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Associations between tumor necrosis factor- and lymphotoxin- polymorphisms and idiopathic recurrent miscarriage

Research Unit of Haematological and Autoimmune Diseases, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia¹ CHU Frahat Hached, Sousse, Tunisia² Faculty of Biological and Pharmaceutical Sciences, Montpellier-1 University, Montpellier, F- 30029 France and³ Department of Medical Biochemistry, College of Medicine and Medical Sciences, Arabian Gulf University, PO Box 22979, Manama, Bahrain

Correspondence should be addressed to W Y Almawi; Email: wassim{at}agu.edu.bh

	Abstract

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Heightened expression of tumor necrosis factor (TNF)- and lymphotoxin- (LT-) was associated with pregnancy complications, including idiopathic recurrent miscarriage (RM). Whereas TNF- and LT- gene polymorphisms affect serum cytokine concentrations, their contribution to RM is controversial. The single nucleotide polymorphisms (SNPs) TNF- (–238G/A, –308G/A) and LT- (+252A/G) were investigated in 350 RM women and 200 control women. Higher frequency of the TNF- –238A, but not the TNF- –308A or the LT-+252G, allele was seen in patients, with comparable frequencies of TNF- –238G/A, TNF- –308G/A, and LT-+252A/G genotypes seen between both groups, except for TNF- –238G/G, which was lower in patients. Regression analysis confirmed the association of the TNF- –238G/A SNP with idiopathic RM, and both TNF- –308A/TNF –238G/LT-+252G and TNF- –308G/TNF- –238A/LT-+252G haplotypes played a susceptible role in idiopathic RM. TNF- –238G/A and –238A/A, and LT-+252G/G genotypes were positively associated only with exclusively early RM. This supports the concept of the association of TNF- (–238G/A) and LT- (+252A/G) polymorphic variants in idiopathic RM.

	Introduction

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Spontaneous miscarriage is a frequent common complication of pregnancy, with an estimated 1–3% of otherwise healthy women reportedly having experienced recurrent miscarriage (RM), defined as three or more consecutive pregnancy losses prior to the 20th week of gestation (Sierra & Stephenson 2006). While anatomic, autoimmune, endocrine, and chromosomal abnormalities were implicated in the etiology of RM, a significant number of RM cases remain idiopathic (Quenby et al. 2002). Previous studies suggested a central role of the T helper (Th)1–Th2 cytokine network in the positive or negative maintenance of pregnancy (Wegmann et al. 1993, Makhseed et al. 2001, Zhu et al. 2005). This was evidenced by the association of increased Th2 cytokines with successful pregnancy, while heightened Th1 activity was indicative of poor pregnancy outcome, both in experimental animals and in humans (Hill et al. 1995). In view of this interplay between (pro-inflammatory) Th1 and (anti-inflammatory) Th2 cells and their respective cytokines in pregnancy, dysregulated immunity stemming from altered Th1–Th2 balance was proposed as a potential mechanism underlying idiopathic RM in the face of unknown causes (Makhseed et al. 2001, Kwak-Kim et al. 2005).

Tumor necrosis factor (TNF) is a pleotropic cytokine mainly secreted by mononuclear phagocytes, natural killer (NK) cells, and antigen-stimulated T cells (TNF-) or lymphocytes (TNF-β or lymphotoxin-, LT-; Hehlgans & Pfeffer 2005). TNF- and LT- exert predominantly pro-inflammatory responses, including apoptosis, in many cell types by binding two distinct cell-surface receptors (TNFR1 and TNFR2; Hehlgans & Pfeffer 2005). Due to their pro-inflammatory and pro-apoptotic capacity, TNF- and LT- were described to mediate several aspects of pregnancy complications, including pre-eclampsia (Anim-Nyame et al. 2003), miscarriage (Babbage et al. 2001), and RM (Rezaei & Dabbagh 2002). Several mechanisms were proposed for the pro-abortogenic effects of TNF- and LT-, including trophoblast invasion and placentation (Kwak-Kim et al. 2005) and induction of the expression of pro-apoptotic genes in human fetal membranes (Garcia-Lloret et al. 2000), which in turn accelerates membrane degradation and thus increases the susceptibility to premature rupture (Fortunato et al. 2001). TNF- was also described to facilitate miscarriage indirectly by activating NK cells or macrophages (Raghupathy et al. 2000). Several polymorphic gene variants of TNF- and LT- were described, including the TNF- –238G/A and –308G/A promoter single nucleotide polymorphisms (SNPs), and the LT-+252A/G SNP (Wilson et al. 1997, Ozaki et al. 2002).

