We also explored the phenotypes anencephaly and spina bifida separately for effects associated with lower and upper quartiles of total choline. Results were similar in magnitude to those for all NTDs combined, but precision was compromised owing to restricted sample size. For anencephaly, the odds ratios for lower and upper quartiles (relative to the 25th–74th percentile) were 1.8 (0.96–3.4) and 0.34 (0.11–1.0). For spina bifida, the odds ratios were 1.8 (0.80–4.0) and 0.60 (0.19–1.8).
We used prospectively collected samples to examine potential associations between several serum nutrients related to one-carbon metabolism and NTD risk. We found no difference in midpregnancy serum folate levels between pregnancies with NTDs and those with no structural malformation. This absence of an association with serum folate might be expected given that women in this study were from a population whose food supply was fortified with folic acid. It is also likely that most women took prenatal supplements containing folic acid as well as other nutrients at the time of serum sampling. However, we have no information on dietary and supplement intake.
We found a strong linear association of total choline with decreased NTD risk. Choline, known primarily in the diet as a component of lecithin, is key to several metabolic processes. Like folate, choline is involved in one-carbon metabolism, it is used for the synthesis of cell membrane phospholipids, and it is a precursor of the neurotransmitter, acetylcholine.20–25 The demand for choline is thought to be higher during pregnancy.24 Our observed association with choline is unlikely to be explained by differential use of prenatal vitamin supplements between case and control mothers because choline is not a typical component of multivitamin supplements.
The findings with choline are supported by previous epidemiologic data indicating that choline intake may be associated with NTD risk.10 Our findings are also supported by known biologic underpinnings. Choline, folate, and methionine are highly interrelated in one-carbon metabolism, and an alteration in one affects the others.22 Thus, choline deficiency could affect folate and homocysteine metabolism.25 Methylation of DNA can be influenced by dietary contributions of methyl donors such as choline, folate, and methionine. A less than optimal methyl-donor supply and DNA methylation has been a suggested area for research efforts for certain human birth defects26 and disruption of embryonic methylation has been demonstrated in experimental systems to be linked to NTDs.27 However, findings from one experimental study of cultured mouse embryos casts some doubt on whether choline or betaine serve as methyl donors during neurulation, in that betaine homocysteine methyltransferase was not expressed until neurulation was almost complete.28
Alternatively, choline may affect growth in early embryogenesis because it is a precursor to phosphatidylcholine, a major component of cell membranes. It has been observed that inhibiting choline uptake and metabolism in mouse embryos results in NTDs29 and that knockouts in genes important for mediating choline to phosphatidylcholine conversion result in early embryonal lethality.30,31 A plausible mechanism could be the following: phosphatidylcholine is needed for cell membrane assembly; cell membrane assembly is in critical demand in a developing fetus; nearly all choline uptake by the embryo is converted to phosphatidylcholine; and embryos at the time of neurulation cannot de novo synthesize phosphatidylcholine because the enzyme phosphatidylethanolamine N-methyl transferase is not active at this stage of development.28 Thus, the latter component places the developing embryo reliant on uptake of choline from the mother.
Another possible mechanistic role mediated by choline for improper neural tube closure may be via apoptosis.25 Regulation of apoptosis is important to the development of the neural tube.32 As an alternative to these proposed mechanisms for choline, it has been observed in mouse model systems that degeneration of exposed embryonic neural tissue releases both neuronal and glial proteins into amniotic fluid and that these proteins increase as gestation proceeds.33 Thus, it is possible that a degenerating neural tube requires increased membrane synthesis owing to a repair response with a consequent reduction in maternal circulating choline levels. We are unable, however, to address this theoretical possibility further in our data.
We know of no previous studies that have directly assessed choline levels in NTD-affected pregnancies. Previous studies have investigated other analytes investigated here, including homocysteine,34–36 folate,3,4,12–16,36 methionine,34 methylmalonic acid,37 vitamin B12,6,12–16,38 and vitamin B6.34 In general, studies have observed elevated NTD risks associated with elevated levels of homocysteine, lowered levels of serum folate, and lowered levels of vitamin B12. Direct comparisons between our results and earlier research is complicated by several factors, including: (1) design differences such as sample collection during pregnancy versus postdelivery, sometimes years postdelivery; (2) variations in vitamin supplement content and use by the studied populations; (3) underlying dietary folic acid fortification differences in study populations; and (4) differences in specificity and sensitivity of analytes measured. For example, Ray and colleagues38 recently observed that low maternal holotranscobalamin was associated with increased risks of NTDs.
Even though the samples in our study were collected during pregnancy, they were nonetheless collected on average 12 weeks after closure of the neural tube. If the resulting error in measurement biases our results, it is likely to result in an underestimate of measured effects. A second limitation is potential degradation of analytes between collection and analysis. Folate may degrade when frozen at higher temperatures (–20°C) than were used for samples in this study (–80°C).39 Such degradation would likely be nondifferential to case and control status and therefore tend to underestimate real effects. Moreover, the average length of time between collection and frozen storage was similar between cases and controls. We explored whether even small differences between cases and controls influenced observed effects with total choline. Analyses that incorporated length of time into models produced even stronger odds ratios. We conducted experiments to explore the stability of total choline at room temperature and found that this analyte was stable for at least 8 days at room temperature. Specimens analyzed in the current study were, on average, at room temperature for only 3 days (range 2–4 days). Other limitations include a lack of information on supplemental and dietary intake of nutrients, a relatively small sample size that reduced power in some comparisons, and the inability to investigate allelic variants of genes involved in the biosynthesis of these nutrients, eg, folate, choline, and B12. Additional study of genetic variants coupled with analyte measures could be informative owing to the known or suspected function of selected genes.
For more than 3 decades, evidence has accumulated to show that periconceptional nutrient intakes—particularly folate—lower risks of NTD-affected pregnancies. Although fortification of the US food supply with folic acid is associated with a decreased prevalence of NTDs,2 there is still a substantial population burden of these serious birth defects. Interestingly, Benevenga40 has recently posited that betaine, a metabolite of choline, should be considered along with folate as a dietary supplement to reduce NTD risk. Our results showing an association with serum levels of choline (the precursor of betaine) offers an additional clue toward understanding the complex etiologies of NTDs in the era of folic acid fortification of the food supply. This result needs to be replicated in other settings, potentially through a designed trial, before firmer inferences can be drawn or recommendations made about choline.
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