The rate of preterm delivery (PTD) remains high in the US at about 17.4% in blacks and 9.8% in whites.1 While previous epidemiological and clinical studies have identified a number of potential risk factors of PTD, the underlying biological mechanisms for these observed associations are poorly understood. Most cases of PTD occurring in the general population cannot readily be explained by any of the known or suspected risk factors.2 In the last two decades, various programmes undertaken specifically to prevent PTD have been largely unsuccessful.3,4 The few that are effective, including treatment of urinary tract infection, cerclage, and treatment of bacterial vaginosis in high-risk women, are not universally effective and apply to only a small percentage of women at risk of PTD.5 A large multicentre randomised clinical trial of treatment of asymptomatic bacterial vaginosis in pregnant women did not reduce PTD.6 Even in a low-income population with prevalent environmental risk factors, a randomised controlled trial of a PTD prevention programme did not significantly reduce the rate of PTD.7 It is generally agreed that the major obstacle to PTD prevention has been our incomplete understanding of its pathophysiology. This underlines the need for future research of PTD to go beyond the classical epidemiological approach and to look beyond traditional risk factors.
The current literature has provided strong evidence of a familial or intergenerational influence on PTD or low birthweight (LBW). A study from Scotland8 found that sisters of women who had delivered preterm LBW infants were more likely to have a preterm infant than the sisters of women who had delivered term growth-retarded infants. A Norwegian study9 suggests no significant association between mother and offspring preterm status. However, a US study showed an increased risk of PTD among women who themselves were born before 37 weeks’ gestation.10 A mother’s own birthweight is also an important determinant of her infant’s birthweight. Infants born to LBW mothers have lower mean birthweight and are more likely to be LBW than those born to normal birthweight mothers, even after accounting for other relevant maternal and infant covariates.9,11–13 A previous history of LBW or PTD is one of the most important risk factors for a subsequent PTD.14 It has also been shown that the risk of PTD increases substantially with the number of previous LBW or preterm infants. Bakketeig and coworkers15 showed that the risk of PTD (defined as < 36 weeks in their study) in the second pregnancy was 14.3% if the first birth was preterm and 28.1% for the third pregnancy if both prior births were preterm. The risk of recurrence did not appear to be affected by the presence of medical complications, the length of the interpregnancy interval, or fetal survival. Our group has demonstrated strong familial aggregation of LBW in both US white and black populations.16 The combined effects of the mother’s birthweight and that of the index child on the risk of PTD or IUGR in the siblings are either additive or interactive. Consistently, a recent study17 based on US white and black populations suggests that recurrence of PTD contributes a notable portion of all PTDs, especially at the shortest gestations. The strong familial or intergenerational influences on PTD or LBW may be attributed to environmental factors, genetic factors, or both.
In contrast to many studies that consider the relative contribution of both genetic and environmental factors to a number of human complex diseases such as cancer, obesity, diabetes, asthma and hypertension, few studies have examined genetic influences on PTD. We hypothesise that PTD is a highly heterogeneous complex entity determined by multiple genetic and environmental factors. As illustrated in Fig. 1, clinical and experimental evidence indicate that most PTDs may result from four pathogenic processes: (1) decidual-chorioamniotic inflammation caused by ascending genitourinary tract or systemic infection; (2) maternal-fetal hypothalamic-pituitary adrenal axis activation caused by stress; (3) uteroplacental vascular lesions caused by coagulopathy, hypertension, and vascular lesions; and (4) susceptibility to environmental toxins. Ultimately, the four pathways converge on final clinical presentations characterised as preterm labour, preterm premature rupture of the membranes (PPROM), or medical induction due to maternal or fetal health threat, all of which lead to PTD. The central hypothesis of our study is that the polymorphisms of candidate genes in the four pathogenic pathways of PTD, independently or interacting with environmental factors, are associated with PTD. We are conducting a molecular epidemiological study to examine the effects of important candidate genes and their potential interactions with environmental factors on PTD among 500 preterm trios including 500 preterm babies and their parents and 500 maternal age-matched term controls. The specific aims are: (1) to perform the transmission/disequilibrium test (TDT) on candidate genes thought to be important in the major biological pathways of PTD (see Table 1); and (2) to conduct a case-control analysis among 500 preterm cases and 500 term controls to examine gene–environment interactions. Major environmental, nutritional and social factors known or suspected to be associated with PTD will be assessed with respect to gene–environment interactions. In the next section, we review the epidemiological and clinical evidence, molecular biology/genetic studies, and major candidate genes for each pathway. We are aware of other potential pathogenic pathways, but their significance is less substantiated by the literature.
