Human Reproduction, Vol. 15, No. 8, 1807-1812,
August 2000
© 2000 European Society of Human Reproduction and Embryology
Increased HLA-DR homozygosity associated with pre-eclampsia
1 Department of Reproductive Medicine and Child Development, Division of Gynaecology and Obstetrics, 2 Stella Maris Foundation and 3 Institute of Immunohematology, University of Pisa, S. Chiara Hospital, Via Roma 55, 56100-Pisa, Italy
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Abstract |
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It is generally accepted that maternal recognition of paternally derived fetal antigens occurs during normal pregnancy and may be beneficial for implantation and maintenance of gestation. Thus, we have investigated the human leukocyte class II DR antigens (HLA-DR), whose role in self and non-self recognition is well known, in women with pre-eclampsia, their partners and in control couples. The HLA-DR antigens were tested in 70 pre-eclamptic primigravidae women and their partners and 70 healthy control couples using the serological Terasaki technique. Our results did not show any particular HLA-DR antigen to be correlated with pre-eclampsia, but a statistically significant increase of only one identifiable HLA-DR antigen, which was presumed to express homozygosity at the HLA-DR locus, in the pre-eclamptic women and their partners: 67.1 versus 7.9% in the control couples (P < 0.000001). The analysis of HLA-DR compatibility between pre-eclamptic women and their partners showed a statistically highly significant increase of the female-to-male compatibility (P = 0.0003) and a lower but significant male-to-female compatibility in comparison with controls (P = 0.014). From our results, it emerges that HLA-DR homozygosity and reduced antigenic disparity seem to be associated to a major risk for pre-eclampsia, which consequently appears to be a `couple's disease'.
Key words: histocompatibility/HLA-DR antigens/homozygosity/pre-eclampsia
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Introduction |
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Pre-eclampsia is a severe gestational pathology that represents an impending danger both for the mother and her fetus. The aetiology of this pathology is still undefined, although it is generally accepted that implantation, which is an outstanding example of successive interactions between two genetically distinct tissues (Edwards, 1995a
,b), is impaired in pre-eclampsia. In fact, abnormal trophoblastic invasion occurs in early pregnancy (Gerretsen et al., 1981; Redman, 1992; Taylor, 1997) and later, the placental endothelial cellular dysfunction is accepted to be a common pathophysiological factor in the development of pre-eclampsia (de Luca Brunori et al., 1988; Rodgers et al., 1988; Roberts et al., 1989; Rappaport et al., 1990; Nisell, 1992; Burrows et al., 1994; Dekker et al., 1995; Dekker and Sibai, 1998).
The placenta acts as an immunological barrier between the mother and the fetal `graft', allowing two antigenically different organisms to tolerate one another. During the course of pregnancy this barrier becomes extremely thin, <2 µm at term, which is only slightly greater than the pulmonary alveolar blood/air barrier (Jones and Fox, 1991). It is clear that any damage to this barrier from various ischaemic risk factors (metabolic, hormonal, genetic, immunological) may be responsible for lesions of the syncytiotrophoblast and villous vessel endothelial cells (Jones and Fox, 1980). Therefore, mixing of maternal and fetal blood is possible, with triggering of a maternal rejection reaction (de Luca Brunori et al., 1994).
The concept that pre-eclampsia may be caused by an abnormal maternal immune response to antigenic challenge by the fetal allograft has been analysed by many authors (Jenkins et al., 1977; Stirrat, 1987; El-Roeiy and Gleicher, 1988; Sibai, 1991; Redman, 1992; Vinatier and Monnier, 1995). Following this immunological hypothesis, we previously undertook an immunohistochemical study, using HLA-DR monoclonal antibody in placentae from pre-eclamptic women. We observed intense and widespread expression of the HLA-DR antigens, absolutely absent in the control placentae (de Luca Brunori et al., 1994). This reaction is similar to that which precedes the hyperacute rejection reaction in transplanted human kidney and heart (Gille, 1980; Caforio et al., 1990).
