HLA-G polymorphisms in couples with recurrent spontaneous abortions

The etiology of a fraction of recurrent spontaneous abortions (RSA) may involve immunological mechanisms. Aberrant profiles of Th1 and Th2 cytokines have been observed which are not present in uncomplicated pregnancies. Studies of classical HLA class I and II antigens in relation to RSA have not been conclusive. Furthermore, these antigens are not expressed in the placenta with the exception of HLA-C. However, HLA-G is expressed on especially invasive cytotrophoblasts and exists in both membrane and soluble forms. HLA-G may be involved in materno-fetal tolerance. Therefore, 61 RSA couples (with three or more spontaneous abortions) and 47 fertile control couples were HLA-G genotyped by direct DNA sequencing and analyzed for specific polymorphisms. No statistically significant differences were observed in the distribution of HLA-G alleles between controls and RSA couples, however, 15% of the RSA women carried the HLA-G*0106 allele compared to 2% of the control women. The 14 bp deletion polymorphism in exon 8 was investigated separately. There were a greater number of heterozygotes for the 14 bp polymorphism in the group of fertile control women than expected, according to Hardy-Weinberg equilibrium. Furthermore, the HLA-G alleles without the 14 bp sequence were prominent in the RSA males in contrast to the RSA women in whom alleles including the 14 bp sequence were frequently observed, especially as homozygotes. These results are discussed in relation to two hypotheses concerning HLA-G and RSA. A hypothesis of HLA-G histo-incompatibility between fetus/placenta and the mother was not supported by the data. Another hypothesis concerned certain HLA-G alleles associated with an altered expression profile of HLA-G isoforms or reduced expression of certain HLA-G isoforms.     

Epidemiology of recurrent spontaneous abortion.

With recent scientific advances leading to better understanding of the immunobiology of recurrent spontaneous abortion (RSA), interest has now focused upon the epidemiology of RSA. A cohort of 214 couples with a history of two or more consecutive abortions were studied for the prevalence of etiologic factors and association with other reproductive failures. The prevalence of causes of RSA in this cohort was compared with etiologic factors among 179 couples with a history of three or more consecutive abortions. The obstetrical histories of 214 women with RSA were analyzed for the total number of pregnancies, live births, stillbirths, spontaneous abortions, ectopic pregnancies, and hydatidiform moles. These numbers were compared with the expected frequency of each in the general population. The prevalence of etiologies among 214 with RSA were as follows: chromosomal-6%, anatomic-1%, hormonal-5%, immunologic-65%, and unexplained-23%. No differences in the prevalence of etiologic factors exist when couples with a history of two or more abortions are compared with three or more abortions. When the number of ectopic pregnancies, molar pregnancies, and stillbirths among 214 women with RSA were compared with the expected numbers, the odds ratios were 2.2 for ectopic pregnancies, 6.0 for molar pregnancies, and 2.3 for stillbirths. These data indicate that no difference in the prevalence of etiologies of RSA exist when couples with two or more abortions are compared with three or more and a comorbidity between RSA and other types of reproductive failure exists.     

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.     

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.     

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.     

基因多态性与复发性自然流产的相关性研究

复发性自然流产（recurrent spontaneous abortions,RSA）是指连续发生2次或2次以上流产者。RSA的病因复杂,包括胚胎染色体异常、免疫功能异常、黄体功能不足、感染、生殖道异常等。但目前仍有部分RSA患者病因及发病机制尚不清楚,遗传背景等因素的改变可能使部分人群易发生RSA[1]。近年来从免疫遗传角度提出了一些新的观点,认为RSA的发生与遗传有关。寻找RSA的易感基因或致病基因,从根本上阐明RSA的发病机理,是RSA治疗和预防的重要途径。     

MAJOR HISTOCOMPATIBILITY COMPLEX LOCATED COMPLEMENT C4 AND STEROID 21-HYDROXYLASE GENE REARRANGEMENTS IN COUPLES WITH RECURRENT SPONTANEOUS ABORTIONS

Major Histocompatibility Complex (MHC) class III located complement C4 and steroid 21-hydroxylase (21OH) genes, which form various deletion and duplication units, were studied by Taq I Restriction Fragment Length Polymorphism (RFLP) in 58 Finnish couples who suffered recurrent spontaneous abortions (RSA). The gene rearrangements found in the RSA couples did not differ from those in the controls.     

Major histocompatibility complex located complement C4 and steroid 21-hydroxylase gene rearrangements in couples with recurrent spontaneous abortions.

