Maternal Mthfd1 disruption impairs fetal growth but does not cause neural tube defects in mice1

Anna E Beaudin; Cheryll A Perry; Sally P Stabler; Robert H Allen; Patrick J Stover

doi:10.3945/ajcn.111.030783

. 2012 Feb 29;95(4):882–891. doi: 10.3945/ajcn.111.030783

Maternal Mthfd1 disruption impairs fetal growth but does not cause neural tube defects in mice^¹^²^³

Anna E Beaudin, Cheryll A Perry, Sally P Stabler, Robert H Allen, Patrick J Stover

PMCID: PMC3302363 PMID: 22378735

Abstract

Background: MTHFD1 encodes C1-tetrahydrofolate synthase, which is a folate-dependent enzyme that catalyzes the formation and interconversion of folate-activated one-carbon groups for nucleotide biosynthesis and cellular methylation. A polymorphism in MTHFD1 (1958G→A) impairs enzymatic activity and is associated with increased risk of adverse pregnancy outcomes, but the mechanisms are unknown.

Objective: The objective of this study was to determine whether disruption of the embryonic or maternal Mthfd1 gene or both interacts with impaired folate and choline status to affect neural tube closure, fetal growth, and fertility in mice and to investigate the underlying metabolic disruptions.

Design: Dams with a gene-trapped (gt) allele in Mthfd1 and wild-type dams were fed a control or folate- and choline-deficient AIN93G diet (Dyets Inc). Litters were examined for gross morphologic defects, crown-rump length, and resorptions. Folate status and amounts of folate-related metabolites were determined in pregnant dams.

Results: Reduced folate and choline status resulted in severe fetal growth restriction (FGR) and impaired fertility in litters harvested from Mthfd1^gt/+ dams, but embryonic Mthfd1^gt/+ genotype did not affect fetal growth. Gestational supplementation of Mthfd1^gt/+ dams with hypoxanthine increased FGR frequency and caused occasional neural tube defects (NTDs) in Mthfd1^gt/+ embryos. Mthfd1^gt/+ dams exhibited lower red blood cell folate and plasma methionine concentrations than did wild-type dams.

Conclusions: Maternal Mthfd1^gt/+ genotype impairs fetal growth but does not cause NTDs when dams are maintained on a folate- and choline-deficient diet. Mthfd1^gt/+ mice exhibit a spectrum of adverse reproductive outcomes previously attributed to the human MTHFD1 1958G→A polymorphism. Mthfd1 heterozygosity impairs folate status in pregnant mice but does not significantly affect homocysteine metabolism.

INTRODUCTION

Maternal folate deficiency is a risk factor for pathologic conditions in pregnancy and fetal development. Biomarkers of impaired folate status, including reduced serum folate and elevated plasma homocysteine, correlate with increased risk of neural tube, cardiac, limb, and jaw defects; preeclampsia; placental abruption; low birth weight; preterm delivery; intrauterine growth restriction; and spontaneous abortion (1–8). Conversely, maternal folic acid supplementation may reduce risk of many of these anomalies (9–15). There is increasing recognition that low folate status interacts with genetic variants that compromise folate metabolism to confer risk of developmental anomalies (16–22).

Folate metabolism provides activated one-carbon groups for the de novo biosynthesis of purines and thymidylate, as well as the remethylation of homocysteine to methionine and synthesis of the universal methyl donor S-adenosylmethionine (Figure 1). Formate is a primary source of one-carbon groups for nucleotide biosynthesis and is generated from serine, glycine, or choline catabolism in the mitochondria (23). In the cytoplasm, the synthetase domain of the trifunctional enzyme C1-THF⁴ synthase (MTHFD1) catalyzes the ATP-dependent biosynthesis of 10-formylTHF, which is the cofactor required for de novo purine biosynthesis. The NADP-dependent cyclohydrase/dehydrogenase domain of MTHFD1 catalyzes the reduction of 10-formylTHF to methyleneTHF, which is used for synthesis of thymidine, serine, or methionine (24) (Figure 1). A common variant of MTHFD1, 1958G→A, results in an arginine to glutamine substitution in the active site of the synthetase domain. MTHFD1 1958G→A has been linked to increased risk of NTDs (25–27), cleft lip and palate (28), congenital heart defects (29), placental abruption (30), unexplained second-trimester pregnancy loss (31), and intrauterine growth restriction (32). The 1958G→A variant was shown to encode a thermolabile enzyme associated with reduced enzymatic activity and reduced incorporation of labeled formate into DNA, which indicated disrupted de novo purine biosynthesis (29). These data suggest that impairments in de novo purine biosynthesis may underlie pathogenesis in response to the MTHFD1 1958G→A variant, but this effect has not been shown experimentally.

FIGURE 1. — Folate-mediated one-carbon metabolism. One-carbon metabolism in the cytoplasm is required for the de novo synthesis of purines and thymidylate and for the remethylation of homocysteine to methionine. Formate is generated by one-carbon metabolism in mitochondria from serine and glycine. MTHFD1 catalyzes the ATP-dependent synthesis of 10-formylTHF as well as the NADP-dependent reduction of 10-formylTHF to methyleneTHF. AdoHcy, S-adenosylhomocysteine; AdoMet, S-adenosylmethionine; DHF, dihydrofolate; DHFR, dihydrofolate reductase; dUMP, deoxyuridine monophosphate; MTHFD1, 10-formyltetrahydrofolate synthetase/methenyltetrahydrofolate cyclohydrolase/methylenetetrahydrofolate dehydrogenase; MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; SHMT1, serine hydroxymethyltransferase; THF, tetrahydrofolate; TYMS, thymidylate synthase.

