Reprod. Update, 5, 249255.
Gill, T.J., III (1983) Immunogenetic aspects of the maternal-fetal interaction. In Wegmann, T.G. and Gill, T.J., III (eds), Immunology of Reproduction. Oxford University Press, Oxford, pp. 5376.
Guilbert, L. (1996) There is a bias against type 1 (inflammatory) cytokine expression and function in pregnancy. J. Reprod. Immunol., 32, 105110.[CrossRef][ISI][Medline]
Hammer, A., Hutter, H. and Gohr, G. (1997) HLA class I expression on the materno-fetal interface. Am. J. Reprod. Immunol., 38, 150157.[ISI][Medline]
Hashimoto, M., Kinoshita, T., Yamasaki, M., Tanaka, H., Imanishi, T., Ihara, H., Ichikawa, Y. and Fukunishi, T. (1994) Gene frequencies and haplotypic associations within the HLA region in 916 unrelated Japanese individuals. Tissue Antigens, 44, 166173.[ISI][Medline]
King, A., Boocock, C., Sharkey, A.M., Gardner, L., Beretta, A., Siccardi, A.G. and Loke, Y.W. (1996) Evidence for the expression of HLA A-C class I mRNA and protein by human first trimester trophoblast. J. Immunol., 156, 20682076.[Abstract]
Margulies, D.H. (1999) The major histocompatibility complex. In: Paul, W.E. (ed.), Fundamental Immunology. 4th edition. Lippincott-Raven Publishers, Philadelphia, pp. 263285.
Nepom, G. and Erlich, H. (1991) MHC class II molecules and autoimmunity. Annu. Rev. Immunol., 9, 493525.[CrossRef][ISI][Medline]
Ober, C., Steck, T., Van der Ven, K., Billstrand, C., Messer, L., Kwak, J., Beaman, K. and Beer, A. (1993) MHC class II compatibility in aborted fetuses and term infants of couples with recurrent spontaneous abortion. J. Reprod. Immunol., 25, 195207.[CrossRef][ISI][Medline]
Opelz, G., Mytilineos, J., Scherer, S., Dunckley, H., Trejaut, J., Chapman, J., Middleton, D., Savage, D., Fischer, O., Bignon, J. et al. (1991) Survival of DNA HLA-DR typed and matched cadaver kidney transplants. Lancet, 338, 461463.[CrossRef][ISI][Medline]
Ota, M., Seki, T., Fukushima, H., Tsuji, K. and Inoko, H. (1992) HLA-DRB1 genotyping by modified PCR-RFLP method combined with group-specific primers. Tissue Antigens, 39, 187202.[ISI][Medline]
Raghupathy, R. (1997) Th1-type immunity is incompatible with successful pregnancy. Immunol. Today, 18, 478482.[CrossRef][ISI][Medline]
Raghupathy, R., Makhseed, M., Azizieh, F., Hassan, N., Al-Azemi, M. and Al-Shamali, E. (1999) Maternal Th1- and Th2-type reactivity to placental antigens in normal human pregnancy and unexplained recurrent spontaneous abortions. Cell. Immunol., 196, 122130.[CrossRef][ISI][Medline]
Roby, K.F., Fei, K., Yang, Y. and Hunt, J.S. (1994) Expression of HLA class II-associated peptide transporter and proteasome genes in placenta and trophoblast cell lines. Immunology, 83, 444448.[ISI][Medline]
Sasaki, T., Yamada, H., Kato, E.H., Sudo, S., Kishida, T., Sasaki, T., Nishigaki, F. and Fujimoto, S. (1997) Increased frequency of HLA-DR4 allele in women with unexplained recurrent spontaneous abortions, detected by the method of PCR-SSP. J. Reprod. Immunol., 32, 273279.[CrossRef][ISI][Medline]
Svejgaard, A., Jersild, C., Nielsen, S. and Bodmer, W.F. (1974) HLA antigens and disease: statistical and genetical consideration. Tissue Antigens, 4, 95105.[ISI][Medline]
Takakuwa, K., Higashino, M., Ueda, H., Yamada, K., Asano, K., Yasuda, M., Ishii, S., Kazama, Y. and Tanaka, K. (1992) Significant compatibility does not exist at the HLA-DQB gene locus in couples with unexplained recurrent abortions. Am. J. Reprod. Immunol., 28, 1216.[ISI][Medline]
Takakuwa, K., Hataya, I., Arakawa, M., Kikuchi, A., Higashino, M., Yasuda, M., Kurabayashi, T. and Tanaka, K. (1999) Possible susceptibility of the HLA-DPB1*0402 and HLA-DPB1*04 alleles to unexplained recurrent abortion. Analysis by means of polymerase chain reaction-restricted fragment length polymorphism method. Am. J. Reprod. Immunol., 42, 233239.[ISI][Medline]
Thomson, G. (1986) Human HLA genetics and disease associations. In Weir, D.M. et al. (eds), Handbook of Experimental Immunology. Volume 3. Blackwell Scientific Publications, Oxford, Chapter 102, pp. 35.
Thomson, G. (1995) HLA disease associations: models for the study of complex human genetic disorders. Crit. Rev. Clin. Lab. Sci., 32, 183219.[ISI][Medline]
Trebichavsky, I. and Nyklicek, O. (1992) Expression of HLA-DR molecules and some other differentiation antigens with early human fetus. Folia Biol., 38, 269276.[ISI]
Van Endert, P.M., Lopez, M.T., Patel, S.D., Monaco, J.J. and McDevitt, H.O. (1992) Genomic polymorphism, recombination, and linkage disequilibrium in human major histocompatibility complex-encoded antigen-processing genes. Proc. Natl Acad. Sci. USA, 89, 1159411597.[Abstract]
Wagenknecht, D.R., Green, K.M. and McIntyre, J.A. (1997) Analyses of HLA-DQ alleles in recurrent spontaneous abortion (RSA) couples. Am. J. Reprod. Immunol., 37, 16.[CrossRef][ISI][Medline]
Wegmann, T.G. (1987) Placental immunotrophism: maternal T cells enhance placental growth and function. Am. J. Reprod. Immunol. Microbiol., 15, 6769.[Medline]
Wegmann, T.G., Lin, H., Guilbert, L. and Mosmann, T.R. (1993) Bidirectional cytokine interactions in the maternal-fetal relationship: is successful pregnancy a TH2 phenomenon? Immunol. Today, 14, 353356.[CrossRef][ISI][Medline]
Woolf, B. (1955) On estimating the relation between blood group and disease. Ann. Hum. Genet., 19, 251253.[Medline]
Submitted on July 8, 2002; resubmitted on November 4, 2002; accepted on January 9, 2003.