HLA-E polymorphism in patients with recurrent spontaneous abortion

Abstract: The aim of this study was to compare the frequencies of five HLA-E alleles in 82 women with recurrent spontaneous abortions with that of 150 random Danish controls. PCR sequence-specific oligonucleotide typing was carried out to detect polymorphism in exons 2 and 3 of the HLA-E gene. In informative samples sequencing of these two exons was also undertaken to confirm the presence of the HLA-E*01031 allele. HLA-E*0101, HLA- E*01032 and HLA-E*01031 were detected with frequencies of 56.7%, 33.6% and 9.6% in controls and 58,5%, 32.9% and 8.5% in patients with recurrent abortion, respectively. No HLA-E*0102 and E*0104 alleles could be detected. Since the HLA-E allele distribution was similar in women with recurrent spontaneous abortion and controls, it is suggested that maternal HLA-E polymorphism per se does not play any role in the pathogenesis of this disorder of pregnancy.     

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.     

The HLA-G genotype is potentially associated with idiopathic recurrent spontaneous abortion

The causes for recurrent spontaneous abortion (RSA) remain unknown in a large proportion of the cases. Human leukocyte antigen (HLA)-G and HLA-E are expressed on invasive trophoblast cells, and are supposed to confer to materno-fetal tolerance. A total of 14 different nucleotide sequences have been described for HLA-G, including one dysfunctional null allele (HLA-G*0105N), while five different sequences have been described for HLA-E. In this study, 78 RSA couples and 52 fertile controls were typed for HLA-G and HLA-E by direct sequencing or single strand conformational polymorphism (SSCP) respectively. The overall analysis showed no significant difference in allele frequencies for either HLA-G or HLA-E between the two groups. However, HLA-G allele frequencies in women who had suffered from five or more RSA differed significantly from fertile controls (P: = 0.001), and from women who had undergone three or four RSA (P: = 0.027). Detailed analysis demonstrated a significant increase in the proportion of the HLA-G alleles *01013 and *0105N in the whole group of RSA women compared with fertile controls (P: = 0.007). When studying the prognostic value of HLA genotyping for pregnancy outcome (n = 41), 31 patients (76%) gave birth to a living child without performing immunotherapy. Seven out of 10 (70%) couples suffering from a further RSA carried the HLA-G*01013 or *0105N allele, compared with 10 out 31 (32%) couples giving birth (P: = 0.06). This study suggests that the HLA-G genotype may be a contributing factor in RSA.     

HLA-E and immunobiology of pregnancy

Recently, it has been suggested that non-classical antigens such as human leukocyte antigen (HLA)-G and HLA-E may interact with KIR receptors of NK cells which results into downregulation of immune response and helps in the maintenance of pregnancy. In the present study, we have investigated HLA-E polymorphism in normal fertile women and recurrent spontaneous aborters to assess the effect of HLA-E alleles on the success of pregnancy. Allele E*0101 was found to be significantly higher in patients with recurrent spontaneous abortion (chi(2) = 4.097 and P = 0.0430). Differential expression, peptide affinity, and stability of E*0101 may be one of the reasons.     

HLA—E与原因不明习惯性流产的关联研究

目的探索HLA-E与原因不明习惯性流产的发生是否存在关联.方法采用地高辛标记的寡核苷酸探针杂交技术,对上海地区53例原因不明习惯性流产病人和156例正常对照进行了HLA-E等位基因检测.结果在病人和对照组中HLA-E*0101均是最常见的等位基因,其基因频率分别为50%和43.09%,其次是E*01032分别为29.25%和33.12%.E*01031则分别占20.75%和23.79%,HLA-E*0102和E*0104在两组标本中均未检出.检出的3个等位基因在两组之间比较均无显著性差异(P＞0.05).结论HLA-E等位基因多态性与原因不明习惯性流产的发生可能没有直接关联.     

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.     

