Probabilistic assessment of the HLA sharing of recurrent spontaneous abortion couples in the Japanese population

In spite of a number of investigations, the concept of human leukocyte antigen (HLA) sharing in recurrent spontaneous abortion (RSA) couples remains controversial. We introduced the basal antigen sharing rate (BSR) by the original mathematical approach using the gene frequency of all HLA specificities derived from our regional control population and applied this parameter for the comparison with RSA couples. RSA couples were classified into three subgroups; primary (3 or more consecutive abortions), secondary (3 or more consecutive abortions after 1 live birth), and potential (2 consecutive abortions) aborters. No significant differences between the HLA class I sharing rates (one or more antigens shared on a single locus) of the all RSA subgroups and the calculated BSRs were observed. In the HLA-DR and DQ loci, on the other hand, the antigen sharing rates of primary aborters were significantly higher than BSRs (p less than 0.01/DR, p less than 0.05/DQ). While potential aborters showed a result similar to that of the primary aborters, no significant antigen sharing of HLA class II was observed in secondary aborters. Our data suggest that BSR is a useful parameter for detection of significant HLA sharing in regional populations and the consecutive abortions that occurred primarily are certainly relevant to HLA class II sharing.     

Association between infertility and spontaneous abortion.

Women experiencing recurrent spontaneous abortion have a higher frequency of infertility than that expected in the general population. To further define the relationships between infertility and spontaneous abortion, the obstetrical histories of 43 women with unexplained secondary infertility were evaluated for the frequency of spontaneous abortion. Of the 88 pregnancies studied, 39 (44%) resulted in spontaneous abortion. Women with unexplained secondary infertility experienced a three-fold increase (P less than 0.0001) in the frequency of spontaneous abortions and half the number of live births (P less than 0.0001) compared with the general population. We conclude that the association between infertility and spontaneous abortion includes a higher frequency of spontaneous abortion among infertile couples as well as a higher prevalence of infertility among recurrent spontaneous aborters compared with the general population.     

Significant compatibility does not exist at the HLA-DQB gene locus in couples with unexplained recurrent abortions.

The polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) method was used for both examining compatibility at the HLA-DQB1 gene locus and determining HLA-DQ antigen polymorphism in spouses of unexplained recurrent abortions. Genomic DNA samples were prepared from peripheral mononuclear cells from patient and control couples. Two hundred and thirty base pair fragments of the second exon of the HLA-DQB genes were selectively amplified. Amplified DNAs were digested with the restriction endonucleases, Fok I, Hae III, Hha I, Rsa I and Sau3A I, and subjected to electrophoresis in a polyacrylamide gel. The RFLPs showed that habitual aborters and their husbands had neither significantly frequent alleles nor shared common alleles at the HLA-DQB locus when compared to the control group. Since significant HLA-DQB compatibility was not observed between the spouses and unexplained recurrent aborters, in order to determine whether or not HLA compatibility is responsible for the genesis of unexplained recurrent abortions, it is imperative to further examine the compatibility between other HLA gene loci.     

Immunogenetic studies of recurrent spontaneous abortions in humans

31 clinically defined couples suffering between 3 and 17 consecutive idiopathic spontaneous abortions have been studied. The female partners demonstrated a lower frequency than expected of serum lymphocytotoxic antibodies. Sharing of HLA-A, B or DR antigens was not significantly increased overall within these couples compared with controls, although two couples did show marked HLA sharing (greater than or equal to 4 antigens shared at HLA-A, B and DR loci). There was increased apparent HLA-A and HLA-B locus homozygosity in these women compared with controls (p less than 0.02 and p less than 0.005, respectively), with no significant apparent homozygosity either at HLA-DR or in the males. An increased prevalence of HLA-A, B and DR antigen phenotypes characteristic of various extended haplotypes was also observed in these couples.     

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

BACKGROUND: 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 significantly 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 significant after correction. CONCLUSIONS: These findings suggest that HLA-DRB1*1502 might be a risk allele for unexplained recurrent abortion in the Japanese population.     