The incidence and frequencies of RM are controlled by genetic factors, and poor pregnancy outcome was demonstrated to be associated with a predominantly Th1 cytokine profile, which was controlled to a large extent by polymorphisms in cytokine genes (Prigoshin et al. 2004). In view of their effect on altered regulation of TNF synthesis, secretion, and activity (Wilson et al. 1997, Louis et al. 1998), TNF- and LT- gene variants were implicated in the pathogenesis of RM (Reid et al. 2001, Daher et al. 2003). Conflicting associations of these gene variants with RM were presented, highlighted by the association of these polymorphisms with RM (Reid et al. 2001, Daher et al. 2003, Costeas et al. 2004), and susceptibility to preterm birth (Engel et al. 2005), while others found no association with RM and related complications (Bates et al. 2002, Pietrowski et al. 2004). In view of the critical role of TNF- and LT- in the maintenance of pregnancy, we investigated the contribution of the TNF- –238G/A and –308G/A, and the LT-+252 A/G to the pathogenesis of RM in 350 women with confirmed RM and 200 multiparous women who served as controls.

	Results

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Study subjects
The demographics and clinical characteristics of the study subjects are summarized in Table 1. Both patients and controls had comparable age (P=0.27) and similar education backgrounds (P=0.134) and alcohol (P=1.00) or tobacco (P=0.35) consumption. Body mass index (BMI) values were higher in patients than in controls (25.8±4.0 vs 24.6±3.8, P=0.01); obese women, defined as having a BMI at least equal to 30 kg/m², were more frequent in patients than in controls (19.7% vs 9.5%, P=0.002). In addition, comparable geographical distribution was seen, with both patients and controls originating from different parts of Tunisia (P=0.385).

TNF- –308G/A, TNF- –238G/A, and LT-+252A/G genotype analysis

View this table: [in this window] [in a new window]   Table 2 Allele and genotype frequencies of tumor necrosis factor- (TNF-) and lymphotoxin- (LT-) polymorphisms.

TNF- –308G/A, TNF- –238G/A, and LT-+252A/G haplotype analysis

View this table: [in this window] [in a new window]   Table 4 Matched odds ratios for recurrent miscarriage (RM) for tumor necrosis factor- (TNF-)–lymphotoxin- (LT-) haplotypes.

Distribution of TNF- –308G/A, TNF- –238G/A, and LT-+252A/G genotypes

View this table: [in this window] [in a new window]   Table 5 Association of tumor necrosis factor- (TNF-)–lymphotoxin- (LT-) gene variants with the stage of pregnancy loss.

	Discussion

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RM is a multifactorial condition, and both acquired and inherited risk factors reportedly contributed to its pathogenesis. Cases were included if they had three or more RM of unknown etiology with the same partner, without any known personal or family history of venous and arterial thromboembolic events. As diabetes was suggested to be a potential complication of RM (Temple et al. 2002), all cases and control subjects were normoglycemic, evidenced by glycated hemoglobin (HbA1c) values 6.00. It was shown by several groups that the classic TORCH agents did not impart significant risk of RM (Paukku et al. 1999, Kavalier 2005), supported by the recent recommendation of The Royal College to abandon TORCH screening in the routine RM workout (Kavalier 2005). In spite of this we included only those negative for the standard TORCH agents, thereby ruling out any possible contribution of bacterial, viral, and toxoplasma infections to the pregnancy outcomes.