An Mthfd1-deficient mouse model that contained a gt vector insertion in the synthetase domain of the Mthfd1 gene, resulting in the loss of 10-THF synthetase activity, has been generated and characterized (33). Homozygous Mthfd1^gt/gt mice are not viable, which shows the requirement for folate-activated formate during embryonic development. However, Mthfd1^gt/+ mice are viable, exhibit 50% decreased Mthfd1 protein levels, and exhibit impaired cellular methylation capacity but enhanced de novo thymidylate biosynthesis (33). In this study, we investigated the interaction of reduced Mthfd1 expression with impaired folate status on embryonic development. Reduced maternal, but not embryonic, Mthfd1 expression impaired fetal growth and fertility, similar to the MTHFD1 1958A→G variant, but did not cause NTDs. The Mthfd1^gt/+ genotype was shown to impair folate status in pregnant mice, which to our knowledge, is an observation not previously reported to be associated with the human MTHFD1 1958G→A polymorphism.

MATERIALS AND METHODS

Experimental animals and diets

All animal experiments were approved by the Cornell Institutional Animal Care and Use Committee according to the guidelines of the Animal Welfare Act and all applicable federal and state laws. Mice were maintained on a 12-h light/dark cycle in a temperature-controlled room. For timed pregnancies, virgin female mice aged 70–120 d were housed overnight with male mice. The next morning, dams were examined for the presence of a vaginal plug. The day of the plug, 0900 was designated as E0.5. Pregnant dams were killed with the use of carbon dioxide–induced asphyxiation, and blood was collected by cardiac puncture. Embryos were harvested at E11.5 or E14.5. Gravid uteri were removed, and all implants and resorptions sites were recorded. Embryos were dissected free of extra embryonic membranes under a dissecting microscope and examined for the presence of NTDs. All yolk sacs were collected for subsequent genotyping. Embryos were fixed for 24 h in 10% neutral buffered formalin, dehydrated in an ethanol series, and stored in 70% ethanol.

The generation and characterization of mice with a gene-trap insertion in the Mthfd1 gene (Mthfd1^gt/+) has been previously described (33). To determine the developmental stage of embryonic lethality in Mthfd1^gt/gt mice, embryos were examined from crosses of heterozygote B6.129P2-Mthfd1^gt mice. Because of embryonic lethality in Mthfd1^gt/gt embryos that occurred before E9.5, we conducted diet studies by using Mthfd1^gt/+and Mthfd1^+/+ mice derived from crosses of 129P2/OlaHsd dams to heterozygote 129P2/OlaHsd–Mthfd1^gt/+ male mice to determine and distinguish the effect of partial loss of Mthfd1 expression at both the maternal and embryonic levels. Dams were randomly assigned to either the experimental FCDD or an AIN93G (control) diet (Dyets) at weaning. Choline was excluded from the experimental diet in addition to folate to increase the requirement for folate-derived one carbons; choline synthesis requires methyl groups in the form of S-adenosylmethionine and provides a source of one-carbon units for homocysteine remethylation through its catabolism (34). Female mice were maintained on a diet from weaning throughout the breeding period and for the duration of gestation until killed. For studies that involved hypoxanthine supplementation, hypoxanthine (Sigma) was dissolved in nanopure water at a concentration of 10 mmol/L and diluted with acidified drinking water to a final concentration of 500 μmol/L. The final concentration of hypoxanthine in drinking water was verified by UV spectroscopy (ϵ = 10.5 mmol/L at λ = 250 nm).

Genotype analysis

Genotyping of the Mthfd1^gt allele was performed as recently described (33). Embryonic sex was determined by using a genotyping protocol described elsewhere (35).

Determination of RBC folate concentrations

Folate concentrations in RBC samples were quantified by using a Lactobacillus casei microbiological assay as previously described (36).

Determination of plasma metabolites

Plasma concentrations of homocysteine, cystathionine, cysteine, α -aminobutyric acid, methionine, glycine, serine, N,N-dimethylglycine, and N-methylglycine were determined by stable-isotope dilution capillary gas chromatography–mass spectrometry as previously described (37, 38).

Statistical analysis

Analyses of FGR incidence and embryonic CR length were conducted by using repeated-measured ANOVA (PROC MIXED or PROC GENMOD; SAS Institute) in which independent variables included the maternal and embryonic Mthfd1 genotype, maternal diet, and embryo sex, and the litter was considered as a repeated measure. Relevant interaction terms were included unless assumptions of variance were violated. Total litter resorptions and implants were analyzed by using ANOVA in which litter was considered as the unit of analysis, and independent variables included maternal Mthfd1 genotype and diet. Because hypoxanthine supplementation was examined only in Mthfd1^gt/+ mice, analyses of the effects of hypoxanthine supplementation on FGR incidence, resorptions, and implants were conducted by comparing Mthfd1^gt/+ mice fed the control, FCDD, or FCDD + 500 μmol hypoxanthine/L by using 1-factor ANOVA as previously described, except that the maternal genotype was not included in the model. Total RBC folate and plasma metabolite concentrations were analyzed by using 2-factor ANOVA with maternal Mthfd1 genotype and diet as independent variables. Post hoc comparisons were analyzed by using Student's t test with Bonferroni correction for multiple testing. Chi-square analyses were used to assess any deviation of genotype distribution from Hardy-Weinberg equilibrium.

RESULTS

Mthfd1gt/gt mice exhibited early embryonic lethality

Mthfd1 is an essential gene in mice (33). To investigated whether Mthfd1^gt/gt embryos developed NTDs in utero, litters were harvested from intercrosses of heterozygous B6.129(P2)C57Bl/6-Mthfd1^gt/+ (N6) mice at various gestational time points, and the genotype distribution of embryos was determined (Table 1; data not shown). There were no Mthfd1^gt/gt embryos recovered at any time point during gestation beginning at E9.5. The genotype distribution from 10 litters harvested at E9.5 differed significantly from Hardy-Weinberg equilibrium (Table 1; P < 0.0001, chi-square analysis). The data indicate that embryonic lethality in Mthfd1^gt/gt embryos occurred before neural tube closure at E9.5.

TABLE 1.