Analysis of HLA-DR types of unexplained recurrent spontaneous aborters in the Japanese population by oligonucleotide-DNA typing

To examine whether unexplained recurrent spontaneous abortion (URSA), defined as 2 or more consecutive spontaneous abortions, is correlated with a particular DR type in the Japanese population, we determined the HLA-DR types of 82 primary aborters and 21 secondary aborters by DNA typing utilizing the polymerase chain reaction (PCR) and hybridization with sequence-specific oligonucleotides (SSOs). The DR gene frequencies of the patient group were compared with those of a normal group at three different levels of DR-definition (27, 13 and 11 DR types). At none of the three levels of comparison was any particular DR type with a frequency differing significantly between the patient and normal groups detected in Japanese URSA patients. Furthermore, we examined whether URSA was correlated with the degree of compatibility of HLA-DR antigen within patients and their husbands. Comparison of the DR compatibility between patients and normal couples was made in two different ways, i.e., comparison of the numbers of couples with mismatches and comparison of the average number of mismatches. For either of these two comparisons, we observed no difference in DR compatibility between patients and normal couples. Our results suggest that URSA is not correlated with any particular DR type and that the condition cannot be explained simply by DR compatibility between husband and wife.     

Human Leukocyte Antigen DQ α Sharing Is Not Increased in Couples With Recurrent Miscarriage

PROBLEM : The results regarding human leukocyte antigen (HLA) DQ a allele sharing in recurrent miscarriage couples are conflicting. The purpose of this study was to determine the frequency of HLA DQ α allele sharing in our unexplained recurrent spontaneous abortion (RSA) patients using modern DNA analytical techniques. METHODS : DNA was extracted from whole blood samples of 1) 51 couples with at least three miscarriages, and 2) 43 fertile couples (with at least seven children and no known history of recurrent miscarriage). The polymerase chain reaction (PCR) was used to amplify the second exon of the HLA DQ α locus on chromosome 6. Genotypes were identified by allele specific hybridization with 12 sequence-specific oligonucleotide probes. RESULTS : 47% of recurrent miscarriage couples and 35% of fertile couples shared no alleles. 47% of recurrent miscarriage couples compared to 58% of fertile couples shared one allele, and 6% of recurrent miscarriage couples and 7% of fertile couples shared two alleles. CONCLUSIONS : Reproductive partners with unexplained recurrent pregnancy loss have no increased frequency of HLA DQ α allele sharing. It is unlikely that HLA DQ α genotyping will be helpful in the management of patients with RSA.     

Influence of Histocompatibility Antigens in Recurrent Spontaneous Abortion Couples and on Their Reproductive Performances

PROBLEM: To determine if human leukocyte antigens (HLA) play any role in the aetiology of recurrent spontaneous abortion (RSA), a substantial group of RSA couples were studied, and their reproductive performances in a 3-year follow-up recorded. METHODS: HLA typing was performed for HLA-A, -B, and DR antigens in both partners of 75 couples with unexplained RSA, and compared with a control group of 30 fertile couples that never experienced abortion. A further 57 couples of this group were studied for their reproductive performance in a 3-year follow-up, and subdivided into three subgroups: 1) couples that achieved successful pregnancy during the follow-up; 2) couples that experienced abortion and no livebirth during the follow-up; and 3) couples that experienced infertility during the follow-up. RESULTS: There were no significant differences for antigen frequency in all the different HLA loci, and HLA antigen sharing between all the RSA couples and controls. Significant increase of sharing for HLA-DR locus was observed in the couples that aborted during the follow-up with respect to the couples that achieved livebirth and controls (P < 0.03 and P < 0.02 respectively), and significantly increased frequency of B44, DR5 antigen combination in the same comparison (P < 0.03). No significant differences were observed in terms of the interval between conceptions in couples without antigen sharing with respect to couples with 1, 2 or more antigens shared, and antigen sharing in Locus A, B or DR. CONCLUSIONS: The results suggest that gene(s) disadvantageous for reproduction may exist between the HLA-B and -DR chromosomal region which influences the pregnancy outcome in RSA couples, and that HLA-antigen sharing itself does not influence the outcome.     

Feto-maternal HLA compatibility does not have a major influence on human pregnancy except for lymphocytotoxin production.