1. Decidual-chorioamniotic-inflammation pathway
Epidemiological and clinical evidence
There is increasing epidemiological and clinical evidence that amniochorionic-decidual infections play a role in PTD. The epidemiological profile of women at risk for PTD overlaps that of women at risk for acquiring sexually transmitted diseases (i.e. poor, young, minority, inner city, unmarried).18 Vaginal pathogens including N. gonorrhea, C. trachomatis, T. vaginalis, and Bacteroides spp., as well as asymptomatic bacteriuria, are found in greater frequency among women with PTD.19 Particularly striking are seven studies (two case-control and five cohort) which reported an increased risk of PTD in women with bacterial vaginosis,20–22 with relative risks ranging from 2.0 to 6.9. However, randomised clinical trials on the efficacy of antibiotic treatment of bacterial vaginosis to prolong the pregnancy have yielded mixed results.6,23–26 Systemic infections are also associated with PTD, including pyelonephritis,27 pneumonia,28 peritonitis29 and periodontal disease.30 These findings suggest that an infection even remote from the uterus can activate an inflammatory process that triggers a uteroplacental response, leading to PTD.
Inflammatory mediators and candidate genes
Pro-inflammatory cytokines (e.g. IL-1, IL-6, TNF) are mediators of inflammation produced by the macrophage/monocyte system in response to bacterial products. They are part of, and stimulate further, the cascade of signals that is the inflammatory response to infection.31–35 The amniotic fluid of patients with PTD and intra-amniotic fluid infections display detectable levels of bacterial-derived endotoxin and IL-1 and TNF.31–40 Levels of these cytokines also correlate with histological chorioamnionitis.33,41 IL-1 and TNF stimulate uterotonin expression including prostaglandin E2 (PGE2)37,38,42–44 and endothelin.45 PGE is a powerful stimulant of myometrial contractions. Clinically, systemic or local administration of PGE2 induces labour. A study of 68 women with preterm labour46 found that amniotic fluid concentrations of prostaglandin E2 were significantly greater in women with PTD and intra-amniotic infection than in women without infection. A more recent study showed an increase in prostaglandin bioavailability before onset of labour.47 The effect of IL-1 and TNF can be further amplified by IL-6, which is secreted by cultured decidual and chorionic cells in response to IL-1 and TNF.45,48,49 Activation of the cytokine network also enhances decidual, fetal membrane, and cervical ECM-degrading protease activity.50–52 The concerted effects of these proteases are efficient degradation of collagen, laminin, elastin, and fibronectin, which are crucial ECM components of the fetal membranes, decidua, and cervix.
In sum, the recent increase in knowledge about infection and PTD has shed new light and raised many questions.53 The inflammation pathways appear to be extremely complex and selection of candidate genes and understanding the contribution of any single gene can be a challenge. Our study focuses on genes that encode IL-1, IL-6, and TNF and examines whether maternal or fetal variant genotypes are associated with increased risk of amniochorionic-decidual infection and PTD.