Early embryonic lethality in Mthfd1^gt/gt embryos¹

	Observed genotype distribution			Expected genotype distribution
Genotype	Male mice	Female mice	Total	Male mice	Female mice	Total
Mthfd1^+/+	11	11	22	7.25	7.25	14.5
Mthfd1^gt/+	17	19	36	14.5	14.5	29
Mthfd1^gt/gt	0	0	0	7.25	7.25	14.5
Total	28	30	58	29	29	58

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Mthfd1^gt/+ mice were intercrossed, and 10 litters were harvested at embryonic day 9.5. The expected genotype distribution was calculated based on Hardy-Weinberg equilibrium. Deviation from the expected genotype distribution was determined by using a chi-square statistic. The observed genotype distribution differed significantly from the expected genotype distribution (P < 0.0001). gt, gene-trapped.

Maternal Mthfd1 disruption and low folate status caused FGR

We examined the effect of reduced Mthfd1 expression and diet status on embryonic and neural tube development in Mthfd1^gt/+ mice. Congenic 129P2/Ola- Mthfd1^gt/+ and Mthfd1^+/+ female littermates were fed either the AIN93G (control) diet or FCDD from weaning through gestation. At 8 wk of age, Mthfd1^+/+ and Mthfd1^gt/+ dams were mated to Mthfd1^gt/+ or Mthfd1^+/+ male mice, respectively, to determine the independent contributions of maternal and embryonic Mthfd1 genotype to embryonic development. Litters were harvested at E11.5 or E14.5 and examined for gross morphologic abnormalities. Genotype distribution in litters examined at E11.5 and E14.5 did not differ significantly from expected values (Tables 2 and 3; P = 0.5 and P = 0.3, respectively; chi-square test). No NTDs were observed in any of the litters examined. However, a proportion of embryos from crosses of Mthfd1^gt/+ dams fed either the FCDD or control diet and Mthfd1^+/+ dams fed the FCDD were either resorbing and/or severely malformed, which suggested severe FGR (Table 2). Malformed embryos were characterized by an irregular and convoluted neuroepithelium, torqued body symmetry, and severely delayed development (Figure 2, AndashD). In many cases, affected embryos were in the process of resorbing (Figure 2, C and D). The timing of embryo loss and/or developmental arrest was variable but appeared to have occurred between E9.5 and E11.5 after neural tube closure. No NTDs were observed in malformed embryos, and the neural tube was completely closed even in embryos that were in the process of resorption.

TABLE 2.

Incidence of NTDs and FGR in E11.5 embryos harvested from crosses of Mthfd1^gt/+ and Mthfd1^+/+ mice fed either a control diet, the FCDD, or the FCDD supplemented with 500 μmol hypoxanthine/L in drinking water¹

Diet and maternal Mthfd1 genotype	No. of litters	Embryonic Mthfd1 genotype	Total no. of embryos	No. of NTDs	FGR incidence²
Control
+/+	12	+/+	18	0	0
		gt/+	22	0	0
gt/+	15	+/+	28	0	3 (2 M, 1 F)
		gt/+	21	0	2 (1 M, 1 F)
FCDD
+/+	15	+/+	30	0	4 (3 M, 1 F)
		gt/+	30	0	1 M
gt/+	12	+/+	35	0	6 (5 M, 1 F)
		gt/+	28	0	6 (2 M, 4 F)
FCDD + 500 μmol hypoxanthine/L
gt/+	15	+/+	26	0	5 (2 M, 3 F)
		gt/+	28	2	10 (7 M, 3 F)

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The main effects of diet, maternal and embryonic Mthfd1 genotype, and sex on FGR incidence were analyzed with the use of ANOVA. Analysis of the effects of hypoxanthine supplementation were analyzed separately with the use of 1-factor ANOVA comparing litters from Mthfd1^gt/+ dams fed the control diet, FCDD, or FCDD + hypoxanthine. Maternal genotype (χ² = 4.42, P = 0.03) and maternal diet (χ² = 5.16, P = 0.02) significantly influenced the incidence of FGR. Differences between observed and expected genotype distributions were analyzed by using a chi-square statistic. There were no differences between observed and expected genotype distributions (P = 0.5, chi-square test). ANOVA showed a trend toward an effect of diet (P = 0.11) when FGR incidence was analyzed in Mthfd1^gt/+ dams fed a control diet, FCDD, or FCDD + 500 μmol hypoxanthine/L. Post hoc analysis did not show any significant comparisons. E, embryonic day; FCDD, AIN93G diet deficient in folate and choline; FGR, fetal growth restriction; gt, gene-trapped; NTDs, neural tube defects.

All values are numbers of embryos with FGR; sex distribution in parentheses.

TABLE 3.

Incidence of NTDs and FGR in E14.5 embryos harvested from crosses of Mthfd1^gt/+ and Mthfd1^+/+ mice fed either a control diet or the FCDD¹

Diet and maternal Mthfd1 genotype	No. of litters	Embryonic Mthfd1 genotype	Total no. of embryos	No. of NTDs	FGR incidence²
Control
+/+	11	+/+	22	0	0
		gt/+	24	0	0
gt/+	17	+/+	22	0	0
		gt/+	28	0	3 (1 F, 2 M)
FCDD
+/+	20	+/+	28	0	3 (2 F, 1 M)
		gt/+	18	0	3 (2 F, 1 M)
gt/+	15	+/+	19	0	0
		gt/+	23	0	0

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The main effects of diet, maternal and embryonic Mthfd1 genotype, and sex on FGR incidence were analyzed with the use of ANOVA. Post hoc comparisons were analyzed by using Student's t test with Bonferroni correction for multiple testing. Differences between observed and expected genotype distributions were analyzed by using a chi-square statistic. There were no differences between observed and expected genotype distributions (P = 0.3, chi-square test). ANOVA showed a significant interaction of maternal Mthfd1 genotype and diet on FGR incidence (χ² = 4.34, P = 0.037). However, post hoc analysis did not reveal any significant comparisons. E, embryonic day; FCDD, AIN93G diet deficient in folate and choline; FGR, fetal growth restriction; gt, gene-trapped; NTDs, neural tube defects.