Major Histocompatibility Complex (MHC) class III located complement C4 and steroid 21-hydroxylase (21OH) genes, which form various deletion and duplication units, were studied by TaqI Restriction Fragment Length Polymorphism (RFLP) in 58 Finnish couples who suffered recurrent spontaneous abortions (RSA). The gene rearrangements found in the RSA couples did not differ from those in the controls.     

Natural killer (NK) cell receptors' repertoire in couples with recurrent spontaneous abortions

PROBLEM: Natural killer (NK) cell receptors (NKRs) have been suggested to protect trophoblast, but their function at the fetomaternal interface remains unknown. To investigate if the outcome of pregnancy depends on women's NKRs, we studied the NKR repertoire in couples with recurrent spontaneous abortions (RSA). METHODS: Twenty-six childless couples with > or = 2 abortions, characterized by alloimmune abnormalities, and 26 control couples were genotyped for five killer immunoglobulin-like receptors (KIR) and two CD94/NKG receptors, known to have as ligands human leukocyte antigen (HLA) class I molecules with trophoblastic expression: inhibitory 2DL1,2,3 and activating 2DS1,4 KIRs, inhibitory NKG2A and activating NKG2C. Detected repertoires of women and partners were compared between the two groups. RESULTS: Less aborters than controls were found to have all three inhibitory KIRs (30.77% versus 69.23%, P = 0.01), some of them had only one inhibitory KIR (19.23% versus 3.85%, P = 0.08) and most of them were lacking inhibitory KIRs possessed by their husbands (57.69% versus 15.38%, P = 0.001). CONCLUSIONS: Women with alloimmune abortions have a limited inhibiting KIR repertoire and such miscarriages may occur because trophoblastic HLA class I molecules are recognized by decidual NK cells lacking the appropriate inhibitory KIRs.     

Semen parameters and sperm morphology in men in unexplained recurrent spontaneous abortion, before and during a 3 year follow-up period

To investigate the role of the ‘male factor’ inthe patho-genesis of recurrent spontaneous abortion (RSA), especiallysperm morphology abnormalities, 120 previously selected coupleswith unexplained RSA were studied for sperm parameters retrospectivelyand prospectively. The patients were subdivided into three subgroups,depending on their reproductive outcome during the 3 years offollow-up study: (i) 48 RSA couples who achieved a successfulpregnancy; (ii) 39 RSA couples who experienced further abortions;and (iii) 33 RSA couples who experienced infertility duringthe follow-up period. A semen analysis was performed twice atthe time of inclusion in the study, and twice again during the3 year follow-up period. No significant differences in semenparameters were observed between the RSA males and fertile controls.Instead, significant differences were observed between the groupof RSA couples who experienced infertility during the follow-upand the other two groups (RSA couples who achieved successfulpregnancy and RSA couples who experienced miscarriages and nolive birth during the follow-up) for sperm concentration (P< 0.01 and P < 0.01 respectively), sperm motility (P <0.01 and P < 0.01 respectively) and sperm morphology abnormalities(P < 0.01 and P < 0.01 respectively). Sperm morphologyabnormalities do not seem to be involved in determining RSA;instead, they are an aetiological factor in determining infertilityin patients, along with the other semen parameters, in the RSAcouple‘s subsequent reproductive life. Semen analysisis an important test in the clinical management of RSA couples.     

A Set of MHC Haplotypes Found Among Finnish Couples Suffering From Recurrent Spontaneous Abortions

PROBLEM AND METHOD: The role of major histocompatibility complex (MHC) genes in the etiology of recurrent spontaneous abortion (RSA) was studied by analyzing the polymorphism of several, at least 14, immunogenetically important MHC genes either by serological or molecular methods in 56 Finnish RSA couples, and in 29 infants born to these families during the follow-up period of two years after the abortions. RESULTS: The haplotype analysis showed that the RSA couples had significantly increased sharing of MHC fragments, compared to the control families. Furthermore, the MHC risk markers for abortions defined 12 different, extended MHC haplotypes that were found in a significantly higher proportion among persons in the RSA group (45%) than in the controls (11%). However, neither of these observations associated with the reproductive success of the study couples. CONCLUSIONS: The results suggest that extended MHC haplotypes, disadvantageous for reproduction, exist in some isolated populations, such as the Finns.     