To understand the mechanism at the basis of such an evident immunological reaction in pre-eclampsia, we undertook the present study to evaluate if pre-eclampsia, like transplant rejection, could be related to the immunological role of the HLA-DR antigens.
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Materials and methods |
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Seventy pre-eclamptic women out of 7000 pregnant women admitted to our Obstetrics Department of Pisa University between 1992–1998, their partners and 70 control couples were examined using the Terasaki serological HLA-DR typing technique. All pre-eclamptic women were selected according to the criteria of the Davey and MacGillivray classification of hypertensive disorders in pregnancy: systolic blood pressure
140 mm Hg, diastolic blood pressure 90 mm Hg on two occasions at least 6 h apart; proteinuria 300 mg/24 h (Davey and McGillivray, 1988). All blood pressure levels were measured at hospital admission of patients and 6 h later for the diagnosis of pre-eclampsia and then measured daily by hourly monitoring using Symphony N-3100–100 (Nellcor Puritan Bennett, Pleasanton, CA, USA); proteinuria was quantified in all pre-eclamptic women by taking urine samples in the first 24 h after admission. The group of pre-eclamptic women included 69 Italian Caucasian couples and one South-American couple. All women were singleton primigravidae, between the 26th and 35th week of gestation, aged 29 years on average; they had no history of hypertension, diabetes, renal diseases, autoimmune disorders or blood transfusions. No consanguineous marriages were included.
The control group included 70 normotensive healthy primigravidae women and their partners, selected after term delivery of live and healthy babies. This control group was made up of Italian Caucasian couples from the same geographical area as the couples in the study group to avoid any difference in ethnic composition between groups.
Aliquots of 9 ml of venous blood samples were taken in 1 ml adenine-citrate-dextrosium (ACD) solution tubes, and examined using the standard lymphocytotoxicity technique for HLA-DR typing (Terasaki and McLelland, 1964). Each couple included in the study and the control group was typed by means of two 72-well plates (Terasaki plates; One Lambda Inc., Canoga Park, CA, USA) differing in sera composition in order to use a very wide serological range.
Twenty pre-eclamptic and 10 control couples, in addition to serological Terasaki typing, were checked by polymerase chain reaction (PCR) and reverse dot blot hybridization technique (Buyse et al., 1993). INNO-LIPA (Milan, Italy) HLA-DRB genotyping at low resolution was used.
In order to make the DR typing data uniform, the results were reported as broad antigen specificities from 1 to 10.
The HLA-DR typing results of the pre-eclamptic and control groups were compared using the 
2 test. All differences were considered to be statistically significant when P < 0.05. The relative risk (RR) was calculated by Woolf's method (Woolf, 1955) with Haldane's modification (Haldane, 1956).
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Results |
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The clinical details of the pre-eclamptic women, regarding the time and kind of delivery, proteinuria, blood pressure and maternal endangering are reported in Table I
. The neonatal outcome is described in Table II, in which the time of delivery reflects the severity of pre-eclampsia and explains the perinatal mortality (38.5% in the babies delivered at or before the 26th week; 0% in the babies delivered at or later than the 35th week).
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The HLA-DR typing did not show any particular antigen clearly associated with pre-eclampsia, but the occurrence of a significant excess of only one HLA-DR antigen in the partners of the couples of pre-eclamptic women versus controls (67.1 versus 7.9%; P < 0.000001) (Tables III and IV
). As we used a wide range of typing sera, we presumed that, for the majority of the cases, the presence of a single detected HLA-DR antigen indicated homozygosity at this locus.
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Table III
shows the HLA-DR typing in the study and control couples, clustered according to the number of DR antigens detected. The couples of pre-eclamptic women were distributed in the groups: I (both homozygous partners), II (homozygous female and heterozygous male), III (heterozygous female and homozygous male) and only two couples belonged to group IV (both heterozygous partners). In the majority of the controls, both partners carried two DR antigens (group IV) and only 10 couples were distributed in groups I, II and III.
