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Keywords:

  • high folate;
  • MTHFR;
  • developmental delay;
  • heart defects;
  • ventricular septal defects

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

BACKGROUND

The incidence of neural tube defects has diminished considerably since the implementation of food fortification with folic acid (FA). However, the impact of excess FA intake, particularly during pregnancy, requires investigation. In a recent study, we reported that a diet supplemented with 20-fold higher FA than the recommended intake for rodents had adverse effects on embryonic mouse development at embryonic days (E)10.5 and 14.5. In this report, we examined developmental outcomes in E14.5 embryos after administering a diet supplemented with 10-fold higher FA than recommended to pregnant mice with and without a mild deficiency of methylenetetrahydrofolate reductase (MTHFR).

METHODS

Pregnant mice with or without a deficiency in MTHFR were fed a control diet (recommended FA intake of 2 mg/kg diet for rodents) or an FA-supplemented diet (FASD; 10-fold higher than the recommended intake [20 mg/kg diet]). At E14.5, mice were examined for embryonic loss and growth retardation, and hearts were assessed for defects and for ventricular wall thickness.

RESULTS

Maternal FA supplementation was associated with embryonic loss, embryonic delays, a higher incidence of ventricular septal defects, and thinner left and right ventricular walls, compared to mothers fed control diet.

CONCLUSIONS

Our work suggests that even moderately high levels of FA supplementation may adversely affect fetal mouse development. Additional studies are warranted to evaluate the impact of high folate intake in pregnant women. Birth Defects Research (Part A), 2013. © 2012 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Maternal periconceptional supplementation with folic acid (FA) decreases the risk for neural tube defects (NTDs) in offspring (Czeizel and Dudás, 1992) and may decrease the risk for congenital heart defects (CHDs) (van Beynum et al., 2010). Embryonic development may also be compromised by disruptions in genes that encode enzymes in the folate metabolic pathway such as methylenetetrahydrofolate reductase (MTHFR) (Wenstrom et al., 2001; Li et al., 2005; Chan et al., 2010). MTHFR synthesizes 5-methyltetrahydrofolate, the primary form of circulatory folate. A polymorphism in MTHFR (677C[RIGHTWARDS ARROW]T) increases the risk for NTD and may increase the risk for CHD or other adverse embryonic outcomes (Junker et al., 2001; Zhu et al., 2006).

Although folate fortification has reduced the prevalence of NTDs in most countries with fortification programs (Blencowe et al., 2010; Lane, 2011), there is some concern regarding the potential undesirable consequences of high folate intake (Dary, 2009; Crider et al., 2011), particularly in individuals who also consume folate supplements (Houghton et al., 2011; Hursthouse et al., 2011). These concerns include increased tumor growth (Cole et al., 2007; Mason et al., 2007; Lawrance et al., 2009), although this has been refuted by other investigations (Gibson et al., 2011; Stevens et al., 2011) and perturbations in immune function (Troen et al., 2006; Halsted, 2008). There is also evidence that high maternal folate concentrations can be associated with greater adiposity and higher insulin resistance in the offspring, which could increase the risk for type 2 diabetes (Yajnik et al., 2008).

