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Cell Biology International (2007) 31, 1396–1399 (Printed in Great Britain)
Tumour necrosis factor-α receptor 1 polymorphisms and serum soluble TNFR1 in early spontaneous miscarriage
Xue‑Wen Yua*, Xu Lib, Yong‑Hui Rena and Xue‑Cheng Lia
aCenter of Maternal and Child Health, First Affiliated Hospital of the Medical School in Xi'an Jiaotong University, Xi'an, China
bCenter of Medical Molecular, First Affiliated Hospital of the Xi'an Jiaotong University Medical School, Xi'an, China


Abstract

Objectives

The study investigated the association of TNFR1 gene polymorphism with early recurrent spontaneous miscarriage (ERSM) in Chinese women, and soluble TNFR1 (sTNFR1) expression in ERSM women.


Keywords: Early recurrent spontaneous miscarriage (ERSM), Tumour necrosis factor receptor 1 (TNFR1), Soluble TNFR1(sTNFR1), Gene polymorphism, Single nucleotide polymorphisms (SNPs).

*Corresponding author. Tel.: +86 29 8532 3223; fax: +86 29 8523 5492.


1 Introduction

Spontaneous miscarriage (SM) is a common clinical problem in reproduction, occurring in approximately 1% of reproductive-aged women. The causes of SM are not well understood, but immune mechanisms are thought to be involved. Tumour necrosis factor-alpha (TNF-α) is a potent pro-inflammatory cytokine and identified in the ovary, oviduct, uterus, and placenta (Terranova et al., 1995). Early studies addressing its functional role in reproduction revealed that injection of TNF-α or endotoxin (lipopolysaccharide, LPS) to pregnant females results in embryonic death (Gendron et al., 1990). TNF-α expression is raised in the uterine epithelium and stroma, and in giant and spongiotrophoblast cells of the placenta in mice exposed to the DNA-damaging agent cyclophosphamide (CP) (Gorivodsky et al., 1998). The TNF-α level is significantly elevated in the amniotic fluid of women with uterine infections and its increased production correlates with the incidence of pre-term labour (Romero et al., 1989), and in the serum and chorionic villi of SM patients (Çolakoğlu et al., 2004; Rezaei and Dabbagh, 2002; Makhseed et al., 2000). More importantly, TNF-alpha production in peripheral blood of non-pregnant women with a history of RSM was increased when blood was cultivated in the presence of phytohemagglutinine (PHA) (Rezaei and Dabbagh, 2002). The elevated maternal TNF-α levels in first-trimester pregnancies complicated by threatened abortion with a poor outcome, in comparison to those of uncomplicated pregnancy (TACP) and TACP with a good outcome, further support the abortogenic role of this cytokine (Vitoratos et al., 2006). In vitro studies show that TNF-α induces apoptosis of cytotrophoblasts, suggesting that aberrant expression of TNF-α may have harmful effects on placental development and function (Knofler et al., 2000). Blockade of TNF-α has prevented stress-induced miscarriages in murine models of abortion (Arck et al., 1997). It has also been shown that TNF-α-activated maternal monocytes bind to LFA-1 on placental syncytiotrophoblasts and induce apoptosis that TNF-α and can directly promote tissue damage in pregnancy (Garcia-Lloret et al., 2000). These observations have implicated TNF-α as a cytokine involved in triggering immunological pregnancy loss (Clark et al., 1999; Raghupathy, 2001), i.e. the death of embryos owing to the failure of defence mechanisms preventing rejection of the semi-allogeneic feto-placental unit. The deleterious effects of TNF-α on pregnancy may be direct and indirect. The TNF-α exerts its biological effect through the binding of two different cell surface receptors: TNF-α receptor 1 or p55 (TNFR1) and TNF-α receptor 2 or p75 (TNFR2) (Hohmann et al., 1989; Brockhaus et al., 1990), which harbour different intracellular domains, mediate unique as well as overlapping functions such as activation of the transcriptional activator NF-κB and programmed cell death. Most signals, however, are transmitted by TNFR1 which can induce apoptosis and inflammation upon binding the transducing protein TRADD to its ‘death domain’. TNFR2 has no death domain and is the most important TNFR in circulating blood leucocytes. It regulates apoptosis in CD8+ cells. TNFRs expressed on the trophoblast modulate cell proliferation and differentiation in normal pregnancy (Huppertz et al., 2001). Regulated TNF-α expression in the developing placenta may be essential at specific stages of pregnancy.

