1 Department of Obstetrics and Gynecology, Niigata University School of Medicine, 1-757, Asahimachi-dori, Niigata, 951-8510, 2 Department of Obstetrics and Gynecology, Nagaoka Chuo General Hospital, 2-1-5, Fukuzumi, Nagaoka City, 940-0034 and 3 Department of Obstetrics and Gynecology, Nagaoka Red Cross Hospital, 297-1, Terashima-cho, Nagaoka City, 940-2101, Japan
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Abstract |
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Key words: anticardiolipin antibody/HLA-DR genotypes/PCRRFLP/pre-eclampsia
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Introduction |
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Materials and methods |
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Measurement of anticardiolipin antibody
Anticardiolipin antibody was detected with an enzyme-linked immunosorbent assay (ELISA) according to a modified method (Loizou et al., 1985). Details of the method are described elsewhere (Yasuda et al., 1995
). Briefly, microtitre plates were coated with cardiolipin and blocked for non-specific binding. Duplicate serum samples were added to the microtitre plates, which were then incubated for 60 min. Affinity-purified, peroxidase-conjugated goat antihuman immunoglobulin G was then added. After 60 min incubation, the plates were washed and o-phenylene diamine and hyperoxide was added as a substrate. The plates were read at 492 nm with a spectrophotometer. Control serum samples with elevated ACA (positive wells) and blank wells with ACA-negative serum (negative wells) were assayed in parallel with each plate. GPL (IgG phospholipid) units in the test serum were estimated according to the standard curve drawn from the titration of the control serum, and the threshold value was 20 GPL units of ACA.
Analyses of HLA-DRB1 Genotypes
Analysis of HLA-DRB1 genotypes was performed according to published methods (Ota et al., 1992). Genomic DNA was isolated by the phenol extraction of sodium dodecyl sulphate (SDS)-lysed and proteinase K-treated peripheral lymphocytes from all individuals.
Genomic DNA was amplified by polymerase chain reaction (PCR) 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, Kyoto, Japan), and PCR reaction buffer (10 mmol/l TrisHCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2), and distilled water, to make a total volume 100 µl in a 1.5 ml Eppendorf tube, was covered with 50 µl of mineral oil to prevent evaporation and subjected to 30 cycles of 1 min for denaturing, 1 min for annealing, and 2 min for extension in an automated PCR thermal cycler (Thermal Cyclic Reactor, Toyobo Engineering Co., Tokyo, Japan). For HLA-DRB1 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 (Table I). The DR7, -9, -10 alleles, which have no suballeles, could be simply typed by the presence of amplified bands as DRB1*0701, DRB1*0901, DRB*1001 respectively.
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HLA-DRB1 genotypes were determined by comparing the restriction fragment patterns to those of amplified DRB1 genes previously reported (Ota et al., 1992).
Statistical analyses
2 analysis with Yates' correction was used to analyse any significance in the difference between the frequency of each DRB1 allele in patients with pre-eclampsia and normal fertile women. Fisher's exact probability test was used with small expected frequencies. P-values, corrected by multiplying by the number of tested alleles (Pc), were also obtained (Svejgaard et al., 1974
). The odds ratio (OR) was calculated with a 95% confidence interval (CI).
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Results |
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There was no significant difference between the patient group with positive ACA and the patient group with negative ACA concerning the frequency of any DRB1 alleles, although that of DRB1*0403 tended to be higher in patients with positive ACA compared with that in patients with negative ACA (OR 2.80, 95% CI 0.6911.3, P = 0.14).
No significant difference was observed between the patient group with negative ACA and normal fertile women concerning the frequency of any DRB1 alleles.
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Discussion |
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Pre-eclampsia is a unique human disorder, although its aetiology has not yet been fully elucidated. Schuiling et al. proposed that pre-eclampsia is the consequence of an unsuccessful attack of the maternal non-specific host-defence on the implanting conceptus (Schuiling et al., 1997). In view of the diversity of maternal immunological response during pregnancy, aberrations in this response have been suggested as likely causes of this pathology occurring during pregnancy (Kilpatrick, 1987
; Redman and Sargent, 1993
; Humphrey et al., 1995
). On the other hand, a genetic basis for pre-eclampsia has also been pointed out (Chesley and Cooper, 1986
; Hayward et al., 1992
).