Several studies have supported the hypothesis that the maternal immune response to incompatible paternal HLA antigens present on the conceptus may influence pregnancy outcome. In order to relate feto-maternal histocompatibility directly to pregnancy course and characteristics, complete HLA-A, B and DR types were obtained from 132 healthy family groups consisting of mothers, fathers and neonates. The distribution of feto-maternal HLA compatibility was heavily skewed towards incompatibility, with 90% of fetuses being mismatched at 2 or 3 loci. There was no segregation distortion of paternal haplotypes, however, and the number of feto-maternal mismatches was close to that expected theoretically. More than 2% of the neonates were perfectly HLA-A, B and DR compatible with their mothers. The degree of feto-maternal HLA disparity showed no significant correlation with sex of neonate, birthweight, placental weight, maternal plasma alpha-fetoprotein or parity of the mother. Feto-maternal HLA disparity did, however, correlate significantly with maternal lymphocytotoxin production, even after allowance was made for parity (P less than 0.01). We conclude that feto-maternal HLA compatibility per se does not have a major influence on pregnancy outcome, and in particular is unlikely to predispose to spontaneous abortion; so an absence of antigen sharing between spouses experiencing recurrent spontaneous abortions should not be regarded in itself as a contraindication to offering immunotherapy to such couples.     

HLA-A2 class I antigens in couples with recurrent spontaneous abortions

Class I human leukocyte antigen (HLA) antigens, locus A and B, were typed in fertile and infertile couples in cases where one of the spouses carried the HLA-A2 antigen. HLA-class I typing data were obtained from 282 participants, 63 fertile couples and 78 infertile couples with recurrent spontaneous abortions (RSA). Locus A antigens were grouped into eight broad specificities (A1, A2, A3, A9, A10, A11, A19, A28) and locus B antigens were grouped, according to HLA epitopes, in two classes (BW4 and BW6). Although the number of cases is small, significant differences in the distribution of locus A antigens were found between HLA-A2-positive (A2+) women from fertile and infertile couples. HLA-A3, A11 and A28 cross-reacting antigens were absent in women from fertile couples and present in women from infertile couples. HLA-A19, which is associated with amino acid triplets of low immunogenicity, was significantly more often observed in A2+ fertile than in infertile women. An excess of the BW4 epitope was found in A2(-) husbands from infertile couples compared to fertile ones. The results of this study support the idea that in the presence of the HLA-A2 molecule the distribution of HLA-A and B loci antigens may be different in fertile couples compared to couples with recurrent spontaneous abortions. It can be suggested that the HLA-A2 molecule, in context with specific genotypes, may contribute to the overall maternal immune response in normal and disturbed pregnancy.     

Recurrent spontaneous abortion

The laboratory diagnosis and clinical management of unexplained recurrent spontaneous abortion (RSA) patients is a controversial issue in contemporary obstetrics. In this report, the results of laboratory investigations and immunotherapy of RSA patients referred to our Center since 1986 are detailed. Our analyses have resulted in grouping RSA patients into primary (1 degree), secondary (2 degrees), and unexplained classifications. Laboratory evaluation criteria included assays for both complement-dependent and complement-independent antipaternal antibodies as well as histocompatibility antigen tissue typing for HLA, A, B, C, and DR antigens. In addition, mixed lymphocyte cultures (MLC) were performed to assess the degree of HLA-D locus compatibility between couples and to test for the presence of MLC inhibitors in maternal blood. Immunotherapy options and the rationale for their use are given and preliminary outcome data are presented from randomized double-blinded, placebo-controlled clinical trials.     

HLA-DR locus and maternal-foetal relation

Antigen HLA-A, B sharing couples have been previously observed in abnormal pregnancies of unknown etiology. We have determined the HLA-A, B, C and DR antigens of 20 couples with recurrent spontaneous abortions (RSAs), 32 control couples and 100 normal controls. The results showed that in couples with more than two idiopathic repeated abortions, there is no significant increase in the HLA antigen sharing couples. But we were able to observe, in the affected couples, a significant increase in HLA-DR antigen sharing as regards the control couples. We also find a significant increase in the DR5 antigen, in both wives and husbands, in couples with repeated abortions of unknown etiology as previously described for the Dw5 antigen.     