Ultrasonographic detection of a live fetus in recurrent spontaneous abortion during the first trimester

To examine whether recurrent spontaneous abortion (RSA) canbe distinguished from repeated sporadic spontaneous abortion,the clinical course of 38 cases with three or more consecutiveand unexplained first trimester RSAs were retrospectively investigatedin this study. For comparison with controls, the clinical coursewas examined of 38 fertile females, who had had sporadic abortions.In 19 (50%) RSAs and 6 (16%) controls, fetal cardiac activitywas demonstrated by ultrasound during the course of pregnancy.The rate of detection of live fetus during pregnancy or at 8weeks ± 7 days gestation, was significantly greater inthe RSA group compared to the control. The rate of vaginal bleedingbefore spontaneous abortion was significantly less in the RSAgroup than in the control group. There was no difference betweenthe two groups in age or gestational age at spontaneous abortion.The patients with RSA were all examined for antiphospholipidantibodies in their sera and these were detected in eight ofthem. However, there was no difference in the rate of positivefetal cardiac activity between the RSA patients who tested positiveor negative for antibody. These results reveal that the clinicalcourse of RSA is very different from the course of sporadicabortion. Although sporadic abortion is a common complicationof pregnancy, RSA is not a random repeated abortion, but rathera separate disease from sporadic abortion in normal fertilefemales.     

Genetic predisposition to idiopathic recurrent spontaneous abortion: contribution of genetic variations in IGF-2 and H19 imprinted genes

PROBLEM: Recurrent spontaneous abortion (RSA) is a common clinical problem with a complex etiology of genetic and non-genetic causes, which remains to be fully determined. IGF-2 stimulates trophoblast invasion, proliferation and maturation of placenta, while H19 RNA suppresses growth. As genomic imprinting plays a critical role in the development of placenta and embryo, our aim was to evaluate the possible role of variations in IGF-2 and H19 imprinted genes as factors of predisposition for RSA. METHOD OF STUDY: A case-control study was conducted to determine the association between IGF-2 and H19 gene polymorphisms and the susceptibility to RSA in 113 couples with RSA and 226 controls. PCR/RFLP were performed to analyze IGF-2 ApaI and H19 HhaI polymorphisms. RESULTS: We found a statistically significant difference in the genotype frequency distribution of IGF-2 ApaI polymorphism between males from couples with RSA and healthy males (chi2(2) = 45.12; P < 0.0001). There were no differences in the genotype and allele distribution of H19 polymorphism frequencies, or for the IGF-2 ApaI polymorphism between female groups. CONCLUSION: The presence of IGF-2 ApaI polymorphism in partners of RSA women could affect IGF-2 level of expression in placenta and embryo and represent a risk factor for RSA susceptibility.     

Thrombophilic gene mutations and recurrent spontaneous abortion: prothrombin mutation increases the risk in the first trimester

PROBLEM: Thrombophilic predisposition may be one of the underlying causes of recurrent spontaneous abortions (RSA). We studied the prevalence of five thrombophilic gene mutations in patients with RSA. METHOD OF STUDY: 102 patients with two or more consecutive abortions and 128 women without miscarriage were analyzed for factor V Leiden mutation (FVL), prothrombin G20210A mutation (PTM), C677T mutation in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, glycoprotein IIIa (GPIIIa) C1565T polymorphism, and beta-fibrinogen G-455A polymorphism by polymerase chain reaction (PCR) techniques. RESULTS: No differences in the prevalence of FVL, MTHFR T/T, GPIIIa and 1-fibrinogen polymorphism were detected. Heterozygous PTM occurred more often in patients with RSA. This effect was significant in a subgroup with abortions exclusively in the first trimester (6.7%, vs. 0.8%, P = 0.027, OR 8.5). CONCLUSIONS: In contrast to the other mutations and polymorphisms, heterozygous PTM is more common in patients with abortions in the first trimester. This might reflect an influence of PTM on pathogenesis of early pregnancy loss.     

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.     

Immunologic characteristics of repeated spontaneous abortions

In 28 couples with spontaneous abortions, data of immunological investigations revealed an elevated frequency of HLA DR compatibility and immunological characteristics defining distinct patterns of immune responsiveness. In the half of women with recurrent spontaneous abortion (RSA) we observed a failure to develop a recognition response to paternal inherited fetal antigens expressed by the lack of classical evidence of in vivo allo-immunization such as antipaternal antibodies, and the absence of the inhibitors of cell-mediated immunity found in maternal blood during pregnancy. In few cases, the paternal cells are inefficient to elicit in vitro maternal cell-mediated lympholysis. In most women with a normal pregnancy occurring after spontaneous abortions or prior to RSA, an immune recognition response was evidenced by the presence of antipaternal antibodies and/or blocking factor acting on in vitro cell-mediated lympholysis. These observations support the hypothesis that immunological process could be the cause of some fetal losses of unknown etiology, through a defective or unsuitable maternal immune response.     