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

PROBLEM: To clarify whether HLA-DP antigens are associated with patient population of unexplained recurrent abortion. METHOD OF STUDY: The frequency of HLA-DPB1 alleles in patients with unexplained recurrent abortion, and the compatibility of HLA-DPB1 alleles between patient couples, were studied using a polymerase chain reaction (PCR)-restricted fragment length polymorphism (RFLP) method. Thirty patients who had a history of unexplained primary recurrent abortion, and their husbands, were typed for HLA-DPB1 genotype. Two hundred and ninety-nine base pair fragments from the second exon of HLA-DPB1 genes were selectively amplified using the PCR-primers. After amplification, the DNAs were digested with restriction endonucleases, and subjected to electrophoresis in a 12% polyacrilamide gel to determine HLA-DPB1 genotype. RESULTS: The frequency of HLA-DPB1*0402 and DPB1*04 alleles in the patient group (n = 30) was significantly increased, as compared to that in the normal fertile women (n = 30). The frequency of HLA-DPB1*04 allele in the patient group was significantly increased, as compared to that in the general population (n = 112). No significant compatibility of HLA-DPB1 alleles could be observed between patient couples and normal fertile couples. CONCLUSION: These findings suggest a possible new class II association with patient population of unexplained recurrent abortion.     

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.     

Peripheral Blood Mononuclear Cells from Women with Recurrent Abortion Exhibit an Aberrant Reaction to Release Cytokines upon the Direct Contact of Human Leukocyte Antigen-G-Expressing Cells

PROBLEM: In search for pathogenesis of recurrent abortion, we examined whether lymphocytes/macrophages from women with recurrent abortion exhibited an aberrant ability to release cytokines upon the direct contact of human leukocyte antigen (HLA)-G. METHOD OF STUDY: The amounts of cytokines released from peripheral blood mononuclear cells (PBMCs) from women with recurrent abortion were compared with those from normal multiparous women or normal nulligravidous women when cocultured with or without HLA-G-expressing target cells. RESULTS: When cocultured with HLA-G-expressing target cells, the amount of interleukin-1β released from PBMCs was increased in recurrent aborters whereas it decreased in both normal multiparous and nulligravidous women. The amount of interleukin-3 released from PBMCs did not differ with or without HLA-G-expressing cells in recurrent aborters, whereas it increased in the presence of HLA-G-expressing cells in normal controls. The amount of tumor necrosis factor-α released from PBMCs was decreased in the presence of HLA-G-expressing cells in both recurrent aborters and normal controls. CONCLUSION: The aberrant reaction of maternal lymphocytes/macrophages in releasing cytokines upon the contact of HLA-G expressed on trophoblasts may impact negatively on trophoblastic growth, which may be pathogenic in recurrent abortion.     

Polymorphism at the HLA-E locus predates most HLA-A and -B polymorphism.

The extensive polymorphism of the classic class I antigens has been well described. In contrast, the nonclassic HLA antigens are distinguished by their low polymorphism. We examine here the HLA polymorphism of the HLA-E locus by examining the DNA sequence of cDNA from nine ethnically diverse individuals. From this analysis, we show that there is no polymorphism in the regions including exon 1 and from exon 4 to exon 8, the 3' untranslated exon. In exons 2 and 3, there are two base substitutions, one of which is at a replacement site and the other silent. The replacement substitution changes an arginine to a glycine at position 107, defining two alleles at the HLA-E locus. Using the PCR on exon 3 from genomic DNA and hybridization with oligonucleotide probes, we have examined 90 HLA-typed individuals to determine the relative frequency of the two alleles in the population and their association with the classical antigens. This analysis showed that these two alleles were present at nearly equal frequencies in the population. Surprisingly, both alleles were found in an essentially random association with all but one HLA-A and -B haplotype. The single exception was to the A1-B8 haplotype, which appeared to be linked to only one of the two alleles. One implication of this random association is that these HLA-E alleles may have existed before most of the HLA-A and B polymorphism. Thus, selection has maintained the HLA-E locus essentially unaltered during a time when considerable polymorphism was being selected for at the HLA-A and -B loci. This finding may also have important consequences in an unrelated bone marrow transplant, where it is predicted that 37% of HLA-A and -B matched donors are mismatched at the HLA-E locus.     