We have been studying the impact of genetic and nutritional perturbations in one-carbon metabolism on pregnancy outcomes in mice. In recent work, we reported that folate intake at 20-fold higher levels than the recommended amount was associated with adverse embryonic outcomes (Pickell et al., 2011). We found that folate supplementation was associated with embryonic delay and growth retardation at embryonic day (E)10.5 and E14.5, as well as thinner ventricular walls in embryonic hearts at E14.5. However, because the very high levels of folate in that study may not have accurately reflected the levels of folate intake relevant for human populations, we repeated the experiments at E14.5 using a diet that had 10-fold higher folate than the recommended level for rodents.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Animal experimentation was conducted according to the guidelines of the Canadian Council on Animal Care and approved by the Montreal Children's Hospital Animal Care Committee. Housing of mice, diets, and timed matings followed the same procedures as described previously (Pickell et al., 2011). Briefly, at weaning, BALB/c Mthfr+/+ or Mthfr+/− female mice were placed on an amino acid-defined control diet (TD.01369; Harlan Teklad, Indianapolis, IN) containing the recommended amount of FA for rodents, 2 mg/kg diet (Reeves et al., 1993), or a FA-supplemented diet (FASD; 20 mg/kg diet; TD.09258; Harlan Teklad) for 6 weeks before mating and throughout pregnancy. Both diets contained 2.5 g/kg choline bitrate, 3.3 g/kg L-methionine, and 1% succinylsulfathiazole, which was used to prevent folate production by intestinal flora. Female mice were mated with Mthfr+/− males and the presence of a vaginal plug the following morning was considered as 0.5 days post coitum. At the time of tissue collection, pregnant mice weighed approximately 25 grams. Based on general values for consumption and weight (Han et al., 2009; Marean et al., 2011), these mice would be ingesting 0.008 mg FA per day on a control diet and 0.08 mg FA per day on the FASD. At E14.5, mice were killed and the embryos and placentas were collected, examined, measured, and genotyped as previously described (Li et al., 2005; Pickell et al., 2009). Implantation sites, both discernible and necrotic, were counted and reported as implantation sites per litter and percent resorption is reported as embryonic loss. Developmental delay was assessed by examining the gross morphology of individual viable embryos and comparing them to normal embryos on a given gestational day based on markers of normal mouse development (Kaufman, 2001). An embryo was considered delayed if it did not exhibit the following E14.5 morphologic landmarks: complete separation of proximal digits, the presence of hair follicles, and discernible elbows. Embryos were examined histologically for heart defects and measured for ventricular wall thickness, as previously reported (Li et al., 2005; Pickell et al., 2009; Chan et al., 2010). Ventricular septal defects (VSDs) were defined by the presence of a gap separating the right and left ventricles of the embryonic heart, which was present on three or more serial sections of the heart. The size of the gap was then measured using AxioVision LE Image software (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA). Total plasma folate and homocysteine were measured as before (Pickell et al., 2011).

Statistical analysis was conducted with SPSS software version 20. ANOVA was performed to assess the effect of diet, genotype or a diet-genotype interaction and was followed by Bonferroni correction. Fisher's exact two-tailed test was used to assess diet or genotype effects when examining categorical data. Any p < 0.05 was considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Effects of Maternal High Folic Acid Intake and Mthfr Genotype on Maternal MetaboliteConcentrations

Maternal total plasma folate concentration increased approximately threefold in response to folate supplementation (Fig. 1A; p = 0.001, ANOVA), indicating that the diet was effective in raising folate levels. Plasma folate levels were modestly lower in Mthfr+/− compared to Mthfr+/+ mice with borderline significance (p = 0.08, ANOVA). We have previously observed significantly lower total plasma folate levels in Mthfr−/− mice (Ghandour et al., 2004). Mthfr deficiency also increased plasma total homocysteine concentration (Fig. 1B; p < 0.01, ANOVA), as expected. Folate supplementation did not affect these levels; this observation is similar to that observed in our previous study with 20-fold higher folate intake (Pickell et al., 2011).

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Figure 1. Maternal total plasma folate (A) and tHcy (B) concentrations in methylenetetrahydrofolate reductase (Mthfr)+/+ and Mthfr+/− mothers fed control diet and folic acid-supplemented diet (FASD), and left (C) and right (D) ventricular wall thickness in the embryos of these four maternal groups. A, B: Values represent mean ± SE, n = 5 per group. Folate supplementation increased total plasma folate (p = 0.001, ANOVA). Mthfr deficiency decreased total plasma folate with borderline significance (p = 0.08, ANOVA). Mthfr deficiency increased plasma tHcy concentration (p < 0.01, ANOVA). C, D: Values represent mean ± SE of the ventricular wall thickness of embryos of each Mthfr genotype, from Mthfr+/+ or Mthfr+/− mothers fed control diet or FASD (4 maternal groups; 7 litters from each group). Number of embryos examined = 29-38 Mthfr+/+, 43-45 Mthfr+/−, and 7-8 Mthfr−/− per dietary group. FASD decreased the thickness of the left ventricular wall (p < 0.001, ANOVA) and of the right ventricular wall (p < 0.01, ANOVA). CD, control diet; FASD, folic acid-supplemented diet; mMthfr, maternal genotype; eMthfr, embryonic genotype.

