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Despite participation in overlapping metabolic pathways, the relationship between choline and vitamin B-12 has not been well characterized especially during pregnancy. We sought to determine the effects of maternal choline supplementation on vitamin B-12 status biomarkers in human and mouse pregnancy, hypothesizing that increased choline intake would improve vitamin B-12 status. Associations between common genetic variants in choline-metabolizing genes and vitamin B-12 status biomarkers were also explored in humans. Healthy third-trimester pregnant women (n=26) consumed either 480 or 930 mg choline/day as part of a 12-week controlled feeding study. Wild-type NSA and Dlx3 heterozygous (Dlx3+/-) mice, which display placental insufficiency, consumed a 1×, 2× or 4× choline diet and were sacrificed at gestational days 15.5 and 18.5. Serum vitamin B-12, methylmalonic acid (MMA) and homocysteine were measured in all samples; holotranscobalamin (in humans) and hepatic vitamin B-12 (in mice) were also measured. The 2× choline supplementation for 12 weeks in pregnant women yielded higher serum concentrations of holotranscobalamin, the bioactive form of vitamin B-12 (~24%, P=.01). Women with genetic variants in choline dehydrogenase (CHDH) and betaine-homocysteine S-methyltransferase (BHMT) had higher serum MMA concentrations (~31%, P=.03) and lower serum holotranscobalamin concentrations (~34%, P=.03), respectively. The 4× choline dose decreased serum homocysteine concentrations in both NSA and Dlx3+/- mice (~36% and~43% respectively, P≤.015). In conclusion, differences in choline supply due to supplementation or genetic variation modulate vitamin B-12 status during pregnancy, supporting a functional relationship between these nutrients., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Dlx3 (distal-less homeobox 3) haploinsufficiency in mice has been shown to result in restricted fetal growth and placental defects. We previously showed that maternal choline supplementation (4X versus 1X choline) in the Dlx3 +/- mouse increased fetal and placental growth in mid-gestation. The current study sought to test the hypothesis that prenatal choline would modulate indicators of placenta function and development. Pregnant Dlx3 +/- mice consuming 1X (control), 2X, or 4X choline from conception were sacrificed at embryonic (E) days E10.5, E12.5, E15.5, and E18.5, and placentas and embryos were harvested. Data were analyzed separately for each gestational day controlling for litter size, fetal genotype (except for models including only +/- pups), and fetal sex (except when data were stratified by this variable). 4X choline tended to increase ( p < 0.1) placental labyrinth size at E10.5 and decrease ( p < 0.05) placental apoptosis at E12.5. Choline supplementation decreased ( p < 0.05) expression of pro-angiogenic genes Eng (E10.5, E12.5, and E15.5), and Vegf (E12.5, E15.5); and pro-inflammatory genes Il1b (at E15.5 and 18.5), Tnfα (at E12.5) and Nfκb (at E15.5) in a fetal sex-dependent manner. These findings provide support for a modulatory effect of maternal choline supplementation on biomarkers of placental function and development in a mouse model of placental insufficiency.