Association of the A/G polymorphism at position 49 in exon 1 of CTLA-4 with the susceptibility to unexplained recurrent spontaneous abortion in the Chinese population

PROBLEM: To investigate whether the A/G polymorphism at position 49 in exon 1 of cytotoxic T lymphocyte antigen-4 (CTLA-4) gene, which delivers a negative signal to T-cell activation, confers the susceptibility to unexplained recurrent spontaneous abortion in the Chinese population. METHOD OF STUDY: A total of 168 patients with unexplained recurrent spontaneous abortion (RSA), who were treated in the Renji Hospital affiliated to the Shanghai Second Medical University, were matched against 117 women with normal pregnancy history. Case-control study to compare the frequency of G/A alleles, AA/AG/GG genotypes and A + (AA + AG) /G+ (GG + AG) phenotypes of CTLA-4 between RSA patients and controls were performed. After amplification of CTLA-4 exon-1 region by polymerase chain reaction (PCR), restriction fragment-length polymorphism (RFLP) was used to detect the polymorphism at position 49 in exon-1 of CTLA-4 gene. Statistical significance was tested by SPSS software. RESULTS: There were dissimilar distributions of G/A alleles, AA/AG/GG genotypes and A+/G+ phenotypes of CTLA-4 between RSA patients and controls. The frequencies of G allele (P = 0.032) and GG genotype (P = 0.011) in RSA patients were significantly higher than those in controls, while the frequencies of AG genotype (P = 0.039) and A + (AA + AG) phenotype in RSA patients were decreased significantly (P = 0.011). CONCLUSIONS: Our findings suggest that A/G polymorphism in exon-1 of CTLA-4 is associated with the immunopathogenesis of RSA, and it confers susceptibility to RSA in Chinese population.     

Association between dopamine D4 receptor (D4DR) Exon III polymorphism and novelty seeking in Japanese subjects

This study was designed to assess the association between novelty seeking and D4DR gene polymorphism in the Japanese population. The 48 bp repeat polymorphism in the third exon of the dopamine D4 receptor gene of 153 normal female students was correlated with personality feature results from the Japanese version of Cloninger's Temperament and Character Inventory. The Novelty Seeking subscale of Exploratory Excitability had a significant association with long alleles of the polymorphic exon III repeat sequence of D4DR. Our results suggest that there is an association between long alleles of the polymorphic exon III repeat sequence of D4DR and the personality traits of the Novelty Seeking subscale of Exploratory Excitability, regardless of racial differences in the frequencies of D4DR exon III repeat polymorphism. Am. J. Med. Genet. 74:501–503, 1997. © 1997 Wiley-Liss, Inc.     

Association between HLA-E *0101 homozygosity and recurrent miscarriage in Egyptian women

The objective was to investigate the frequency of human leucocyte antigen (HLA)-E alleles in Egyptian women with and without recurrent miscarriage (RM) to evaluate their role on the maintenance of pregnancy. A case-control study was adopted. HLA-E gene polymorphism typing was carried out by restriction fragment length polymorphism for 108 women with RM and 120 fertile female controls. The frequency of HLA-E *0101 allele was higher in patients with RM and HLA-E*0103 allele was higher in fertile controls, and the difference was statistically significant (P=0.003, P(c)=0.006). HLA-E*0101/0101 genotype was the most frequent genotype in patients (45.4%), followed by HLA-E*0101/0103 (44.4%) and finally HLA-E*0103/0103 genotype (10.2%). The difference in the frequency of HLA-E*0101/0101 homozygous genotype in patients with RM compared with that in the fertile controls was statistically significant (OR=2.02, 95% CI=1.13-3.62, P=0.011, P(c)=0.033). We found an increased frequency of homozygosity for HLA-E*0101 in Egyptian women with RM. HLA-E*0101 homozygosity may thus be a risk factor for RM.     

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.     

HLA-G polymorphism in a Polish population and reproductive failure.

