In this study, pregnant women’s levels of RCF, vitamin B12 and tHcy were measured generally in early second trimester pregnancy. This was in a time period when exposure to vitamin supplementation or fortification was rare. Mothers of children with birth defects did not have lower RCF or B12 levels or higher tHcy levels than mothers of unaffected children. Significantly higher B12 concentrations were found in mothers of cases with cleft palate only, all musculoskeletal defects and musculoskeletal malformations. Significantly higher B12 concentrations were also found in the combined group of midline defects. These findings may be due to chance given the number of comparisons that were made. They are of interest because low B12 has been shown to be an independent risk factor for NTDs (Molloy et al. 2009
). Our results strongly suggest that low B12 is unlikely to be an important risk factor for defects other than NTDs. This is particularly noteworthy in the case of oral clefts where folate has been suggested to play a role because it suggests that the effect of folate, if it is a factor, is not mediated by the methylation pathway in which folate and B12 interact.
Two main studies examined the role of multivitamins containing folic acid in reducing the risk of all birth defects excluding NTDs and both suggested a significant reduction with prenatal vitamin supplementation (Botto et al. 2004
; Czeizel 1998
). Our findings do not support this.
Many studies have investigated a role for folic acid on its own or as part of a multivitamin preparation in the possible prevention of individual congenital malformations other than NTDs but results have been inconsistent (Botto et al. 2000
; Bower et al. 2006
; Canfield et al. 2005
; Czeizel 1998
; Hayes et al. 1996
; Shaw et al. 2000
; Shaw et al. 1995a
; Shaw et al. 1995b
). Among the studies that examined vitamin supplement use and the prevention of individual defects at least four showed a significant reduction in orofacial clefts in vitamin supplement users; but all examined multivitamin supplements that included folic acid in doses ranging from 0.2 mg to 10 mg.(Czeizel et al. 1996
; Itikala et al. 2001
; Shaw et al. 1995a
; Tolarova and Harris 1995
). Johnson and Little (2008)
conducted a systematic review and meta-analysis to assemble evidence on the role of folate in the aetiology of orofacial clefts. This review of 22 studies included some of the studies listed above. They concluded that there is no strong evidence for an association between oral clefts and folic acid alone but that multivitamins may protect against oral clefts. Furthermore, in contrast to the effect on NTD prevalence, the fortification programme in Canada between 1998 and 2000 had no effect on the rate of orofacial clefts (Ray et al. 2003
In studies showing a risk reduction for congenital heart defects, urinary tract defects and limb deficiency defects, the periconceptional exposure was also with a multivitamin but, with the exception of the Hungarian randomized controlled trial which used 0.8 mg, the dose of folic acid was not reported (Botto et al. 2000
; Czeizel 1998
; Li et al. 1995
; Werler et al. 1999
; Yang et al. 1997
). A recent study which looked at severe congenital heart defects in Canada pre and post fortification of grain with folic acid, found that there was a significant decrease in birth prevalence of both conotruncal and non-conotruncal defects post fortification (Ionescu-Ittu et al. 2009
Contradictory findings on vitamin use with regard to the prevention of specific birth defects could occur for many reasons including differences in dose, constituents, frequency and timing of consumption of the vitamin supplementation. This is not an issue in the present study as we measured maternal blood levels in early pregnancy in the era before periconceptional vitamin supplementation was recommended. Our study shows that low maternal folate or B12 levels or elevated tHcy were not associated with the occurrence of congenital defects other than NTDs.
The strengths of this study are as follows: it is an unselected cohort relating to children born with congenital defects; our study samples were taken from a population at a time when women were not exposed to periconceptional vitamin supplementation; there was no termination of pregnancy and control subjects were matched by gestational, temporal and storage variables likely to affect the metabolite levels of cases and controls. Limitations of the study include: even in a large prospective study like this, sufficient numbers of cases were not available for all types of birth defects limiting our power to investigate some defects. We have a limited amount of data on the defects and no data were available on risk factors for birth defects including family history, maternal medical history and demographic information.
The link between low maternal folate levels and NTDs has been clearly established and our research group and others have also found NTD pregnancies to be associated with low maternal levels of B12 independent of folate status (Molloy et al. 2009
). This is not surprising given the critical role B12 plays in folate metabolism. Folate and B12 status are important determinants of plasma homocysteine and raised total homocysteine levels in maternal blood and amniotic fluid are associated with an increased risk of NTD pregnancies (Mills et al. 1995
; Steegers-Theunissen et al. 1995
). Thus, it is important to determine whether B12 might play a role in the etiology of other malformations. Our data indicate that B12 levels were not lower in women whose conceptuses had other types of defects. In fact they were significantly higher for some defects. Even if we account for the non-independence of our defect groups in our statistical correction for multiple comparisons, our findings for B12 would no longer be significant. On the other hand, it is worth noting that all of the unadjusted positive findings occurred in the B12 analysis and all showed an increased risk with higher B12. This was not an a priori hypothesis but the finding that higher B12 levels were associated with increased malformation rates is important and requires investigation. There is a possibility that the serum B12 concentration may be a surrogate marker for a diet that is rich in animal food products that also contain high concentrations of an unidentified risk factor. The higher serum B12 levels in cases could also be the result of genetic factors that impair transport of B12 into tissues. Thus further investigation is needed especially in light of suggestions that B12 be added to fortification programmes.
We found no studies that measured vitamin levels during pregnancies affected by malformations other than NTDs for comparison with our results. Our findings show that within the physiological range of folate and homocysteine there was no association between levels and malformation risk. This does not, however, preclude the possibility that raising folate levels to the pharmacologic range could prevent some malformations as the effect could well be dose-dependent. Overall this study suggests that fortification with folic acid or vitamin B12 may not have a major beneficial effect in the prevention of congenital malformations other than NTDs.