Our results were reminiscent of a previous study, in which the TNF- –238G/A, but not the –308G/A, SNP were associated with RM (Bertolaccini et al. 2001). This lack of association of the TNF- –308G/A SNP with increased RM risk was also reported for Caucasian (Baxter et al. 2001, Bertolaccini et al. 2001, Pietrowski et al. 2004, Prigoshin et al. 2004) and Iranian women (Kamali-Sarvestani et al. 2005), and in apparent partial disagreement with the study of Costeas et al. (2004). A recent meta-analysis (Daher et al. 2003) confirmed the lack of association of the TNF- –308G/A SNP with RM, and suggested instead that interferon- (874T/T) and IL-10 (–1082G/G) gene variants represented stronger RM inherited risk factors. While the differences in ethnic background between the study groups, in the selection of RM cases, and in the sample size cannot be overlooked, it should also be noted that the differences in cytokine production induced by gene polymorphisms is not always indicative of immune/inflammatory events that occur at the local maternofetal interface during pregnancies/miscarriage, as was suggested (Babbage et al. 2001), since the cytokines (which are generally short acting) exert their effects both in a paracrine and autocrine manner.

Increased prevalence of two of the TNF- –308G/A/TNF- –238G/A/LT-+252A/G haplotypes, –308A/–238G/+252G and –308G/–238A/+252G (out of the possible maximum of eight haplotypes), was seen among RM cases, after controlling for potentially confounding variables. This was in apparent disagreement with the study of Baxter et al. (2001) who failed to find an association between RM and TNF--LT- haplotypes. The differences may be reconciled by ethnic differences, patient selection, and the low number of subjects included in their study (76 cases and 69 controls, compared with the 350 RM cases and 200 controls analyzed here), which may have missed the detection of susceptible (and possibly protective) haplotypes.

A dominant role of the innate, rather than the adaptive, immune system in RM was proposed previously (Sacks et al. 1999, Borzychowski et al. 2005), highlighted by defective HLA expression, and the detection of large granular lymphocytes and monocytes cellular infiltrates in RM (Sacks et al. 1999, Babbage et al. 2001). Altered immunity in RM was dominated by the Th1/Th2 hypothesis, which proposes that the fetus escapes maternal-derived T-cell responses through skewing the Th0 differentiation toward Th2 pathway (Saito et al. 1999), which in turn dampens (harmful) Th1 immunity (Wegmann et al. 1993, Makhseed et al. 2001, Zhu et al. 2005). We propose here that normal pregnancy, and hence RM, should be viewed as inflammation state, in addition to the widely adopted altered Th1–Th2 (Saito et al. 1999), in which pro-inflammatory mediators and cells act locally at the implantation site, and also more systemically by amplifying the immune/inflammatory responses at the fetomaternal junction (Sargent et al. 2006). Insofar as TNF activates NK cells, which subsequently damage the placenta (Raghupathy 1997), it was interesting to find that high NK cell activities were seen prior to conception (Aoki et al. 1995), and early in gestation (Yamada et al. 2001), and that normal pregnancy was not the result of altered Th1/Th2 or Tc1/Tc2 cell ratios, but rather decreased NK1/NK2 and NKT1/NKT2 cell ratios (Borzychowski et al. 2005).

While this study clearly demonstrated an association between TNF- –238G/A and LT-+252A/G polymorphisms and idiopathic RM, several limitations to these findings are noteworthy. With regard to possible functional role of TNF- and LT- polymorphisms, varied cytokine gene polymorphism and cytokine production was noted between patient groups and healthy subjects (Koss et al. 2000), thereby necessitating the assessment of the relationship between gene polymorphisms and TNF-–LT- production in RM. In addition, the association of other genetic polymorphisms with RM, through LD with TNF-–LT- polymorphisms, should not be overlooked. Further study aiming at addressing these two points is required to characterize more precisely the role of the TNF system in RM.