The TNFR1 gene has been mapped to 12p13 and consists of 10 exons. Single-nucleotide polymorphisms (SNPs) exist in the TNFR1 gene (Pitts et al., 1998a,b). Genetic variations in the promoter region may influence TNFR1 production, thus determining individual susceptibility to disease. An SNP in exon 1 at nucleotide position +36 is described (A36G or Pro12Pro). An association between this polymorphism and inflammatory diseases or anaemia of chronic disease was found in a Caucasian population (Glossop et al., 2005; Waschke et al., 2005). On the basis of this rationale, the two SNPs in the promoter and exon 1 region of the TNFR1 gene have been investigated for their possible correlation with early recurrent spontaneous miscarriage (ERSM), as well as soluble TNFR1 (sTNFR1) with ERSM.

2 Materials and methods

2.1 Subjects

One hundred and eighty-eight non-pregnant women who had experienced early recurrent spontaneous miscarriages (ERSM, two or more consecutive spontaneous miscarriages at 6–10weeks of gestation) before the study, referred from the First Affiliated Hospital of Xi'an Jiaotong University Medical School, were identified from the maternal care clinics of First Affiliated Hospital (Xi'an, China) as an ERSM group, between August 2004 and March 2006. The average age of these women was 28.29years. In the women studied, chromosomal rearrangements, uterine anomalies, thyroid dysfunction, autoimmune disorders, and infection with rubella, toxoplasma, cytomegalovirus and herpes virus were excluded. Normal non-pregnant women (n=138), who had a successful pregnancy without any other adverse pregnancy outcomes, were included in the control group. Their average age was 28.68years. The women were in good general health and gave written informed consent before participation. The study was approved by the Department of Science and Research of the First Affiliated Hospital of Xi'an Jiaotong University Medical School.

2.2 Genomic DNA isolation

Fresh peripheral blood samples of about 2ml were obtained from each patient and stored in EDTA tubes at −20°C. When required, blood samples were thawed at room temperate and the genomic DNA isolated using an AxyPrep Whole Blood Genomic DNA Miniprep Kit procedure (Axygen Biosciences, Union City, USA). Briefly, this is a standard ethanol-based procedure for the isolation of genomic DNA from whole blood, and performed as directed by the manufacturer.

2.3 Geno-typing

DNA fragments containing one base substitution (AGA to AGC) at position −383 in the promoter region of the TNFR1 gene were amplified by polymerase chain reaction (PCR) using a 2×PCR Master Mix kit (MBI Fermentas, USA). The reaction was carried out in a final volume of 25ml containing: 100ng of genomic DNA; 10pmol of each primer; 12.5μl of 2×PCR Master Mix. The sense and anti-sense primers to detect TNFR1 gene polymorphism were 5-TTA TTG CCC CTT GGT GTT TGG TTG-3 and 5-TTG TGA CGG AGT GAG AAG GGG AGG-3, respectively. Template DNA was amplified by 35 cycles by the following steps: denaturation at 95°C for 30s; annealing at 60°C for 60s; and extension at 72°C for 60s. The PCR products were digested with BglII (Takara), which can recognise A to C substitution and electrophoresed in 2% agarose gel. The A allele completed a BglII restriction site generating fragments of 240bp and 130bp from the PCR product. The C allele destroyed the restriction site and gave products that remained intact.

Amplification of a 183bp fragment containing the A36G polymorphism in exon 1 of the TNFR1 gene was performed using the following primer sequences: forward: 5′-GAG CCC AAA TGG GGG AGT GAG AGG-3′; and reverse: 5′-ACC AGG CCC GGG CAG GAG AG-3′. The reaction conditions were as above. The annealing temperature was 58°C. After amplification of DNA fragments containing the CCA to CCG substitution at Pro12 in exon 1 of TNFR1 and the PCR products were digested with MspA1I (New England BioLabs Ltd), which can recognise A to G substitution. The polymorphic MspA1I site was detected as 108bp and 75bp.