All values are numbers of embryos with FGR; sex distribution in parentheses.

FIGURE 2. — Maternal *Mthfd1* disruption affected embryonic development. A normal (A) and a growth-restricted (B) *Mthfd1^+/+* littermate isolated from an *Mthfd1^gt/+* female mouse fed the FCDD are shown. An *Mthfd1^+/+* embryo (C) and *Mthfd1^gt/+* embryo (D) exhibiting growth restriction, torqued body symmetry, and highly irregular neuroepithelial organization (arrows) isolated at embryonic day 11.5 from an *Mthfd1^gt/+* female mouse fed the FCDD are shown. A normal *Mthfd1^+/+* embryo (E) and an *Mthfd1^gt/+* littermate with exencephaly (F) are shown. The extent of defect is indicated by arrows. Embryos were uncovered in a litter isolated from an *Mthfd1^gt/+* female mouse fed the FCDD and supplemented with 500 μmol hypoxanthine/L. FCDD, AIN93G diet deficient in folate and choline; gt, gene-trapped.

Surprisingly, embryonic Mthfd1 genotype did not influence the incidence of FGR in litters derived at E11.5 (P = 0.85) because approximately equal numbers of Mthfd1^gt/+ and Mthfd1^+/+ littermates were affected by FGR (Table 2). However, both the maternal Mthfd1 genotype and maternal diet significantly influenced the FGR incidence ([χ² = 4.42 (P = 0.03) and χ² = 5.16 (P = 0.02), respectively]. There was also a trend toward an effect of embryonic sex on FGR incidence (χ² = 3.31, P = 0.068), with more male mice affected than female mice (14 male mice compared with 8 female mice). There were no FGR-affected embryos uncovered in 12 litters isolated from Mthfd1^+/+ dams fed the control diet. Approximately 10% of embryos isolated from Mthfd1^gt/+ dams fed the control diet or Mthfd1^+/+ dams fed the FCDD exhibited FGR. The highest incidence of FGR occurred in litters derived from Mthfd1^gt/+ female mice fed the FCDD, and ∼20% of embryos exhibited FGR. The additive effect of compromised maternal folate and choline status and maternal Mthfd1 genotype on FGR incidence suggested that these 2 factors did not interact in FGR pathogenesis.

Litters examined at E14.5 had significantly fewer FGR-affected embryos compared with litters examined at E11.5 (χ² = 3.65, P = 0.056; Table 3). No NTDs were observed in any embryos examined at E14.5. The embryonic Mthfd1 genotype did not affect FGR incidence in embryos at E14.5. A significant interaction was observed between maternal Mthfd1 genotype and maternal diet for the number of FGR-affected embryos at E14.5 (χ² = 4.34, P = 0.037), although a subsequent post hoc analysis did not reveal any significant comparisons. No FGR-affected embryos were uncovered at E14.5 from Mthfd1^+/+ dams fed the control diet or Mthfd1^gt/+ dams fed the FCDD. The absence of FGR-affected embryos at E14.5 in litters derived from Mthfd1^gt/+ female mice fed the FCDD indicated that affected embryos from these litters did not survive past E11.5 and were resorbed.

Hypoxanthine supplementation of Mthfd1gt/+ female mice did not rescue FGR

MTHFD1 provides one-carbon moieties for de novo purine biosynthesis in the cytoplasm, and embryonic fibroblasts that lack MTHFD1 expression are auxotrophic for purines (39). Therefore, we investigated whether maternal supplementation with hypoxanthine, which is a precursor for purine biosynthesis through the folate-independent purine salvage pathway, would rescue FGR in litters derived from Mthfd1^gt/+ dams maintained with the FCDD. Pregnant dams were supplemented with 500 μmol hypoxanthine/L in drinking water from the time of the vaginal plug until being killed at E11.5. Hypoxanthine supplementation did not affect the genotype distribution of embryos derived from Mthfd1^gt/+ dams fed FCDD (Table 2). Surprisingly, hypoxanthine supplementation of Mthfd1^gt/+ dams fed the FCDD did result in an almost 50% increase in the incidence of FGR compared with that in unsupplemented Mthfd1^gt/+ dams, although the comparison was NS (28% compared with 19%, respectively; χ² = 4.32, P = 0.11). Although FGR was observed in both Mthfd1^+/+ and Mthfd1^gt/+ embryos derived from hypoxanthine-supplemented dams, the increased incidence of FGR in these litters could be attributed to a doubling of the incidence of FGR in Mthfd1^gt/+ embryos compared with that in Mthfd1^+/+ littermates (10 compared with 5 embryos, respectively; 36% compared with 19%, respectively). In addition, 2 of 28 Mthfd1^gt/+ embryos derived from hypoxanthine-supplemented Mthfd1^gt/+ female mice exhibited exencephaly (Figure 2, E and F), whereas no NTDs were observed in Mthfd1^+/+ embryos from the same litters (Table 2).

Maternal Mthfd1 disruption, low folate status, and hypoxanthine supplementation influenced embryo length

To determine the effect of Mthfd1 disruption on embryonic growth, we measured CR length in individual embryos derived from the crosses previously described. CR lengths of embryos at E11.5 were not significantly affected by the embryonic Mthfd1 genotype, maternal Mthfd1 genotype, or maternal diet in the absence of hypoxanthine supplementation (Table 4). However, hypoxanthine supplementation significantly affected CR lengths at E11.5. Analysis of average CR length in Mthfd1^gt/+ dams fed the control diet, FCDD, or FCDD + 500 μmol hypoxanthine/L showed a significant effect of maternal diet (F = 5.37; P = 0.01). Post hoc analysis indicated that the average CR length of embryos derived from hypoxanthine-supplemented Mthfd1^gt/+ dams was significantly shorter than those from Mthfd1^gt/+ dams fed the control diet (5.1 compared with. 6.2 mm, respectively; P = 0.0078) but not from those unsupplemented Mthfd1^gt/+ dams fed the FCDD (P = 0.37). The average CR length of embryos at E14.5 was also unaffected by embryonic Mthfd1 genotype. However, there was a trend toward a significant interaction of maternal Mthfd1 genotype and diet (F = 2.9, P = 0.09) on CR lengths of embryos at E14.5. Embryos derived from Mthfd1^gt/+ dams fed the FCDD had decreased CR lengths compared with those of embryos derived from Mthfd1^+/+ dams fed the FCDD at E14.5 (10.7 compared with 11.5 mm, respectively; P = 0.05; Table 4).