FOETO-MATERNAL COMPATIBILITY IN HLA-DR, -DQ,AND -DP LOCI IN FINNISH COUPLES SUFFERING FROM RECURRENT SPONTANEOUS ABORTIONS

The polymorphism of Major Histocompatibility Complex (MHC) class II genes DRB, DQA, DQB, and DPA was studied by Taq I Restriction Fragment Length Polymorphism (RFLP) in recurrent spontaneous abortions (RSA). The study group consisted of 35 primary abortion (PA) couples (no children) and 15 secondary abortion (SA) couples (1-2 children before abortions). We found no increase in DR-DQ compatibility between the mother and the foetus in the Finnish RSA group. In contrast to findings in some other populations, foeto-maternal incompatibility was increased in the PA group. Thus, our results do not support the theory that increased MHC class II compatibility is a cause of abortions as such. The Finns are a small and relatively isolated population with a unique gene inheritance. Thus, one can speculate that, if the human MHC class II is in the linkage with disadvantageous ‘fertility genes’, and these genes might nonetheless still be clustered in only a few MHC haplotypes among the Finns. This would be the reason, that DR-DQ sharing is not seen. The presence of rare HLA alleles, such as DR2 and DR6, among the aborters also supports this. In addition, this study extends our previous findings on MHC class III in regards to PA and SA couples differing immunogenetically from each other. In MHC class II, this was most obvious in the DPA1 locus. The vast majority of SA women were heterozygous for the two most common DPA1 alleles (14.0kb and 13.5kb), resulting in significantly smaller chances for a DPA1 mismatched foetus to occur in the SA group than in the controls or in the PA women.     

原因不明习惯性流产与HLA相关性的初步研究

探讨ＨＬＡ与原因不明习惯性流产的关系。方法用血清学方法对６４例３次以上ＲＳＡ患者及其配偶进行ＨＬＡ抗原检测,并与１００例正常群体比较。结果ＨＬＡ－Ａ、－Ｂ、－ＤＱ位点抗原频率在ＲＳＡ病人组与正常对照组中无明显差异,而且ＨＬＡ－ＤＲ７抗原频率在ＲＳＡ夫妇中则明显增高,为２１．８８％,Ｐ〈０．０１ＲＲ值为２．８。     

Recurrent Spontaneous Abortion: Histocompatibility Between Partners,Response to Immune Therapy,and Subsequent Reproductive Performance

PROBLEM: Immunological factors may account for previously unexplained cases of recurrent abortion. METHOD: After screening 76 couples for causes of recurrent spontaneous abortion and measuring maternal antipaternal immunity, 23 primary spontaneous recurrent aborters were immunized once with their husbands' leukocytes. Testing for antipaternal cytotoxicity was repeated in 21 couples. Seroconversion was significantly less frequent in couples who shared more than one human leukocyte antigen [one of five (20%) versus 13 of 16 (81%), P < .02]. RESULTS: Twelve of 16 women (75%) who became pregnant had live children and five of those have had a second live child. All 12 women who achieved successful pregnancies had become antipaternal cytotoxic antibody-positive after immunization, whereas all four patients who had repeat abortions had failed to seroconvert (P < .001). However, this relationship is not necessarily causative, as the successful group also tended to have fewer previous abortions and less human lymphocyte antigen sharing. CONCLUSION: Except for transient illness after immunization, one moderately small for gestational age baby and one premature labor at 32 wk, no complications were observed after immunization.     

HLA and red blood group antigens in pregnancy disorders

Total of 356 women with various types of pregnancy disorders as well as their husbands were classified in four groups regarding the type of the disorder as follows: 1. Recurrent spontaneous abortions (RSA) of unknown etiology (N = 105) and RSA - primary aborters only (N = 84); 2. Blighted ovum (N = 80); 3. Rh immunization in pregnancy (N = 90); 4. ABO immunization in pregnancy (N = 47). Two groups of couples were used as controls: 1. Couples randomly taken from forensic medicine cases of paternity evaluation (N = 104); 2. Couples having two or more children with HLA immunization in pregnancy (N = 78). The couples from all groups were typed for red blood group antigens of ABO, Rhesus, MNSs, Kell, Duffy, Lewis, Kidd and P systems and also for HLA antigens. Significantly higher frequency of antigen HLA-A9 was found in women with RSA (corr. p = 0.0003) and in women with pregnancy disorders caused by Rh immunization (corr. p = 0.0136). In couples with RSA the degree of HLA compatibility was significant (p = 0.0048) and the reactivity of spouses in MLR was significantly decreased (p = 0.0001). Significantly, more low responders in MLR were also found among the women with RSA as compared to the controls (p = 0.0217). Two possible pathologic mechanisms may explain the association between HLA antigens and RSA: 1. immunological defects which are linked to HLA-D/DR region causing malfunction of immunosuppressive mechanisms during pregnancy; 2. endocrinological defect which is linked to HLA region as 21-OH hydroxylase deficiency gene.     