2. Stress and activation of the hypothalamic-pituitary-adrenal (HPA) axis
Epidemiological and clinical evidence
Epidemiological factors commonly associated with maternal stress are also associated with PTD.54,55 The incidence of PTD is increased among unmarried and poor mothers,56 African-Americans even after controlling for socio-economic status,57,58 patients with major stressful events,54,59 patients with elevated psychological scores for anxiety,60 and those subjectively reporting increased stress and anxiety.61 A recent study demonstrated an inverse correlation between levels of psychosocial and physiological stress and cervical length.62 The link between fetal stress and PTD is suggested by the increase in placental vascular lesions and intrauterine growth retardation among patients delivering preterm without infections or overt pre-eclampsia.63,64
Stress mediators and candidate genes
When individuals are under internal and/or external stress, they undergo a cascade of neuroendocrine responses. Corticotropin-releasing hormone (CRH) is the major hypothalamic regulator of the mammalian stress response. In addition to expression in the central nervous system, CRH is also expressed by trophoblasts in placenta and chorion, as well as by amnion and decidual cells.65–71 Plasma CRH levels rise during the second half of pregnancy, peak during labour, and rapidly decline postpartum.72–75 It has been suggested that activation of the fetal HPA axis drives a CRH-mediated ‘placental clock’ that triggers the onset of parturition at term.11,72,73,76 Similar HPA axis-modulated pathways also appear to be capable of triggering stress-induced PTD. A few studies showed that maternal CRH levels rise precociously among women who deliver prematurely.73,77
Parturition appears to be induced by CRH in two pathways. CRH mediates pituitary adrenocorticotropin (ACTH) secretion. The latter enhances adrenal cortisol secretion.78,79 Moreover, hypothalamic-induced activation of the fetal HPA axis is associated with increase in fetal ACTH and cortisol.80 Thus, activation of the maternal or fetal HPA axis would lead to increased levels of cortisol which, in turn, would result in enhanced placental CRH production,81 which leads to enhancement of prostanoid production by isolated amnion, chorion, and decidual cells.68–71,81,82 Prostaglandins act as direct uterotonins, but also enhance myometrial receptivity by increasing oxytocin receptors83 and formation of gap junctions.84 Prostaglandins also elicit cervical change by enhancing ECM turnover.85 CRH also appears to induce parturition by stimulating the secretion of DHEAS from the fetal adrenal gland.76 DHEAS is the obligate precursor of placenta oestrone (E1), oestradiol (E2), and oestriol (E3).86 Oestrogens interact with myometrium to enhance gap junction (connexin 43) formation,87 oxytocin receptor,88 prostaglandin activity,89 myosin light chain kinases (MLCK) and calmodulin expression.90
In sum, current data suggest that both maternal and fetal stress with resultant activation of the HPA axis appears to be an important pathogenic pathway of PTD. The relative contribution of maternal and fetal genes in this pathway has not been evaluated. Our study chooses the gene encoding CRH and investigates both maternal and fetal CRH gene polymorphisms in relation to the risk of PTD. We are also interested in whether there are interactions between maternal CRH genotype and psychosocial stressors before and during pregnancy in relation to PTD.
3. Uteroplacental vasculopathy
Epidemiological and clinical evidence
The potential importance of a vascular pathway to PTD has recently been emphasised.91 Decidual haemorrhage presenting as vaginal bleeding in the first and subsequent trimesters is associated with a threefold increased adjusted relative risk for PTD due to preterm labour with intact membranes.92 Hager et al.93 observed that vaginal bleeding in more than one trimester carried the highest identifiable risk of PPROM with an odds ratio of 7.4. Ekwo et al.94 found an adjusted odds ratio of > 100 for PTD among women who experienced vaginal bleeding in more than one trimester when their previous pregnancy had been complicated by PPROM. When subchorionic haemorrhage is detected by ultrasound, the risk of PTD as well as stillbirth, miscarriage, and abruptio placentae are increased.95 The normal function of placental vessels depends on the balance of procoagulant and anticoagulant mechanisms for damage repair and maintenance of blood fluidity. Pregnancy induces marked changes in the coagulation system and may increase the risk of thromboembolic events, especially among pregnant women who have acquired or have genetic risk factors for thrombosis.96 Below we review one condition that may affect such risk.