TABLE 4.

CR lengths in E11.5 and E14.5 embryos harvested from crosses of Mthfd1^gt/+ and Mthfd1^+/+ mice fed either a control diet or FCDD or Mthfd1^gt/+ mice fed FCDD supplemented with 500 μmol hypoxanthine/L in drinking water¹

Diet	Maternal Mthfd1 genotype	Embryonic Mthfd1 genotype	CR length (E11.5)	CR length (E14.5)
Control	+/+	+/+	6.17 ± 0.24	11.14 ± 0.23
	—	gt/+	5.7 ± 0.2	11.27 ± 0.25
	gt/+	+/+	6.21 ± 0.22	11.38 ± 0.27
	—	gt/+	6.15 ± 0.33	11.35 ± 0.23
FCDD	+/+	+/+	5.81 ± 0.16	11.55 ± 0.17
	—	gt/+	5.93 ± 0.16	11.55 ± 0.23
	gt/+	+/+	5.57 ± 0.14	10.7 ± 0.35
	—	gt/+	5.67 ± 0.27	10.7 ± 0.21
FCDD + 500 μmol hypoxanthine/L	gt/+	+/+	5.25 ± 0.23	ND
	—	gt/+	4.90 ± 0.26	ND

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All values are means ± SEs. n = 41–58 embryos/group. The main effects of maternal and embryonic Mthfd1 genotype and diet and relevant interactions were analyzed with the use of ANOVA with litter as a random variable. Post hoc comparisons were analyzed by Student's t test with Bonferroni correction for multiple testing. Analyses of the effects of hypoxanthine supplementation were analyzed separately in a 1-factor ANOVA that compared litters from Mthfd1^gt/+ dams fed the control diet, an FCDD, or an FCDD + hypoxanthine. ANOVA showed a trend toward a significant interaction of maternal genotype and diet on average CR length at E14.5 (F = 2.9, P = 0.09). Post hoc analysis showed that the average CR length of embryos derived from Mthfd1^gt/+ dams fed the FCDD was significantly shorter than that of embryos derived from Mthfd1^gt/+ dams fed the control diet (P = 0.05). ANOVA showed a significant effect of maternal diet on CR length (F = 5.27; P = 0.01). Post hoc analysis revealed that CR lengths in the FCDD + 500 μmol hypoxanthine/L group were significantly shorter than those in the control group (P = 0.0078) but not those in the FCDD group (P = 0.37). CR, crown-rump; E, embryonic day; FCDD, AIN93G diet deficient in folate and choline; gt, gene-trapped; ND, not determined.

Effect of maternal Mthfd1 disruption and diet on fertility

To examine the effect of maternal Mthfd1 disruption and maternal diet on fertility, total implants and resorptions were examined in litters isolated at E11.5 or E14.5 from Mthfd1^gt/+ and Mthfd1^+/+ dams fed the control diet or FCDD and Mthfd1^gt/+ dams fed the FCDD and supplemented with 500 μmol hypoxanthine/L (Tables 5 and 6). The analysis of total litter implants included viable embryos, FGR-affected embryos that were either viable or in the process of being resorbed, and complete resorptions. Maternal diet significantly affected the total number of fetal implants in litters derived at E11.5; surprisingly, there were significantly more implants in litters derived from female mice maintained on the FCDD than in litters derived from female mice maintained with the control diet (5.4 compared with 3.6 implants, respectively; F = 9.59, P = 0.004; Table 5). Total fetal implants were also slightly higher in litters of Mthfd1^gt/+ dams than in litters of Mthfd1^+/+ dams (5.0 compared with 4.0 implants, respectively; F = 2.82, P = 0.099). Total fetal implants in litters isolated from Mthfd1^gt/+ dams fed the FCDD + 500 μmol hypoxanthine/L were significantly lower than from unsupplemented Mthfd1^gt/+ dams fed the FCDD (3.8 compared with 6.3 implants, respectively; P < 0.01) and were similar to those in litters of Mthfd1^gt/+ dams maintained with the control diet (3.8 compared with 3.7 implants, respectively). Litters derived at E14.5 from dams maintained with the FCDD had slightly fewer fetal implants that did litters derived from dams fed the control diet (3.3 compared with 4.3 implants, respectively; F = 3.06, P = 0.08; Table 6). There was no independent effect of maternal Mthfd1 genotype on the number of fetal implants in litters derived at E14.5.

TABLE 5.