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Effects of Maternal High Folic Acid Intake and Mthfr Genotype on Reproductive Outcomes and Embryonic Heart Development

Eleven to 14 litters were examined for reproductive outcomes at E14.5 for each of the four groups: Mthfr+/+ or Mthfr+/− pregnant mice fed control diet or FASD. Mthfr genotype of viable embryos did not vary significantly from expected Mendelian ratios (45 Mthfr+/+ and 49 Mthfr+/− from control diet Mthfr+/+ dams; 26 Mthfr+/+, 37 Mthfr+/− and 15 Mthfr−/− from control diet Mthfr+/− dams; 44 Mthfr+/+ and 60 Mthfr+/− from FASD Mthfr+/+ dams; and 26 Mthfr+/+, 42 Mthfr+/− and 16 Mthfr−/− from FASD Mthfr+/− dams). Mothers fed FASD during pregnancy had a higher number of implantation sites compared withmothers fed control diet (Table 1; p < 0.05, ANOVA).

Table 1. Effects of Control Diet and FASD, with or without Maternal MTHFR Deficiency, on E14.5 Reproductive Outcomes
Maternal dietControl dietFASD
  • a

    Dietary effect on number of implantation sites, p < 0.05, ANOVA.

  • b

    Dietary effect on embryonic loss, p < 0.01, ANOVA.

  • c

    Genotype effect on number of embryos with delay in control diet mothers, p < 0.05, Fisher's exact test.

  • d

    Dietary effect on number of embryos with delay in Mthfr+/+ mothers, p < 0.05, Fisher's exact test.

  • e

    Dietary effect on number of embryos with VSD, p < 0.05, Fisher's exact test.

  • FASD, folic acid-supplemented diet; MTHFR, methylenetetrahydrofolate reductase; E, embryonic day; VSD, ventricular septal defect.

Maternal genotype Mthfr+/+ Mthfr+/− Mthfr+/+ Mthfr+/−
Total litters examined13111414
Average number of implantation sitesa8.23 ± 0.67.7 ± 0.69.5 ± 0.48.5 ± 0.4
Total number of viable embryos947810484
Average number of viable embryos per litter7.23 ± 0.67.09 ± 0.67.42 ± 0.66.0 ± 0.4
Embryonic loss (%)b    
 Number of embryos resorbed/number of  implantation sites per litter × 10013.15 ± 2.57.8 ± 2.522.78 ± 5.127.28 ± 5.5
Embryonic delay    
 Number of delayed embryos4 (3 litters)11c(4 litters)15d (7 litters)12 (6 litters)
Embryonic crown-rump length (mm)10.92 ± 0.110.73 ± 0.1310.63 ± 0.0910.71 ± 0.14
Embryonic weight (mg)200.8 ± 4.5190 ± 8.2185 ± 4.0193.6 ± 7.6
Placental weight (mg)92.3 ± 1.696.4 ± 3.891.4 ± 2.395.7 ± 3.3
Embryonic VSDe    
 Number of embryos with VSD4 (4 litters)4 (2 litters)8 (4 litters)9 (4 litters)
 Number of embryos sectioned45 (7 litters)45 (7 litters)41 (7 litters)39 (7 litters)

FASD was associated with a higher incidence of embryonic loss (p < 0.01, ANOVA). This diet also increased the number of delayed embryos in Mthfr+/+ mothers (p < 0.05, Fisher's exact test). Mthfr+/− mothers fed control diet also had higher numbers of delayed embryos compared to wild-type mothers on this diet (p < 0.05, Fisher's exact test). No diet-genotype interactions were observed for any of the parameters examined.

