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Background Suboptimal folate intake, a risk factor for birth defects, is common even in areas with folate fortification. A polymorphism in methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), R653Q (MTHFD1 c.1958 G > A), has also been associated with increased birth defect risk, likely through reduced purine synthesis. Objective We aimed to determine if the interaction of MTHFD1 synthetase deficiency and low folate intake increases developmental abnormalities in a mouse model for MTHFD1 R653Q. Methods Female Mthfd1S+/+ and Mthfd1S+/- mice were fed control or low-folate diets (2 and 0.3 mg folic acid/kg diet, respectively) before mating and during pregnancy. Embryos and placentas were examined for anomalies at embryonic day 10.5. Maternal 1-carbon metabolites were measured in plasma and liver. Results Delays and defects doubled in litters of Mthfd1S+/- females fed low-folate diets compared to wild-type females fed either diet, or Mthfd1S+/- females fed control diets [P values (defects): diet 0.003, maternal genotype 0.012, diet × maternal genotype 0.014]. These adverse outcomes were associated with placental dysmorphology. Intrauterine growth restriction was increased by embryonic Mthfd1S+/- genotype, folate deficiency, and interaction of maternal Mthfd1S+/- genotype with folate deficiency (P values: embryonic genotype 0.045, diet 0.0081, diet × maternal genotype 0.0019). Despite a 50% increase in methylenetetrahydrofolate reductase expression in low-folate maternal liver (P diet = 0.0007), methyltetrahydrofolate concentration decreased 70% (P diet

BACKGROUND Moderately high folic acid intake in pregnant women has led to concerns about deleterious effects on the mother and fetus. Common polymorphisms in folate genes, such as methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (MTHFD1) R653Q, may modulate the effects of elevated folic acid intake. OBJECTIVES We investigated the effects of moderate folic acid supplementation on reproductive outcomes and assessed the potential interaction of the supplemented diet with MTHFD1-synthetase (Mthfd1S) deficiency in mice, which is a model for the R653Q variant. DESIGN Female Mthfd1S+/+ and Mthfd1S+/- mice were fed a folic acid-supplemented diet (FASD) (5-fold higher than recommended) or control diets before mating and during pregnancy. Embryos and placentas were assessed for developmental defects at embryonic day 10.5 (E10.5). Maternal folate and choline metabolites and gene expression in folate-related pathways were examined. RESULTS The combination of FASD and maternal MTHFD1-synthetase deficiency led to a greater incidence of defects in E10.5 embryos (diet × maternal genotype, P = 0.0016; diet × embryonic genotype, P = 0.054). The methylenetetrahydrofolate reductase (MTHFR) protein and methylation potential [ratio of S-adenosylmethionine (major methyl donor):S-adenosylhomocysteine) were reduced in maternal liver. Although 5-methyltetrahydrofolate (methylTHF) was higher in maternal circulation, the methylation potential was lower in embryos. The presence of developmental delays and defects in Mthfd1S+/- embryos was associated with placental defects (P = 0.003). The labyrinth layer failed to form properly in the majority of abnormal placentas, which compromised the integration of the maternal and fetal circulation and presumably the transfer of methylTHF and other nutrients. CONCLUSIONS Moderately higher folate intake and MTHFD1-synthetase deficiency in pregnant mice result in a lower methylation potential in maternal liver and embryos and a greater incidence of defects in embryos. Although maternal circulating methylTHF was higher, it may not have reached the embryos because of abnormal placental development; abnormal placentas were observed predominantly in abnormally developed embryos. These findings have implications for women with high folate intakes, particularly if they are polymorphic for MTHFD1 R653Q.

Introduction Chorioallantoic fusion is essential for development of the labyrinth layer of the mouse placenta. However, events that occur after chorioallantoic attachment remain poorly described, partly due to difficulties of conducting ex vivo analysis of the placenta. Herein, we report conditions for ex vivo culture of the developing murine placenta. Methods Mesometrial halves of decidua containing pre-attachment chorions were cultured alone or with explants of allantoides from stage-matched controls and analyzed by confocal and immunofluorescence microscopy. Expression and levels of marker genes associated with specific placental cell types were measured by in situ hybridization and qRT-PCR, respectively. Results After 24 h (hr) of co-culture, a mosaic pattern of eGFP + and eGFP − cells were found when explants of pre-attachment chorions from eGFP + embryos were co-cultured with stage-matched allantoides from eGFP − embryos or vice versa. In addition, proliferation increased in the allantoic region and folds formed on the chorionic plate. PECAM positive cells derived from the allantois were found in the chorionic region. Levels of the SynT-II marker, Gcm1 , significantly increased at 24 h, although expression of Gcm1 , was only found in explants co-cultured with an allantois at 12 h and 24 h. In addition, though levels of Tpbpα was not altered by co-culture with an allantois, Tpbpα was only detected in explants co-cultured with an allantois for 24 h. Discussion Our data show that chorioallantoic fusion and events associated with initiation of labyrinth layer formation can be modeled ex vivo , and reveal a previously unsuspected requirement of chorioallantoic fusion for Tpbpα expression.

Members of the transmembrane emp24 domain (Tmed)/p24 family of proteins are required for transport of proteins between the endoplasmic reticulum and the Golgi. One member of this family, Tmed2/p24β1, is expressed during placental development in mice and its expression is required for normal development of the labyrinth layer. Although TMED2 is conserved in humans, little is known about its expression and function in human placenta. We examined TMED2 expression in human placenta between 5.5 and 40 weeks of gestation and showed that TMED2 is expressed in syncytiotrophoblast, cytotrophoblast, and stromal cells. We also found high levels of TMED2 expression in BeWo but not in JEG-3 choriocarcinoma cell line. We used the BeWo cell line to determine TMED2 subcellular localization in placental cells and show its co-localization with the endoplasmic reticulum Golgi intermediate compartment. Our findings show conservation of TMED2 expression in human placenta and suggest that this protein may also play a role during placental development in humans.

s / Placenta 34 (2013) A1–A99 A84 insufficiency and adverse fetal outcomes in the T2DM HFD-NZO mice. Translational studies in the use of Tacrolimus in the pre-pregnancy glycemic control and immuno-conditioning for women with T2DM may be deemed beneficial in the prevention of adverse pregnancy outcomes. http://dx.doi.org/10.1016/j.placenta.2013.06.247 P2.82. IS TMED2 ESSENTIAL IN THE CHORION FOR NORMAL INTERACTION BETWEEN THE ALLANTOIS AND THE CHORION IN MICE? Wenyang(Dominic) Hou , Didem Sarikaya , Loydie Jerome-Majewska 1,2 Department of Human Genetics, McGill University, Montreal, Canada; Montreal Children Hospital Research Institute, Montreal, Canada; Organismic and Evolutionary Biology Department, Harvard University