Resorptions and implants in litters derived at E11.5 from Mthfd1^gt/+ and Mthfd1^+/+ dams fed a control diet or the FCDD¹

Diet and maternal Mthfd1 genotype	No. of litters	No. of total implants	No. of implants per litter	No. of total resorptions	No. of resorptions per litter
Control
+/+	10	34	3.4 ± 0.6²	1	0.1 ± 0.1
gt/+	15	56	3.7 ± 0.6	7	0.8 ± 0.32
FCDD
+/+	16	74	4.4 ± 0.5	10	0.87 ± 0.23
gt/+	13	82	6.3 ± 0.7	10	1.3 ± 0.45
FCDD + 500 μmol hypoxanthine/L
gt/+	15	59	3.8 ± 0.5	17	1.06 ± 0.36
P-diet effect	—	—	0.004	—	0.0454
P-genotype effect	—	—	0.099	—	0.0760
P-diet × genotype effect	—	—	NS	—	NS

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The main effects of Mthfd1 genotype and diet and interactions of genotype and diet on implantation rates and log-transformed resorption rates were analyzed with the use of ANOVA. Post hoc comparisons were analyzed with the use of Student's t test with Bonferroni correction for multiple testing. Analysis of implants and resorptions for the FCDD + 500 μmol hypoxanthine/L group was completed separately by comparing Mthfd1^gt/+ dams fed a control diet, an FCDD, or an FCDD + 500 μmol hypoxanthine/L with the use of ANOVA. The number of implants in the FCDD + 500 μmol hypoxanthine/L group was significantly different from that of the FCDD-alone groups (P < 0.01) but not from that of the control group (P > 0.05). E, embryonic day; FCDD, AIN93G diet deficient in folate and choline; gt, gene-trapped.

Mean ± SE (all such values).

TABLE 6.

Resorptions and implants in litters derived at E14.5 from Mthfd1^gt/+ and Mthfd1^+/+ dams fed a control diet or the FCDD¹

Diet and maternal genotype	No. of litters	Total implants	No. of implants per litter	Total no. of resorptions	No. of resorptions per litter
Control
+/+	11	43	4.5 ± 0.6²	6	0.55 ± 0.25
gt/+	17	55	4.1 ± 0.3	14	0.82 ± 0.18
FCDD
+/+	22	55	3.3 ± 0.3	17	0.77 ± 0.25
gt/+	12	30	3.4 ± 0.4	11	0.91 ± 0.40
P-diet effect	—	—	0.08	—	NS
P-genotype effect	—	—	NS	—	NS
P-diet × genotype effect	—	—	NS	—	NS

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Main effects of the Mthfd1 genotype, diet, and interaction of genotype and diet on implantation rates and log-transformed resorption rates were analyzed with the use of ANOVA. Post hoc comparisons were analyzed with the use of Student's t test with Bonferroni correction for multiple testing. E, embryonic day; FCDD, AIN93G diet deficient in folate and choline; gt, gene-trapped.

Mean ± SE (all such values).

Analysis of resorptions included both complete resorptions as well as FGR-affected embryos that were in the process of resorbing. Examination of total resorptions in litters derived at E11.5 revealed a significant effect of maternal diet (χ² = 2.89, P = 0.045; Table 5). More resorptions occurred in response to the FCDD than to the control diet (1.06 compared with 0.52 resorptions per litter, respectively). Maternal Mthfd1 genotype did not significantly affect resorption rates; however, there was a trend toward a higher number of resorptions in litters derived from Mthfd1^gt/+ dams than from Mthfd1^+/+ female mice (1.02 compared with 0.57 resorptions per litter, respectively; χ² = 3.15, P = 0.0760). Only one resorption was shown in 10 litters derived from Mthfd1^+/+ dams fed the control diet, whereas almost 50% of litters isolated from crosses of Mthfd1^gt/+ dams fed the FCDD included at least one resorption. There was no effect of either the maternal Mthfd1 genotype or maternal diet on total resorptions in litters derived at E14.5 (Table 6). Hypoxanthine supplementation of Mthfd1^gt/+ dams fed the FCDD did not affect the number of resorptions.

Maternal Mthfd1 disruption and folate status influenced markers of folate metabolism

To understand the metabolic alterations associated with impaired fetal growth and fertility, folate status and levels of folate metabolites were determined in RBCs or plasma isolated from pregnant dams on sacrifice at E11.5. The FCDD was associated with a significant decline in maternal RBC folate concentrations measured at E11.5 (Table 7; F = 234, P < 0.0001). RBC folate concentrations were reduced by ∼65% with the FCDD relative to the control diet (8 compared with 23 fmol/μg protein, respectively). Mthfd1 deficiency was also associated with a significant reduction in RBC folate concentrations in pregnant dams maintained with both diets (F = 46.7, P < 0.0001). However, Mthfd1 genotype influenced RBC folate concentrations to a greater extent in mice maintained on the control diet than on the FCDD (gene × diet interaction: F = 31.25, P = 0.01). Post hoc analysis showed that, although the Mthfd1 genotype significantly influenced RBC folate concentrations in dams maintained with the control diet (P < 0.0001), RBC folate concentrations did not differ significantly between Mthfd1^gt/+ and Mthfd1^+/+ dams maintained on the FCDD (6.5 compared with 10.2, respectively; P = 0.1; Table 7).

TABLE 7.

Red blood cell folate concentrations and amounts of plasma metabolites in pregnant Mthfd1^gt/+ and Mthfd1^+/+ dams at E11.5 fed a control diet or the FCDD¹

	Diet
	Control		FCDD
Metabolite	+/+	gt/+	+/+	gt/+	P-diet effect	P-genotype effect	P-diet × genotype effect
Folate (fmol/μg)	27.9 ± 1.1	18.3 ± 1.2	10.2 ± 0.9	6.5 ± 0.5	<0.0001	<0.0001	0.0118²
Homocysteine (μmol/L)	4.9 ± 0.9	5.7 ± 1.3	15.5 ± 3.1	12.8 ± 2.9	0.0008	NS	NS
Cysteine (μmol/L)	183 ± 15	183 ± 16	192 ± 12	184 ± 17	NS	NS	NS
Cystathionine (μmol/L)	997 ± 190	694 ± 101	1211 ± 129	1204 ± 214	0.03	NS	NS
α-Aminobutyric acid (μmol/L)	5.3 ± 0.8	4.9 ± 1.1	5.7 ± 0.5	5.1 ± 1.1	NS	NS	NS
Methionine (μmol/L)	54.0 ± 8.6	33.5 ± 3.4	51.4 ± 3.6	56.8 ± 10.6	NS	NS	0.08³
Glycine (μmol/L)	93.3 ± 12.1	103 ± 13	91.0 ± 14.0	80.2 ± 11.6	NS	NS	NS
Serine (μmol/L)	115 ± 9	94 ± 11	130 ± 10	127 ± 17	0.02	NS	NS
Dimethylglycine (μmol/L)	7.25 ± 0.67	8.11 ± 0.53	4.64 ± 0.51	3.61 ± 0.4s4	<0.0001	NS	NS
Methylglycine (μmol/L)	0.54 ± 0.05	0.56 ± 0.06	0.50 ± 0.05	0.45 ± 0.03	NS	NS	NS