On the basis of the above-mentioned premises, several researchers have attempted to ascertain the possible existence of an association between antigens of the HLA system and the development of pre-eclampsia using a serological method for determining HLA alleles. In an earlier study it was reported that women who were homozygous for HLA-A and -B had more severe pre-eclampsia (Redman et al., 1978). Simon et al. noted the association of HLA DR4 with the risk of recurrence of hypertension in pregnancy (Simon et al., 1988
), and Kilpatrick et al. also pointed out that HLA-DR4 common to both the mother and fetus had an association with the pathogenesis of pre-eclampsia (Kilpatrick et al., 1990
). We have also reported the possible association between HLA-DR antigens and pre-eclamptic women using a serological method (Takakuwa et al., 1996
).
It is suggested, however, that serologically defined HLA Class II antigens may be unreliable, and that genotyping of the HLA antigen system is necessary to elucidate the relationship between the HLA Class II antigen system and various diseases (Mytilineos et al., 1990; Opelz et al., 1991
). Opelz et al. reported that up to 25% of serological HLA-DR typing assignments were incorrect when compared with to a more precise DNARFLP method in a large, multicentre study (Opelz et al., 1991
). They noted that the ambiguity of serological HLA-DR typing was due to the difficulty in handling B lymphocytes and the wide cross-reaction of typing reagents for HLA-DR antigens. In this context, we adopted a PCRRFLP method, which enabled us to determine the HLA Class II genotypes very accurately, to investigate the association between pre-eclampsia and the HLA-DR antigen system.
Moreover, there is support for the suggestion that autoimmune factors such as antiphospholipid antibodies or lupus anticoagulants are generative factors in pre-eclampsia (Branch et al., 1985; El-Roeiy et al., 1991
; Milliez et al., 1991
; Yasuda et al., 1993
, 1995
). Gleicher and El-Roeiy have postulated a reproductive autoimmune failure syndrome in which autoimmune factors, such as lupus anticoagulants or antiphospholipid antibodies, are implicated in the genesis of unexplained recurrent fetal miscarriages as well as pre-eclampsia (Gleicher and El-Roeiy, 1988
).
Recently, it has been noted that pre-eclampsia is associated with inherited or acquired thrombophilia (Kupferminc et al., 1999). Wetzka et al. pointed out that an increased expression of cyclooxygenase 1 could be involved in the pathophysiology of pre-eclamptic changes within the placental bed, which possibly caused a decreased prostacyclin:thromboxane A2 ratio followed by an increased formation of thrombi (Wetzka et al., 1997
); antiphospholipid antibodies are considered to be one of thrombophilic factors in patients with pre-eclampsia.
Recent investigations adopting a genotyping method disclosed that a significantly high frequency of HLA Class II alleles exist in patients having recurrent fetal miscarriage positive for ACA. Christiansen et al. reported that HLA-DR3 phenotypes were significantly more frequent in ACA-positive recurrent miscarriage patients compared with healthy controls in Danish and Czech population (Christiansen et al., 1998). We reported that the frequency of DR*0403 was significantly higher in patients having recurrent fetal miscarriage positive for ACA, while DR*0101 was lower compared with the normal fertile women group (Hataya et al., 1998
).
In this context, we divided the patients with severe pre-eclampsia into two groups according to positivity for ACA, and compared the frequency of each HLA-DRB1 allele in patient populations with that of normal fertile women in this study. As a result, the frequency of HLA-DRB1*0403 and -DRB1*04 was found to be significantly higher in patients positive for ACA compared with normal fertile women, although the significance was not so marked (OR 7.18, 95% CI 1.6731.0, P < 0.05, Pc not significant: OR 2.90, 95% CI 1.167.23, P < 0.05, Pc not significant respectively). The patients in the current study were not included in our previous study (Hataya et al., 1998) and the current results suggest the same immunogenetic background between patients with recurrent fetal miscarriage positive for ACA and patients with severe pre-eclampsia positive for ACA.
On the other hand, the frequency of each HLA-DRB1 allele was not significantly different in patients with severe pre-eclampsia negative for ACA compared with normal fertile women. Wilton et al. described absence of an association between maternal HLA-DRB gene and pre-eclampsia/eclampsia (Wilton et al., 1990, 1991
), and results obtained in this study are concordant with their results, as far as the patients with negative ACA are concerned.
Recently, Kilpatrick et al. reported that HLA and tumour necrosis factor genes are closely related to patient population with pre-eclampsia (Kilpatrick, 1996, 1999
). Thus, further investigations of the frequency of the HLA antigen alleles in connection with immune response genes are warranted to determine new association between the antigen system and pre-eclamptic patients.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 3, 1999; accepted on August 26, 1999.