Evaluation of immunological infertility.

To evaluate the frequency with which immunological factors are associated with infertility, 92 couples with unexplained infertility were studied by using assays to detect anti-sperm antibodies (ASA), human leukocyte antigen tissue types (HLA), lymphocytotoxic antibodies (LCA), and inhibitors of mixed lymphocyte culture (MLC) and mouse blastocyst assay (MBA). Seventy-three of the women had blood assayed for antiphospholipid antibodies (APA). The frequencies of APA, ASA, LCA, inhibitors of MLC and MBA, as well as HLA associations previously reported to be related to infertility (B locus blanks, single DQ locus in offspring, greater than 1 DR sharing between mates) among 92 infertile couples were compared with those observed among 41 fertile control couples. No significant differences in the frequencies of APA, ASA, MBA, and HLA associations between fertile and infertile couples were observed. Fertile couples demonstrated the presence of wife anti-husband LCA and MLC inhibitors more frequently than did infertile couples (51% vs. 8%, P = 0.0001 and 29% vs. 4%, P = 0.002, respectively). While APA, ASA, HLA tissue typing, LCA, and MLC and MBA have been previously reported as being markers of autoimmune and alloimmune responses relating to reproductive outcome, the current data suggest that more specific markers are necessary to diagnose immunological components of infertility.     

HLA antigen-sharing in couples with repeated spontaneous abortions and the birthweight of babies in successful pregnancies

Previously, several groups reported an increase in HLA antigen-sharing in couples suffering from unexplained repeated spontaneous abortions. It was felt necessary to find out if HLA sharing could have any effect on children born after a successful pregnancy. The birthweight figures of children of 76 couples with repeated spontaneous abortions were analyzed. The results show a significantly lower birthweight in babies born from those couples, presenting a high incidence of HLA antigen-sharing, particularly concerning class II antigens.     

HLA-DR and DQ antigens and anticardiolipin antibodies in women with recurrent spontaneous abortions.

IgG anticardiolipin antibodies (ACL) have been shown to occur in a high proportion of women with repeated unexplained miscarriages. Forty-nine women with unexplained recurrent spontaneous abortions (RSA), previously assayed for the presence of ACL by an enzyme-linked immunoabsorbent assay, were typed for HLA-DR and DQ antigens by the classical microlymphocytotoxicity test. Twenty-five women were positive for ACL and 24 were negative. HLA-DR7 was found in 24.5% of 49 habitually aborting women vs. 28% of healthy controls; but the DR7 frequency was 40% in ACL positive patients vs. 8.3% in ACL negative patients (P = 0.011). These results show that in the Italian population an association between HLA-DR7 antigen and ACL is present in women with unexplained RSA, suggesting that HLA-DR genes might control the susceptibility to specific autoantibody production.     

Increased frequency of complement C4 'null' alleles in recurrent spontaneous abortions.

Typings for major histocompatibility antigens HLA A, B, C and DR and for complement C4A, C4B and factor B were performed for 59 Finnish couples experiencing at least three consecutive recurrent spontaneous abortions (RSA). Forty-one of them were primary abortion (PA) couples with no children and 18 were secondary abortion (SA) couples who had one or two children before abortions. HLA sharing in A and B loci was slightly but significantly increased (15%, P less than 0.05) among RSA couples compared to the controls, as was DR sharing among SA couples compared to PA couples (50% versus 22%, P less than or equal to 0.05). The most interesting new finding, however, was the statistically significant increase of complement C4 functionally silent, i.e. C4 'null', alleles in RSA couples. C4 is a duplicated gene and its products differ in their functions in the complement cascade. C4A null alleles were equally increased in PA wives and in PA husbands (32%, P less than or equal to 0.05) compared to the controls (18%) and C4B null alleles in SA wives (56%, P less than or equal to 0.05) and in SA husbands (50%) compared to the controls (29%). Therefore, the offspring of RSA couples have a significantly increased risk of inheriting several null alleles. The majority, 95% (P less than 0.001) of PA couples and 83% of SA couples, had at least one C4A or C4B null in their phenotypes compared to 66% among Finnish controls.