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All values are means ± SEs. Independent effects of the Mthfd1 genotype, diet, and interaction between Mthfd1 genotype and diet were determined by using 2-factor ANOVA. Post hoc comparisons were analyzed by using Student's t test with Bonferroni correction for multiple testing. E, embryonic day; FCDD, AIN93G diet deficient in folate and choline; gt, gene-trapped.

Red blood cell folate concentrations did not differ significantly between Mthfd1^gt/+ and Mthfd1^+/+ dams maintained on the FCDD (6.5 compared with 10.2 fmol/μg; P = 0.1), although all other comparisons were significant (P < 0.001).

Mthfd1^gt/+ dams fed the control diet differed significantly from Mthfd1^+/+ dams fed the control diet (P = 0.05) and from Mthfd1^+/+ dams fed the FCDD (P = 0.03).

The FCDD significantly influenced several markers of homocysteine metabolism (Table 7). Plasma homocysteine was elevated ∼3-fold in all dams maintained on the FCDD, independent of the Mthfd1 genotype (5.3 compared with 14.2 μmol/L, respectively; P = 0.0008). The FCDD was also associated with significantly elevated cystathionine and serine concentrations (Table 7; P = 0.03 and P = 0.02, respectively), and significantly decreased concentrations of dimethylglycine (P < 0.0001) in all mice. The Mthfd1 genotype did not affect concentrations of any of the metabolites measured. However, there was a trend toward a significant interaction of the maternal Mthfd1 genotype and diet on plasma methionine concentrations (F = 3.24, P = 0.08). Plasma methionine concentrations in Mthfd1^gt/+ dams fed the control diet were decreased relative to those of Mthfd1^+/+ dams fed the control diet (33.5 compared with 54.0 μmol/L, respectively; P = 0.05) and Mthfd1^+/+ dams fed the FCDD (33.5 compared with 51.4 μmol/L, respectively; P = 0.03).

A comparison of plasma metabolite profiles of Mthfd1^gt/+ dams fed the control diet, FCDD, or FCDD + 500 μmol hypoxanthine/L revealed a significant effect of diet on serine (F = 3.63, P = 0.04), dimethylglycine, and methylglycine concentrations (P < 0.0001 for both comparisons) and a trend toward an effect of diet on methionine (F = 3.03, P = 0.06; see Supplemental Table 1 under “Supplemental data” in the online issue). However, a post hoc analysis showed that, in each of these cases, hypoxanthine supplementation did not significantly affect plasma metabolite profiles compared with those of Mthfd1^gt/+ mice fed the FCDD.

DISCUSSION

The gene-trap insertion in Mthfd1^gt/+ mice resulted in a 50% decrease in 10-formylTHF synthetase activity, which was similar to the reduction of synthetase activity reported for the MTHFD1 1958G→A variant (33). In this study, the metabolic disruption observed in Mthfd1^gt/+ mice adversely affected embryonic development and fertility and recapitulated some of the reproductive outcomes associated with the human 1958G→A polymorphism, including FGR and pregnancy loss. Maternal Mthfd1^gt/+ genotype and/or reduced dietary folate and choline status caused FGR, whereas the embryonic Mthfd1^gt/+ genotype had no effect on fetal growth. However, complete loss of Mthfd1 expression in Mthfd1^gt/gt embryos resulted in early fetal loss before neurulation, which is consistent with human studies that have demonstrated that the 1958G→A SNP is not in Hardy-Weinberg equilibrium, with the 1958A genotype being underrepresented relative to expectations (25). Together, these data confirm that the generation of one-carbon groups in the cytoplasm by MTHFD1 is essential for early embryonic development, and that partial loss of MTHFD1 activity at the maternal level is sufficient to affect fetal growth and development. The maternal Mthfd1^gt/+ genotype did not affect serum homocysteine concentrations, which suggests that purine biosynthesis is the primary metabolic disruption, as observed by others (29, 40).

In the absence of hypoxanthine supplementation, neither the maternal nor embryonic Mthfd1^gt/+ genotype caused NTDs. These data may be inconsistent with some reports in human populations that showed that the MTHFD1 1958G→A polymorphism was a maternal risk factor for NTDs. Hypoxanthine supplementation of Mthfd1^gt/+ pregnant female mice fed the FCDD did not ameliorate impaired fetal growth but resulted in 2 of 28 Mthfd1^gt/+ embryos that exhibited exencephaly. Hypoxanthine supplementation also resulted in a 2-fold increase in the incidence of FGR in Mthfd1^gt/+ embryos and significantly reduced CR length in both Mthfd1^+/+ and Mthfd1^gt/+ embryos. Together, these findings suggested that either hypoxanthine supplementation negatively impacted fetal growth or rescued earlier fetal loss in litters derived from Mthfd1^gt/+ female mice fed the FCDD. To our knowledge, there is no available evidence to suggest that hypoxanthine is embryotoxic after implantation. However, hypoxanthine supplementation of very early stage (preimplantation) mouse embryos cultured in vitro has been shown to cause arrest at the 2-cell stage via a mechanism unrelated to folate metabolism or DNA synthesis (41, 42). Notably, we observed the highest frequency of implants in litters derived from Mthfd1^gt/+ dams fed the FCDD, which was normalized by hypoxanthine supplementation to numbers comparable to those fed the control diet. We also observed the highest number of resorptions in litters derived from Mthfd1^gt/+ dams fed the FCDD. Therefore, it is possible that regulation of early embryonic development and implantation was affected by diet and genotype-related changes in hypoxanthine availability, and these effects were distinct from the effects of hypoxanthine on embryonic development after implantation. Christensen et al (42) have previously demonstrated purine auxotrophy in immortalized Mthfd1-null mouse embryonic fibroblasts lines; therefore, it is likely that Mthfd1 deficiency at both the maternal and embryonic levels may have affected early fetal survival by impairing purine biosynthesis. The small litter size observed on the 129P2Ola background may have precluded detection of a significant fetal loss of Mthfd1^gt/+ embryos as genotype distributions did not deviate significantly from expected values for any of the groups examined. Despite this limitation, the occurrence of NTDs and increased frequency of FGR in Mthfd1^gt/+ embryos derived from hypoxanthine-supplemented female mice raises the possibility that hypoxanthine supplementation rescued early fetal loss and that embryonic de novo purine biosynthesis may be crucial during neural tube closure and/or after the development of a definitive placenta. Additional investigation of the mechanism or mechanisms by which hypoxanthine supplementation affects fetal growth is warranted. In particular, this study was limited because it did not assess the effects of hypoxanthine supplementation in dams fed the control diet. However, these data indicate that maternal hypoxanthine supplementation increases risk of NTDs in folate-deficient Mthfd1^gt/+ dams. Although hypoxanthine supplementation did not affect total resorptions, the 2 NTD cases may have resulted from a rescue of early fetal loss of Mthfd1^gt/+ embryos.