Association of IL-1β and IL-6 gene polymorphisms with recurrent spontaneous abortion in a Chinese Han population

As certain cytokines may play a role in unexplained recurrent spontaneous abortion (RSA) and also some cytokine gene polymorphisms may affect the level of cytokine production, the aim of this study was to investigate the relationship between Chinese RSA and polymorphisms of the genes coding for interleukin (IL)-1β (-31C/T, -511C/T, +3954C/T) and IL-6 (-634C/G). Women (n = 162) with at least three consecutive spontaneous abortions and 156 ethnically matched healthy women with at least one successful pregnancy were included. Genotypes were determined using restriction fragment length polymorphism analysis of polymerase chain reaction products. No significant differences were found in the IL-1β-31T, -511T and +3954T distributions between the RSA group and the control group. On the other hand, the frequencies of the IL-6-634GG genotype and -634G allele were significantly decreased in the RSA group versus the control group (genotype: P = 0.0003; allele: P = 0.002), suggesting the IL-6-634C/G polymorphism might be a possible genetic protective factor for RSA.     

Variations in the thrombomodulin and endothelial protein C receptor genes in couples with recurrent miscarriage

BACKGROUND: Recurrent miscarriage (RM) has been suggested to be caused by mutations in genes coding for various coagulation factors resulting in thrombophilia. Mouse models indicate that genes involved in the protein C anticoagulant pathway are essential for normal embryonic development. Loss of function of two of these genes, thrombomodulin (TM) and endothelial protein C receptor (EPCR), causes embryonic lethality in mice. The aim of this study was to determine whether variations in the human TM or EPCR genes are associated with an increased risk for RM. METHODS: Forty-six RM patients and 191 controls were screened for mutations in TM and EPCR using denaturing high-performance liquid chromatography (DHPLC). The partners of 40 RM patients were also screened. RESULTS: One exonic and one intronic variation in TM and two exonic and two intronic sequences in EPCR were detected. Four variants were common in both patients and controls. A previously identified truncating mutation in EPCR, suggested to have a role in pregnancy complications, was identified in two patients and one control. A novel deletion in the 3'UTR region of TM was detected, but its significance remains unsolved. CONCLUSIONS: These data suggest that mutations in the TM or EPCR genes are not a major cause of RM, although they may exert a modifier effect in combination with other variants.     

Tumour Necrosis Factor B Gene Polymorphism in Relation to Complotype in Couples with Spontaneous Abortions and in Control Families

NcoI restriction fragment length polymorphism (RFLP) of the tumour necrosis factor B (TNFB) gene gives two allelic fragments of 5.5 and 10.5 kb, corresponding to the TNFB*1 and TNFB*2 alleles, respectively. The frequencies of these alleles were analysed in 121 healthy Finns and 56 Finnish couples suffering from recurrent spontaneous abortions (RSA). In the healthy Finns the frequency of TNFB*1 was 37% and that of TNFB*2 63%, of which the frequency of TNFB*1 was significantly increased compared with the Danish population. No deviation was seen between the RSA couples and the Finnish controls. TNF genes are located in major histocompatibility complex class III region close to complement (BF, C4A and C4B) genes. Some complotypes associated most often with the TNFB*1 (S01, S30, S02) and some (S31, F320) with the TNFB*2 allele in the healthy Finns. The combination of the TNFB and the C4 'null' allele differed between the RSA couples and the Finnish controls.     

The factor V Leiden mutation in Japanese couples with recurrent spontaneous abortion.

Thrombosis of placental vessels can be a major cause of recurrent spontaneous abortion (RSA). The factor V Leiden (FVL) mutation, a single point mutation in the factor V gene, is the most common genetic predisposition to thrombosis in European countries and the United States. However, even among Caucasian populations, the association between the FVL mutation and RSA is still controversial. The objectives of the present study were to investigate the prevalence of the FVL mutation in Japanese women who have experienced RSA and to clarify the contribution of the FVL mutation to recurrent miscarriages. A total of 52 Japanese women with a history of three or more consecutive idiopathic first trimester miscarriages and 41 of their male partners were studied. The control group consisted of 55 parous women without obstetric complications. Peripheral blood cell DNA was examined for the presence of the FVL alleles by polymerase chain reaction with Mnl I restriction fragment length polymorphisms. None of the 52 women with RSA and the 41 partners carried the mutation. We also found no subject carrying the FVL alleles in the control group. These results suggest that the FVL alleles are not concentrated in women with RSA at least to clinically significant levels and that there is no apparent association between the FVL mutation and RSA in our Japanese population.