2.4 Measurement of soluble TNFR1 in serum

Serum was obtained from clotted venous blood samples by centrifugation at 2000×g for 20min, and then stored at −80°C until use. Level of sTNFR1 in serum samples was determined using a commercially available enzyme-linked immuno-sorbant assay (ELISA) kit (Rapid BioLab, Calabasas, CA, USA). This is a standard ‘sandwich’ ELISA and was performed according to the manufacturer's instruction using the human recombinant standard. This assay has a lower limit of detection for sTNFR1 at 0.5ng/ml. Concentration of the sTNFR1 in 5μl sample was determined by generation of a standard curve for comparison.

2.5 Statistical analysis

SPSS for Windows v11.5 was used for statistical analysis. Genotype and allele frequencies of the ERSM group and the controls were examined using a chi-squared exact test. Comparison of sTNFR1 levels in sera between the ERSM group and the control group was performed using Student’s t-test. The threshold for statistical significance was predefined at a <0.05.

3 Results

3.1 TNFR1−383 polymorphism

The haplotype frequencies and carrier frequencies of the promoter region of the TNFR1 gene are summarized in Table 1. The allele distribution reveals an increased frequency of the TNFR1 AC and TNFR1 CC genotype frequencies in the ERSM group as compared to normal controls (11.7% versus 8.7%; 5.32% versus 2.9%), this did not reach statistical significance (χ2=2.05; p>0.05). Observed in the ERSM group was an increase in the TNFR1 −383 (A to C) allele when compared to normal controls (11.33% versus 7.25%), however not a significant difference (χ2=3.35; p>0.05).


Table 1.

Genotype and allele frequencies for BglII of TNFR1 in women with early recurrent spontaneous miscarriage and normal women

GroupsAA (%)AC (%)CC (%)A (%)C (%)
ERSM156 (82.98)22 (11.70)10 (5.32)334 (88.83)42 (11.17)
Controls122 (88.41)12 (8.70)4 (2.90)256 (92.75)20 (7.25)
χ22.0502.85
p>0.05>0.05


3.2 TNFR1+36 polymorphism

The allele frequency of the +36 (A to G) polymorphism was 9.17% in women with ERMS and 11.72% in the control women. No significant difference was found between the women with ERMS and controls (X2=0.81; p>0.05). The subjects were categorised into three genotypes: AA, AG, and GG. Frequencies of genotypes of TNFR1 MspA1I in the women with ERSM and controls were 84.40% and 78.13% AA homozygotes; 12.84% and 20.31% AG heterozygotes; and 2.75% and 1.56% GG homozygotes, respectively. There was no significant difference between women with ERSM and controls (Table 2). Therefore, there was no evidence of an association between the TNFR1 promoter region and exon 1 polymorphism and ERSM.


Table 2.

Genotype and allele frequencies of TNFR1 MspA1I in women with early recurrent spontaneous miscarriage and normal women

GroupsAA (%)AG (%)GG (%)A (%)G (%)
ERSM92 (84.40)14 (12.84)3 (2.75)198 (90.83)20 (9.17)
Controls100 (78.13)26 (20.31)2 (1.56)226 (88.28)30 (11.72)
χ22.6270.81
p>0.05>0.05


3.3 Serum sTNFR1 level in women with ERSM

Using the ELISA kit, serum levels of sTNFR1 were detectable in any sample of women with or without ERSM. However, serum levels of sTNFR1 were 1.84±0.54ng/ml from non-pregnant women who had experienced a prior first pregnancy loss, and 1.62±0.38ng/ml from non-pregnant women who had a successful pregnancy. Compared to the normal group, women with ERSM produced a significantly higher concentration of serum sTNFR1 (mean difference −0.213; 95% confidence intervals −0.422–0.004; t=−2.033; p=0.045<0.05). Serum levels of sTNFR1 were significantly associated with pregnancy outcome.