All the embryos from seven litters, chosen at random from each of the four maternal groups, were examined for CHDs and ventricular wall thickness. Embryos from mothers fed FASD had a significantly higher number of VSDs (17 of 80 embryos) compared with those from mothers fed control diet (8 of 90 embryos) (Table 1; p < 0.05, Fisher's exact test). The majority of defects were membranous VSD with gaps ranging in size from 10 to 80 μm in diameter (∼40 μm on average). Mean gap size was not significantly different between mice on control diet and FASD (data not shown). Embryonic genotype did not seem to influence VSD incidence because embryos with defects were observed in Mthfr+/+, Mthfr+/−, and Mthfr−/− embryonic groups (3 Mthfr+/+, 3 Mthfr+/−, and 2 Mthfr−/− with VSD from control diet dams; and 4 Mthfr+/+, 11 Mthfr+/−, and 2 Mthfr−/− with VSD from FASD dams). However, the number of embryos with defects was too small to assess statistical significance.

Among the total of 42 embryos from control diet (n = 15) and FASD (n = 27) dams that showed developmental delay, 12 embryos also displayed VSDs, whereas the other 30 did not. The total number of Mthfr−/− embryos obtained from the matings was 31, but only five of these Mthfr−/− embryos were developmentally delayed; however, four of these five delayed embryos also had VSDs.

Left and right ventricular wall thickness was measured in Mthfr+/+, Mthfr+/−, and Mthfr−/− embryos from Mthfr+/+ and Mthfr+/− mothers fed control diet and FASD. All embryos from seven litters of each of the four maternal groups were examined. Embryos from mothers fed FASD had significantly thinner walls in both left (Fig. 1C; p < 0.001, ANOVA) and right (Fig. 1D; p < 0.01, ANOVA) ventricles compared with embryos from mothers fed control diet. Analysis without Mthfr−/− embryos did not affect statistical significance.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Concern has been expressed in recent years regarding the potential adverse consequences of high folate intake, resulting from food fortification with FA and/or vitamin supplementation (Crider et al., 2011). In a recent study (Pickell et al., 2011), we demonstrated that high intake of folate disrupted mouse embryonic development when we fed pregnant mice a diet supplemented with 20-fold higher levels of folate (40 mg/kg diet) than the recommended levels for rodents. In this study, to assess a lower level of folate supplementation, we administered a diet supplemented with 10-fold higher than recommended levels of folate (20 mg/kg diet), to Mthfr+/+ or Mthfr+/− pregnant mice and examined E14.5 embryonic development and heart defects. The human equivalent of this dose would be 4 mg/day FA because the recommended intake is 400 μg/day.

Supplementation with FASD resulted in ∼threefold higher concentrations of total plasma folate compared to control diet-fed mothers, indicating that the diet was effective. After food fortification with FA, serum folate levels have risen approximately 2.5-fold in the U.S. population (Pfeiffer et al., 2012) and 30% in Australia (Brown et al., 2011). Consequently, the increase in blood folate in our mice is comparable to the increase in human populations, although the actual amounts of FA supplied to mice in this study are much higher than those in human diets. The daily recommended intake of FA for mice (0.4 mg/kg body weight) is ∼70-fold higher than the recommended intake for humans (0.006 mg/kg body weight). Rodents may be able to handle the higher doses of FA better than humans because dihydrofolate reductase activity is considerably higher in rats compared to humans (Bailey and Ayling, 2009). Plasma total homocysteine levels were not affected by diet in this study or in our previous study with even higher folate (Pickell et al., 2011). They may have reached a plateau as suggested by human studies with higher plasma folate (Refsum et al., 2006). These findings are noteworthy because they suggest that plasma homocysteine is not a good biomarker for the high FA intake that could be associated with adverse reproductive outcomes.

