Molecular genetic studies of HLA‐DRB1 alleles in patients with unexplained recurrent abortion in the Japanese population

Human Reproduction Vol.18, No.4 pp. 728±733, 2003 DOI: 10.1093/humrep/deg188 Molecular genetic studies of HLA-DRB1 alleles in patients with unexplained recurrent abortion in the Japanese population Koichi Takakuwa1,3, Hiroshi Adachi1, Isao Hataya2, Keisuke Ishii1, Masaki Tamura1 and Kenichi Tanaka1 1 Department of Obstetrics and Gynecology, Niigata University School of Medicine, Niigata and 2Department 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, 9518510, Japan. E-mail: BACKGROUD: Recently, evidence that HLA antigens are markers for recurrent spontaneous abortion has gained increased attention. Although the association between HLA class II antigens and patients with unexplained recurrent abortion was elucidated by a large population study in a Caucasian population, such analyses have been conducted in only a small Japanese population. The aim of the present study was to determine whether HLA-DR antigens are associated with patient populations with unexplained recurrent abortion in the Japanese population. METHODS: HLA-DRB1 genotypes were determined using a PCR-restriction fragment length polymorphism (PCR-RFLP) method in 93 patients with unexplained recurrent abortion (79 primary recurrent aborters and 14 secondary recurrent aborters) and in 115 normal fertile women. The rate of possession of each HLA-DRB1 genotype was compared among the three populations. RESULTS: The rate of possession of the HLA-DRB1*1502 in patients with secondary recurrent abortion was signi®cantly higher (P < 0.01 after correction for multiple comparisons) compared with the control, fertile women. The rate of possession of HLA-DRB1*1502 was also higher in patients with primary recurrent abortions than in controls, but the difference was not statistically signi®cant after correction. CONCLUSIONS: These ®ndings suggest that HLA-DRB1*1502 might be a risk allele for unexplained recurrent abortion in the Japanese population. Key words: genotype/HLA-DR/PCR-RFLP/unexplained recurrent abortion Introduction As antigens expressed on the surface of fetal or placental tissues possibly induce the allo-immune response of the mother, recurrent spontaneous abortionÐespecially that of unknown aetiologyÐhas been assumed to be caused by an immunological defect that elicits maternal allogeneic reactions against the fetus (Gill, 1983). 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 signi®cant compatibility between patient couples compared with normal fertile couples 728 (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 signi®cant compatibility of HLA-DQA1 and DQB1 alleles between patients and aborted fetuses using a PCR-sequence-speci®c 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 signi®cant 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. ã European Society of Human Reproduction and Embryology Recurrent abortion and HLA-DR Materials and methods Patients and controls The HLA-DR antigen genotypes were analysed in 93 patients with unexplained recurrent abortion. All patients underwent three or more consecutive ®rst-trimester spontaneous abortions with the same partner. Among the 93 patients, 79 had no other pregnancy history (primary recurrent aborters), and the remaining 14 had experienced one delivery prior to consecutive abortion (secondary aborters). None of the patients indicated abnormalities in these systemic analyses, such as chromosomal abnormalities, MuÈllerian anomalies, hormonal de®ciencies, infectious diseases, metabolic disorders or autoimmune abnormalities. A total of 115 normal fertile women who had experienced at least two full-term deliveries, and had never had abortions, were studied as a control population. All individuals in this study were Japanese, and all provided their informed consent. Analyses of HLA-DRB1 genotypes Analyses of HLA-DRB1 genotypes were performed using the PCRRFLP 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 ampli®ed using the PCR procedure with 2.5 units of Taq DNA polymerase (Takara Co. Ltd, Kyoto, Japan). The reaction mixture, which contained 1 mmol/l each of the PCR 3¢ and 5¢ primers, 1 mg of genomic DNA, 10 ml of dNTP mixture (Takara Co. Ltd), a PCR reaction buffer (10 mmol/l Tris±HCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2), and distilled water, to make a total volume of 100 ml in a 500 ml Eppendorf tube, was covered with 50 ml 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-speci®c primers, DR1, DR2, DR4, DR7, DR9, DR10 and DRw52-associated (DR3, -5, -6 and -8) antigenspeci®c primers, were used to obtain only the ampli®ed 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 b1 domain exons), were simply typed by the presence of ampli®ed bands as DRB1*0701 or 0702, DRB1*0901 and DRB*1001 respectively. After ampli®cation, aliquots (6 ml) of the reaction mixture, together with an appropriate restriction buffer and restriction enzymes, were incubated for 1±3 h. AvaII and PstI were used for digestion of the ampli®ed 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 ampl (...truncated)