4 Discussion

Recurrent spontaneous miscarriage is a clinical entity with many potential aetiologies. Recent investigation has focused on the potential cell-mediated immune mechanism that may be involved in RSM in women. High levels of TNF-α has been detected in women with RSM (Rezaei and Dabbagh, 2002; Arslan et al., 2004). Pregnancy failure is associated with excessive production of TNF-α. Soluble TNFR1 could function as anti-TNF by forming high affinity complexes with TNF. Bioactive TNF is slowly released from these complexes in physiological concentrations which are inherent for normal pregnancy and in a sustained cytokine balance in pregnancy. The study shows that in non-pregnant ERSM women elevation of levels of sTNFR1 in serum were significant. Previously, we have described that the level of serum soluble TNFR1 was elevated in a group of spontaneous abortion women (Yu et al., 2005). Usually elevation of sTNFR1 is a result of inflammatory reaction. To date, the immortalised villous cells and cytotrophoblasts of first trimester placentas shed TNFR1 (Knofler et al., 2000). Constitutive shedding of TNFR1 from villous trophoblasts in vivo may therefore be unfavourable when TNF- α levels exceed those of binding proteins and result in the programmed cell death of the trophoblast, or other detrimental effects to the placenta. In comparison, Chernyshov et al. (2005) reported a significant decrease in TNFR1 levels in spontaneous abortion compared with normal pregnancy in the first trimester. These contradictory results are partly explained by the use of different assay methods and inclusion criteria of the patients into the study or control groups who may have unknown subclinical immunologic abnormalities (i.e. antiphospholipid antibodies, systemic lupus erythemathosus). We hope that it is possible to explain these unusual changes by specific mechanisms of pregnancy.

Although the causes leading to miscarriage have not been fully defined, the pro-inflammatory cytokine TNF-α and its receptor 1 are associated with the pathogenesis of miscarriage. Thus, we hypothesise that polymorphism in the gene encoding TNFR1 could confer genetic susceptibility to miscarriage. In the cohort of subjects examined in the present case-control study, we observed association of TNFR1 −383 and +36 single nucleotide polymorphisms (SNPs) with ERSM. To our knowledge, this is the first genetic association study on the role of TNFR1 gene polymorphisms in ERSM women, although the lack of differences between non-pregnant ERSM women and normal non-pregnant women regarding TNFR1 gene polymorphism was observed here. Previous studies have demonstrated that mutations in the extra-cellular domain of TNFR1 lead to increased receptor shedding and surface expression, and were associated with the phenotype of inherited periodic fever syndromes (McDermott et al., 1999). The investigated polymorphism in the first exon of the TNFR1 gene does not affect the amino acid sequence of the TNFR1 protein. Alternatively, there is evidence that even if a polymorphism in a coding region does not result in an amino acid change, or if it is not in a coding sequence, it can still affect gene function by altering the stability, splicing, or localisation of the mRNA (Cartegni et al., 2002). The association between TNFR1+36 polymorphism and susceptibility to Crohn's disease has been defined. The different phenotypical expressions for Crohn's disease depend on the polymorphism (Waschke et al., 2005). We excluded the role of the −383 and +36 polymorphism of the TNFR1 gene as a major susceptibility factor for ERSM in Chinese women, and TNFR1 expression was increased in women with ERSM. Functional data and further genetic mapping will be important to fully understand the importance of TNFR1 gene mutations in the pathogenesis of ERSM.

The A–C substitution in the promoter region of the TNFR1 gene seems to be rare in Caucasians: the frequency of the C allele was shown to be 0.7% in 152 healthy Caucasians (Pitts et al., 1998b). In present studies of Chinese women, the frequency of the C allele was 7.25% in controls, and 11.17% in ESM women, suggesting the presence of ethnic differences.

In summary, our data does not provide evidence that TNFR1 gene polymorphism is etiologically important for ERSM in Chinese women; we found significantly raised sTNFR1 levels in non-pregnant ERSM women compared with those with normal pregnancies. Our result suggests that pregnancy failure is associated with an increase of sTNFR1. We cannot conclude, however, if an elevated sTNFR1 level is causative or a result of the abortion process in women with ERSM. A further study including patients with a good and bad outcome is required to clarify if the maternal serum sTNFR1 level in patients with threatened abortion has a prognostic value in prediction of the pregnancy outcome.

Acknowledgements

We thank Dr Xiang Wang and Ping Zhou from the Centre of Medical Molecular of First Affiliated Hospital, Xi'an Jiaotong University Medical School, for their kind help with the collaborative trials.

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Received 9 May 2007/3 June 2007; accepted 6 June 2007

doi:10.1016/j.cellbi.2007.06.005


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)