	Materials and Methods

Top Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Study subjects
This was a retrospective case–control study, performed at the Maternity Center of Hôpital Farhat Hached of Sousse, Tunisia. Data collection procedures were the same for patients and control subjects. Cases comprised 350 fertile women with a history of three to six unexplained consecutive pregnancy losses with the same partner, which had occurred by clinical definition from the beginning of pregnancy to the end of the 30th week of gestation. Patients were referred from local hospitals and/or gynecology consultants to the Department of Obstetrics and Gynaecology at CHU Farhat Hached (Sousse, central Tunisia), for RM workup. The gestational age at the time of spontaneous abortion was calculated on a clinical basis and was defined as the time between the date of the first day of the last normal menstrual period and the date of the first clinical symptom related to pregnancy loss. Exclusion criteria included preclinical miscarriages, anatomical abnormalities, endocrine disorders including diabetes mellitus (HbA1c levels <6.00), autoimmune disease, arterial hypertension, liver function abnormalities, drug abuse, previously known systemic disease, and personal and/or a family history of thromboembolism. Additional exclusion criteria included abnormal thyroid function, thyroid antibodies (antithyroglobulin and antithyroid peroxidase antibodies) identified even in the absence of abnormal thyroid function, hyperprolactinemia prior to luteal phase defects (a normal luteal phase of at least 12 days and a plasma progesterone level above 25 ng/ml), erythroblastosis fetalis (Rh disease), immune thrombocytopenic purpura, and fetomaternal alloimmune thrombocytopenia. Chromosomal aberrations and Rh incompatibility were ruled out before inclusion in the study. In addition, all subjects included in the study were negative for the TORCH agents, Toxoplasma gondii, rubella, cytomegalovirus (CMV), herpes simplex viruses (HSV-1 and HSV-2), varicella zoster virus (VZV), and human immunodeficiency viruses (HIV-1 and HIV-2), by indirect ELISA. Transvaginal ultrasound examination was done to confirm spontaneous miscarriage (no heartbeat detection).

Pregnancy losses were classified as early (5–10 weeks) and late (11–30 weeks; Table 1). Control subjects comprised 200 healthy women (with a combined total of 1223 successful pregnancies) who were examined in the Outpatient Department of Gynaecology and Obstetrics of the University Hospital of Sousse, as a routine checkup following uncomplicated pregnancy. Controls were matched with patients according to a number of risk factors (smoking, alcohol consumption, oral contraceptive use); age distribution was comparable between RM cases (mean age 28.9±5.9 years) and normal fertile controls (mean age 28.4±3.8 years; Table 1). Patients’ and controls’ venous blood was collected from the study participants in EDTA tubes (for DNA extraction), and all subjects were required to sign an informed consent prior to entering the study, which was conducted after all institutional ethics requirements were met.

TNF- and LT- gene polymorphisms
Total genomic DNA was isolated from leukocyte-rich interphase layer of EDTA anticoagulated blood by the phenol–chloroform method, was dissolved in nuclease-free water, and stored at 4 °C pending assay. TNF- –308G/A and –238 G/A and LT-+252A/G gene polymorphism were determined by PCR restriction fragment length polymorphism analysis as described previously, using the following primers: TNF- –308G/A: sense, 5'-GAG GCA ATA GGT TTT GAG GGC CAT-3' and anti-sense, 5'-GGG ACA CAC AAG CAT CAA G-3'; TNF- –238G/A: sense, 5'-AAA CAG ACC ACA GAC CTG GTC-3' and anti-sense, 5'-CTC ACA CTC CCC ATC CTC CCG GAT C-3'; LT-+252A/G: sense, 5'-CTC CTG CAC CTG CTG CCT GGA TC and anti-sense, 5'-GAA GAG ACG TTC AGG TGG TGT CAT. Genotype determination was made following digestion with specific restriction endonuclease (BamHI for TNF- –238G/A and NcoI for TNF- –308G/A and LT-+252A/G).

Statistical analysis
Statistical analysis was performed on SPSS v. 13.0 statistics software (SPSS Inc., Chicago, IL, USA). Quantitative data were presented by their mean (±S.D). Pearson's ² and Fisher's exact tests were used to compare the allele and genotype frequencies. LD analysis, the non-random association between two loci, was calculated using the HLAStat 2000 software. Haplotype frequency was determined using the H-Plus haplotype estimation software (http://cougar.fhcrc.org/hplus/). Logistic regression analysis, performed in a monovariate then multivariate way, taking the control women as the reference group, was performed in order to determine the crude OR and 95% CI associated with the risk of recurrent idiopathic pregnancy loss. Statistical significance was set at P<0.05.

	Acknowledgements

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The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

Received 12 July 2007
First decision 9 November 2007
Accepted 9 November 2007

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