REPRODUCTION RESEARCH Associations between tumor necrosis factor-a and lymphotoxin-a polymorphisms and idiopathic recurrent miscarriage W Zammiti, N Mtiraoui, H Khairi 1 , J-C Gris 2 , W Y Almawi 3 and T Mahjoub Research Unit of Haematological and Autoimmune Diseases, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia, 1 CHU Frahat Hached, Sousse, Tunisia, 2 Faculty of Biological and Pharmaceutical Sciences, Montpellier-1 University, Montpellier, F- 30029 France and 3 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@agu.edu.bh Abstract Heightened expression of tumor necrosis factor (TNF)-a and lymphotoxin-a (LT-a) was associated with pregnancy complications, including idiopathic recurrent miscarriage (RM). Whereas TNF-a and LT-a gene polymorphisms affect serum cytokine concentrations, their contribution to RM is controversial. The single nucleotide polymorphisms (SNPs) TNF-a (K238G/A, K308G/A) and LT-a (C252A/G) were investigated in 350 RM women and 200 control women. Higher frequency of the TNF-a K238A, but not the TNF-a K308A or the LT-aC 252G, allele was seen in patients, with comparable frequencies of TNF-a K238G/A, TNF-a K308G/A, and LT-aC252A/G genotypes seen between both groups, except for TNF-a K238G/G, which was lower in patients. Regression analysis confirmed the association of the TNF-a K238G/A SNP with idiopathic RM, and both TNF-a K308A/TNF K238G/LT-aC252G and TNF-a K308G/TNF-a K238A/LT-aC252G haplotypes played a susceptible role in idiopathic RM. TNF-a K238G/A and K238A/A, and LT-aC252G/G genotypes were positively associated only with exclusively early RM. This supports the concept of the association of TNF-a (K238G/A) and LT-a (C252A/G) polymorphic variants in idiopathic RM. Reproduction (2008) 135 397–403 Introduction Spontaneous miscarriage is a frequent common compli- cation 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 impli- cated 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 under- lying 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-a) or lymphocytes (TNF-b or lymphotoxin-a, LT-a; Hehlgans & Pfeffer 2005). TNF-a and LT-a 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-a and LT-a 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-a and LT-a, 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-a was also described to facilitate miscarriage indirectly by activating NK cells or macrophages (Raghupathy et al. 2000). Several poly- morphic gene variants of TNF-a and LT-a were described, including the TNF-a K238G/A and K308G/A promoter q 2008 Society for Reproduction and Fertility DOI: 10.1530/REP-07-0322 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org