Hyperhomocysteinaemia (HHC) and candidate genes
HHC is indicative of disrupted homocysteine metabolism. It occurs in the rare hereditary homocystinuria but more commonly results from a combination of vitamin B12 or folate deficiency and mutations in the gene encoding the enzyme methylenetereahydrofolate reductase (MTHFR). The missense mutation (C677T) of MTHFR gene has been associated with reduced MTHFR activity and modestly increased plasma homocysteine concentrations, particularly in persons with plasma folate levels below the median.97 Homozygosity for the MTHFR mutation is found in 10–20% of the population, but this mutation varies significantly in populations.98–100 A second common mutation in the MTHFR gene (A1298C) has recently been identified.101 A significant interaction appears to exist between the C677T and A1298C mutations.101 HHC has been associated with increased risk of thromboembolism102,103 and of coronary heart disease.104,105 Moreover, even mildly elevated plasma homocysteine (about 30% above normal controls) has been identified as an independent risk factor for numerous vascular disorders, including cerebrovascular,106 cardiovascular, and peripheral vascular disease.107 Studies have also shown that HHC-inducing interaction between MTHFR mutation and low folate intake accounts for a substantial portion of neural tube defects.108 Particularly pertinent to this study are recent reports linking HHC to increased risk of pre-eclampsia,97,109–111 recurrent miscarriage,112,113 and placental abruption or infarction.113,114
In sum, uteroplacental vasculopathy appears to be an important pathogenic pathway of PTD. HHC (as a result of MTHFR mutation and/or low folate intake) may be an important underlying condition. Our study investigates whether MTHFR gene polymorphisms affect the risk of uteroplacental vasculopathy and PTD, and assess potential interaction of MTHFR gene polymorphisms with low folate intake on the risk of uteroplacental vasculopathy and PTD. It is noted that genes encoding other metabolic enzymes may also affect HHC levels. For example, B12-dependent methionine synthase (MS), an enzyme that catalyses the remethylation of homocysteine to methionine, also plays an important role in the remethylation pathway of homocysteine.115 Thus, in addition to MTHFR gene, other homocysteine metabolism genes may be of future interest.
4. Genetic susceptibility to environmental toxins
Humans are exposed to a variety of reproductive toxicants. A growing body of evidence demonstrates an association between environmental and occupa-tional exposures and adverse reproductive outcomes. Exposures studied include cigarette smoking,116,117 caffeine consumption,118 pesticides119,120 and organic solvents and related compounds.121–126 Nevertheless, not all women who are exposed have adverse reproductive outcomes. It is speculated that the reproductive risk associated with exposure to endogenous or exogenous chemicals may be modified by genetic variation in metabolic detoxification activities.127 The metabolic detoxification process involves two parts: phase I, in which the original non-polar compound becomes polar and reactive, and phase II, in which the transformed polar compound is conjugated with certain endogenous functional groups such as glutathione, sulphate, glucuronide, and amino acids; thus, the end product becomes a stable hydrophilic compound that can easily be excreted.128 In humans, a significant proportion of these metabolic genes are polymorphic. As multiple alleles exist at loci encoding chemical-metabolising enzymes, the expression of different host susceptibility phenotypes may explain the considerable variability in pregnancy outcomes associated with environmental toxins. For example, the cytochrome P450 family serves as the major enzyme system in phase I metabolism. CYP1A1 is a well studied phase I enzyme, and its polymorphism has been associated with individual cancer susceptibility.129,130 The glutathione S-transferases (GSTT1 and GSTM1) are the major phase II enzymes. Our study of a Chinese population131 showed that the GSTT1 deletion genotype significantly modified the risk of increased sister chromatid exchange among workers exposed to benzene. In combined phase I and phase II enzyme disorders, a 40-fold increased risk of tobacco smoke-induced lung cancer was observed in individuals with susceptible CYP1A1 and GSTM1 genotypes,132,133 which suggests that phase I and phase II enzymes have a synergistic effect.
In summary, available data support the hypothesis that a woman’s reproductive risk is related to both her environmental exposures and her genetic susceptibility to adverse effects of these exposures. Our study will focus on nine metabolic genes known to lead to genetic differences in metabolic detoxification capacity: GSTM1, GSTT1, CYP1A1, CYP2D6, CYP2E1, NAT2, NQO1, ALDH2, and EPHX.