To investigate whether human leukocyte antigen (HLA)-G gene polymorphism is associated with reproductive failure in a Polish population, we sequenced exons 2-4 of the HLA-G gene in 58 couples with three recurrent spontaneous abortions (RSAs) in the first trimester of pregnancy and 58 fertile control couples. We identified 12 different HLA-G alleles. Neither allele was found to be associated with an increased risk of RSA in the population. HLA-G allele sharing was similar in couples with RSA and in control fertile couples. All cases and controls were also genotyped for the -725C>G polymorphisms in the promoter region and the 14-bp insertion deletion in the 3' untranslated region of the HLA-G gene. The frequencies of both variants in RSA women and control fertile women were similar. These results suggest that HLA-G gene polymorphism does not influence the risk of RSA in the Polish population, but further studies are needed in this regard.     

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.     

Recurrent miscarriage and variant alleles of mannose binding lectin, tumour necrosis factor and lymphotoxin alpha genes.

Variant alleles of the mannose binding lectin (MBL) gene are associated with increased susceptibility to infection and polymorphisms of tumour necrosis factor and lymphotoxin alpha genes (TNF, LTA) are associated with increased severity of infection. Studies have associated recurrent miscarriage with low serum mannose binding lectin concentrations and premature membrane rupture and preterm delivery with elevated maternal and fetal levels of TNF and the TNF (- 308) polymorphism. In this study the frequencies of variant MBL, TNF and LTA alleles in 76 Caucasian couples with idiopathic recurrent miscarriage were compared with those in 69 Caucasian control couples with no history of miscarriage and at least one previous live birth. A new assay based on hybridization to immobilized sequence-specific oligonucleotides (SSO) was used to rapidly detect nine MBL, two TNF and two LTA sequence variants. The assay genotyped all the structural and promoter MBL variants known to influence serum MBL concentrations. This assay was more reliable than restriction digestion or nested allele-specific PCR for the structural variants at codon 54 or 52, respectively. Reliability for codon 57 alleles was not assessed because of the low frequency in this population. The MBL haplotype frequencies in antenatal controls were similar to those reported in other control populations. The frequencies of structural variant MBL genes and of low, medium and high MBL level haplotypes were similar in the recurrent miscarriage and control couples. The TNF and LTA haplotype frequencies were similar in the recurrent miscarriage and control couples. In this carefully defined population no association has been found between recurrent miscarriage and variant alleles of the MBL, TNF or LTA genes.     

HLA sharing in couples with recurrent abortion

To investigate the interactions between HLA region and recurrent abortion we examined the HLA-A and HLA-B antigen frequencies, the degree of HLA sharing, and the incidence of anti-HLA antibodies in 18 recurrent abortion and in 23 control couples. HLA antigens with low distribution (less than 25% of phenotype frequencies in the general population) represent 38.5% of the HLA antigens shared between recurrent abortion partners. No antibodies against partners' HLA antigens were detected in recurrent abortion women, while such antibodies were present in 39.1% of control women (p = 0.002).     

Association Between Major Histocompatibility Antigen and Reproductive Performance

ABSTRACT: Many studies have both supported and refuted an association between HLA antigens and reproductive performance. To clarify these discrepant results, HLA antigens from 59 couples experiencing recurrent spontaneous abortions and 79 couples with unexplained infertility were compared with 51 fertile couples. Patients with recurrent spontaneous abortions were classified as either primary (no children) or secondary (abortions after having children or stillbirths) aborters, and patients with unexplained infertility were classified as primary (never pregnant) or secondary (previously pregnant) infertiles. The amount of antigenic disparity, homozygosity, and the probability of producing a heterozygotic offspring were analyzed for each group. Significantly more disparities at combined HLA loci and at DR loci were observed when childbearing controls were compared with primary aborters. Significant disparity between controls and secondary aborters was at the DQ locus. Total homozygosity as well as homozygosity at DR and DQ loci were significantly increased among primary aborters, but not secondary aborters, and at the B locus among secondary, but not primary infertile couples. Significant association in probability of heterozygote production was seen at the DQ locus in patients with primary infertility. These results indicate that controversy involving association of HLA and reproductive performance can be explained by properly classifying recurrent spontaneous aborters and unexplained infertiles.