The distribution of HLA-DR homozygosity in the study group (Table IV) was either in both partners, 26/70 cases (37.1%), or only in women, 22/70 cases (31.4%), or only in men, 20/70 cases (28.6%). No significant difference in homozygosity frequency was found between the partners in the couples of pre-eclamptic women.
Out of the two couples of pre-eclamptic heterozygous partners, the first was HLA-DR identical (female DR 6,7; male DR 6,7) while the second shared one DR specificity (female DR 2,5; male DR 4,5).
Pre-eclampsia was extremely severe for perinatal mortality and/or maternal endangering (eclampsia, pleural and pericardial effusion) in 24/26 couples in whom both partners were homozygous and also in the above mentioned couple of heterozygous but DR identical partners (female DR 6,7; male DR 6,7).
We have also analysed the antigenical variety (i.e. the number of different DR antigens in the couple) and the antigenical disparity (i.e. the DR specificities not shared by the partners) (Table V). From this table, it emerges that parents associated with pre-eclamptic pregnancies generally possess a smaller total number of different HLA-DR specificities than control couples.
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The HLA-DR sharing between partners of the study and control groups did not show any significant difference (21 versus 25 couples, not significant; Table III
).
The analysis of the HLA-DR compatibility between partners, female to male (i.e. when the DR of the female is compatible with that of the male) and male to female (i.e. when the DR of the male is compatible with that of the female), showed a statistically significant increase in the female-to-male compatibility (RR 30.13, P = 0.0003) and a lower but significant increase of male-to-female compatibility (RR 4.6, P = 0.014) in comparison with controls (Table III).
In the 20 couples of pre-eclamptic women and 10 control couples checked by PCR in addition to the Terasaki method, the results of the biological molecular technique confirmed the serological data (Tables VI, VII).
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Discussion |
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Several authors studying the HLA in pre-eclampsia have obtained various and sometimes conflicting results. However, different parameters were evaluated: grading of pre-eclampsia, HLA-DR typing techniques, HLA classes I or II, different ethnic groups with different HLA-DR specificities, different inclusion criteria and environmental conditions (Redman et al., 1978
; Johnson et al., 1988; Simon et al., 1988; Kilpatrick et al., 1989, 1990, 1999; Hayward et al., 1990; Hoff et al., 1990, 1992; Brostoff et al., 1991; Illeni et al., 1994; Takakuwa et al., 1997, 1999). In our study, we did not find any particular HLA-DR specificity associated with pre-eclampsia. However, we did find a highly significant increase in HLA-DR homozygosity compared to controls in both partners of the couples with pre-eclamptic pregnancies.
This finding is consistent with a previous observation (Redman et al., 1978) that led to the suggestion that increased HLA compatibility between couples might contribute to pre-eclampsia as a result of an impaired ability to generate a protective maternal immune response. This attractive hypothesis predicts increased male-to-female compatibility, but the data reported here are characterized by an exceptionally high degree of female-to-male compatibility. Moreover, our data are at variance with several other studies (reviewed by Kilpatrick et al., 1999).
Although different geographical populations were investigated and minor differences in disease definitions applied, the reason(s) for this discrepancy is not obvious and cannot readily be explained.
Nevertheless, from our data, two observations have emerged: (i) pre-eclampsia could be a couple's disease because of the occurrence of HLA-DR homozygosity in one or both partners of the couples associated with pre-eclamptic pregnancies; (ii) pre-eclampsia appears to occur more frequently in couples with a smaller total number of different HLA-DR specificities.
The discussion is still open and further studies are needed to clarify the enigma.
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Acknowledgments |
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We wish to thank Professor M.Cristina Mazzilli, Associate Professor of Human Genetics, Department of Experimental Medicine and Pathology, University `La Sapienza' of Rome, Italy and her co-worker Barbara Mora, MD, for their immuno-genetical interpretation and critical review of data.
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Notes |
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4 To whom correspondence should be addressed at: Lung'Arno Simonelli 3, 56100-Pisa, Italy. E-mail: g.arcidiacono{at}obgyn.med.unipi.it
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Submitted on October 26, 1999; accepted on May 18, 2000.
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