Results
General characteristics, lifestyle, biomarkers as folate, homocysteine and vitamin concentrations and foetal outcomes, of case mothers and control mothers were summarized in Table 1 . The interval after index pregnancy, BMI, maternal COD incidence, ethnicity, use of alcohol, smoking and folic acid supplement use were comparable between both groups. Case mothers had a significantly higher age (p=0.009) with a mean age of 31,8 years in case mothers and 30,9 years in control mothers. A positive family history for COD was more common in case mothers. A significant difference was found in education level between cases and controls, where a low and high education level was more common in the case mothers. Homocysteine and folate concentrations were comparable between the groups. Birth weight of offspring was significantly lower in the group of case mothers compared to control mothers. Preterm birth was significantly more common in case mothers. The COD phenotypes of case children (n = 306) comprised aortic valve stenosis (n = 7), atrioventricular septal defect (n = 29), perimembranous ventricular septal defect (n = 84), pulmonary valve stenosis (n=42), coarctation of the aorta (n = 33), hypoplastic left heart syndrome (n = 12), transposition of the great arteries (n = 50), tetralogy of Fallot (n = 38) and others (n = 11). Two hundred thirty four of the 306 cases were defined as isolated COD cases. Seventy three cases were non-isolated and had another major structural congenital anomaly besides COD. Forty two of the 72 non-isolated cases had a genetic syndrome, i.e. trisomy (n = 27), deletion 22q11 (n=5), Noonan syndrome (n=4), Beckwith-Wiedemann syndrome (n=1), CHARGE syndrome (n=1), Saethre-Chotzen syndrome (n=1), Alagille syndrome (n=1), Kartagener syndrome (n=1) and Turner syndrome (n=1). The distribution of maternal TL (T/S ratio) among the controls, COD cases and VSD cases were depicted in Figure 2. Maternal age revealed a significantly inverse correlation with TL (R = -0.13; p=<0.001). There was no correlation between the homocysteine concentrations in plasma and TL (R = 0.037; p=0.32) (Figure S1 ). A positive family history for COD was not significantly associated with TL. A multivariable logistic model was used to determine the independent association of maternal TL with COD risk in offspring (Table 2A ). Model 1 and model 2 showed no significant associations between shorter maternal TL, per decrease in standard deviation of TL, and the risk of COD offspring (crude OR 1.10 (95%CI 0.95-1.27), p= 0.22). Model 1: adjOR 1.07 (95%CI 0.92-1.24), p=0.37 and Model 2: adjOR 1.07 (95%CI 0.92-1.24), p=0,40. The syndromal and non-syndromal COD group showed also a comparable non-significant association, crude OR 1.15 (95%CI 0.84-1.58), p=0.36 and crude OR 1.09 (95%CI 0.93-1.27), p=0.28, respectively. In addition, we analysed the association between TL and VSD (n = 113), and demonstrated that a shorter maternal TL is associated with a significantly increased risk of VSD in their offspring (crude OR 1.29 (95% CI 1.04-1.61), p=0.02, per standard deviation TL decrease). In Model 1 the association remained significant (adjOR 1.25 (95% CI 1.01-1.55), p=0.04)(Table 2B ). In Model 2 the association slightly attenuated (adjOR 1.24 (95% CI 1.00-1.55). After stratification, the syndromal VSD group showed no association between shorter maternal TL and the risk of VSD in offspring (crude OR 1.28 (95% CI 0.88-1.89), p=0.20). Whereas, the non-syndromal VSD group showed a significant association between shorter maternal TL and the risk of VSD in offspring (crude OR 1.29 (95% CI 1.02-1.66), p=0.04). In model 1 and model 2 this association remained, albeit not significant (adjOR 1.26 (95% CI 0.99-1.62), p= 0.06 and adjOR 1.28 (95% CI 1.00 -1.64), respectively)(Table 2B ). Adjustment of the associations for homocysteine concentrations did not substantially affect the association between TL and the risk of COD and VSD offspring.