The effect of the Mthfd1^gt/+ genotype on adverse fetal outcomes was most strongly associated with maternal genotype, just as the risk of poor pregnancy outcomes associated with the human MTHFD1 1958G→A polymorphism is strongly linked to the maternal genotype (25, 31). However, maternal hypoxanthine supplementation did not ameliorate FGR associated with combined maternal Mthfd1^gt/+ genotype and dietary folate and choline deficiency. These data indicate that the underlying cause of FGR in neurulation-stage embryos may be distinct from that of earlier fetal loss or that maternally derived free nucleotides may be more available to the fetus before formation of the definitive placenta. The timing of FGR in affected embryos coincided with the development of the definitive placenta in mice. Impairments in maternal de novo purine biosynthesis may have contributed to poor placental development in litters of Mthfd1^gt/+ dams, whereas elevated homocysteine in response to the FCDD may have contributed to impaired placental function in Mthfd1^+/+ female mice fed the FCDD. Thus, Mthfd1 disruption may impact embryonic development via several different mechanisms that produce a wide range of developmental anomalies, similar to those associated with the MTHFD1 1958G→A polymorphism in humans.

In a previous study, plasma folate concentrations were not affected in Mthfd1^gt/+ male mice (33). In this study, we observed a significant reduction in RBC folate concentrations in pregnant Mthfd1^gt/+ female mice. Thus, exacerbated maternal folate deficiency may have also contributed to poor reproductive outcomes in Mthfd1^gt/+ dams and may be a potential mechanism whereby polymorphisms in the human MTHFD1 gene confer risk of NTDs. Indeed, the effects of the maternal Mthfd1^gt/+ genotype on fetal growth and fertility mirrored the effects of the FCDD in Mthfd1^+/+ dams, as well as previously reported effects of folate deficiency during pregnancy (43). These data imply that disruption of Mthfd1 may confer risk of adverse pregnancy outcomes by impairing both folate status and metabolism.

Supplementary Material

Supplemental data

supp_95_4_882__index.html^{(724B, html)}

Acknowledgments

We thank Sylvia Allen, Rachel Slater, Dina Diskina, Donald Anderson, and Martha Field for technical assistance.

The authors’ responsibilities were as follows—AEB: study concept and design, data collection and analysis, and manuscript preparation, editing, and revision; AEB, CAP, SPS, and RHA: data collection; and PJS: study concept and design and manuscript editing and revision. None of the authors had a conflict of interest.

Footnotes

⁴

Abbreviations used: CR, crown-rump; E, embryonic day; FCDD, AIN93G diet deficient in folate and choline; FGR, fetal growth restriction; gt, gene-trapped; MTHFD1, 10-formyltetrahydrofolate synthetase/methenyltetrahydrofolate cyclohydrolase/methylenetetrahydrofolate dehydrogenase; NTD, neural tube defect; RBC, red blood cell; THF, tetrahydrofolate.

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Supplemental data

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[bib12] 12.Godwin KA, Sibbald B, Bedard T, Kuzeljevic B, Lowry RB, Arbour L. Changes in frequencies of select congenital anomalies since the onset of folic acid fortification in a Canadian birth defect registry. Can J Public Health 2008;99:271–5 [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Maternal Mthfd1 disruption impairs fetal growth but does not cause neural tube defects in mice123

Anna E Beaudin

Cheryll A Perry

Sally P Stabler

Robert H Allen

Patrick J Stover

Abstract

INTRODUCTION

FIGURE 1.

MATERIALS AND METHODS

Experimental animals and diets

Genotype analysis

Determination of RBC folate concentrations

Determination of plasma metabolites

Statistical analysis

RESULTS

Mthfd1gt/gt mice exhibited early embryonic lethality

TABLE 1.

Maternal Mthfd1 disruption and low folate status caused FGR

TABLE 2.

TABLE 3.

FIGURE 2.

Hypoxanthine supplementation of Mthfd1gt/+ female mice did not rescue FGR

Maternal Mthfd1 disruption, low folate status, and hypoxanthine supplementation influenced embryo length

TABLE 4.

Effect of maternal Mthfd1 disruption and diet on fertility

TABLE 5.

TABLE 6.

Maternal Mthfd1 disruption and folate status influenced markers of folate metabolism

TABLE 7.

DISCUSSION

Supplementary Material

Acknowledgments

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Maternal Mthfd1 disruption impairs fetal growth but does not cause neural tube defects in mice^¹^²^³