The 10-fold higher FASD in this study increased the incidence of embryonic loss and embryonic delay, and affected heart development (increased VSD and decreased ventricular wall thickness). The negative impact of increased folate intake on reproductive outcomes had also been observed with the 20-fold higher FASD. We found no evidence for embryonic Mthfr genotype effects on developmental outcomes; this is consistent with the evidence that on E14.5, the embryo depends on maternal 5-methyltetrahydrofolate because it is the primary source of folate that can cross the placenta (Henderson et al., 1995). VSD incidence in embryos from Mthfr+/+ dams was ∼ two-fold higher in mice receiving the 20 mg/kg FASD (8 of 41) compared to mice on control diet (4 of 45) in this study, whereas it was ∼ five-fold higher in mice receiving the 40 mg/kg FASD (7 of 65) compared to mice on control diet (1 of 45) in our previous study (Pickell et al., 2011). However, there was no statistically significant difference in the frequency of VSDs between the two studies (p = 0.26, Fisher's exact test).

Although we had previously shown that some adverse outcomes could be ameliorated in Mthfr+/− dams fed 20-fold higher FASD, we observed no such interactions between maternal Mthfr genotype and 10-fold higher FASD for any of the outcomes examined in this study. This is likely due to the extremely high folate levels used in the previous study, which could suggest that very high FA supplementation may be less harmful to individuals with certain genetic variants.

We also note that although we had previously observed increased VSD incidence in Mthfr+/− mothers fed control diet, we did not detect a similar genotype effect on VSD in this study. We did, however, observe a maternal Mthfr genotype effect on embryonic delay in control diet mice, suggesting that Mthfr deficiency can manifest itself in multiple ways. Adverse outcomes could thus encompass resorption, developmental delay, or heart malformations and NTDs, among other anomalies. The common finding of developmental delays due to Mthfr deficiency, or to low dietary folate, may be a critical factor that contributes to the other phenotypes. In particular, a delay in overall development could result in the failure of cardiac neural crest cells to migrate in a timely fashion during the formation of the septum and thereby result in VSDs (Waldo et al., 1998; Hutson and Kirby, 2007). As mentioned above, four of the five Mthfr−/− embryos that were developmentally delayed also showed a VSD; on the other hand, the majority of developmentally delayed mice with other genotypes did not have a VSD.

Although 70% of small VSDs in human infants usually resolve on their own during the first 2 years of life, large VSDs are known to cause failure to thrive, especially if compounded by other anomalies (Turner et al., 2002; Rudolph, 2009). Similarly, in rodents, small VSDs can resolve whereas the closure of larger VSDs may be less common (Fleeman et al., 2004). It would be of interest to conduct a study in our mouse model postnatally to determine whether the VSD or the thinner ventricular walls are present at birth or whether they can resolve prenatally or in the early postnatal period.

Our findings suggest that, in mice, even a diet supplemented with only 10-fold higher than recommended levels of intake may have some detrimental effects on development. A study examining the effect of FA supplementation in different mouse models of NTDs showed that FA supplementation with 10 mg/kg diet could have a negative impact on neural tube closure depending on genetic variation and the length of FA exposure (Marean et al., 2011). Current recommendations for women of childbearing age are 0.4 mg daily of FA (Hursthouse et al., 2011) although much higher doses (4 mg; Centers for Disease Control, 1991; and up to 5 mg, New Zealand Ministry of Health, 2003) have been suggested for women who have had a previous complicated or NTD pregnancy, or for women who are themselves affected with an NTD, women who are on insulin treatment for diabetes, or those who are taking medications known to affect folate metabolism. These latter doses are 10-fold higher than the 0.4 mg daily requirement mentioned above. Our findings are consistent with the suggested upper limit of 1 mg (Rogovik et al., 2010), although additional studies should be undertaken to establish a safe daily dose in all pregnant women.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
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