The placental epigenome regulates processes that affect placental and fetal development, and could be mediating some of the reported effects of maternal choline supplementation (MCS) on placental vascular development and nutrient delivery. As an extension of work previously conducted in pregnant mice, the current study sought to explore the effects of MCS on various epigenetic markers in the placenta. RNA and DNA were extracted from placentas collected on embryonic day 15.5 from pregnant mice fed a 1X or 4X choline diet, and were subjected to genome-wide sequencing procedures or mass-spectrometry-based assays to examine placental imprinted gene expression, DNA methylation patterns, and microRNA (miRNA) abundance. MCS yielded a higher (fold change = 1.63-2.25) expression of four imprinted genes ( Ampd3 , Tfpi2 , Gatm and Aqp1 ) in the female placentas and a lower (fold change = 0.46-0.62) expression of three imprinted genes ( Dcn , Qpct and Tnfrsf23 ) in the male placentas (false discovery rate (FDR) ≤ 0.05 for both sexes). Methylation in the promoter regions of these genes and global placental DNA methylation were also affected ( p ≤ 0.05). Additionally, a lower (fold change = 0.3; P unadjusted = 2.05 × 10 -4 ; FDR = 0.13) abundance of miR-2137 and a higher (fold change = 1.25-3.92; p < 0.05) expression of its target genes were detected in the 4X choline placentas. These data demonstrate that the placental epigenome is responsive to maternal choline intake during murine pregnancy and likely mediates some of the previously described choline-induced effects on placental and fetal outcomes., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Background: Fetal growth is dependent on placental nutrient supply, which is influenced by placental perfusion and transporter abundance. Previous research indicates that adequate choline nutrition during pregnancy improves placental vascular development, supporting the hypothesis that choline may affect placental nutrient transport. Objective: The present study sought to determine the impact of maternal choline supplementation (MCS) on placental nutrient transporter abundance and nutrient metabolism during late gestation. Methods: Female non-Swiss albino mice were randomly assigned to the 1×, 2×, or 4× choline diet (1.4, 2.8, and 5.6 g choline chloride/kg diet, respectively) 5 d before mating ( n = 16 dams/group). The placentas and fetuses were harvested on gestational day (E) 15.5 and E18.5. The placental abundance of macronutrient, choline, and acetylcholine transporters and glycogen metabolic enzymes, and the placental concentration of glycogen were quantified. Choline metabolites and docosahexaenoic acid (DHA) concentrations were measured in the placentas and/or fetal brains. Data were stratified by gestational day and fetal sex and were analyzed by using mixed linear models. Results: At E15.5, MCS downregulated the placental transcript and protein abundance of glucose transporter 1 (GLUT1) (-40% to -73%, P < 0.05) and the placental transcript abundance of glycogen-synthesizing enzymes (-24% to -50%, P ≤ 0.05). At E18.5, MCS upregulated GLUT3 protein abundance (+55%, P = 0.016) and the transcript abundance of glycogen-synthesizing enzymes only in the female placentas (+36% to +60%, P < 0.05), resulting in a doubling ( P = 0.01) of the glycogen concentration. A higher placental transcript abundance of the transporters for DHA, choline, and acetylcholine was also detected in response to MCS, consequently altering their concentrations in the placentas or fetal brains ( P ≤ 0.05). Conclusions: These data suggest that MCS modulates placental nutrient transporter abundance and nutrient metabolism in late gestation of mouse pregnancy, with subsequent effects on nutrient supply for the developing fetus., Competing Interests: Author disclosures: ST(C)K, JHK, JY, ZW, XJ, JSH, HRK, JTB, MSR, and MAC, no conflicts of interest., (© 2017 American Society for Nutrition.)

Impairments in placental development can adversely affect pregnancy outcomes. The bioactive nutrient choline may mitigate some of these impairments, as suggested by data in humans, animals, and human trophoblasts. Herein, we investigated the effects of maternal choline supplementation (MCS) on parameters of fetal growth in a Dlx3 +/- (distal-less homeobox 3) mouse model of placental insufficiency. Dlx3 +/- female mice were assigned to 1X (control), 2X, or 4X choline intake levels during gestation. Dams were sacrificed at embryonic days E10.5, 12.5, 15.5, and 18.5. At E10.5, placental weight, embryo weight, and placental efficiency were higher in 4X versus 1X choline. Higher concentrations of hepatic and placental betaine were detected in 4X versus 1X choline, and placental betaine was positively associated with embryo weight. Placental mRNA expression of Igf1 was downregulated by 4X (versus 1X) choline at E10.5. No differences in fetal growth parameters were detected at E12.5 and 15.5, whereas a small but significant reduction in fetal weight was detected at E18.5 in 4X versus 1X choline. MCS improved fetal growth during early pregnancy in the Dlx3 +/- mice with the compensatory downregulation of Igf1 to slow growth as gestation progressed. Placental betaine may be responsible for the growth-promoting effects of choline., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Introduction: Normal placental vascular development is influenced by inflammatory, angiogenic and apoptotic processes, which may be modulated by choline through its role in membrane biosynthesis, cellular signaling and gene expression regulation. The current study examined the effect of maternal choline supplementation (MCS) on placental inflammatory, angiogenic and apoptotic processes during murine pregnancy., Method: Pregnant dams were randomized to receive 1, 2 or 4 times (X) the normal choline content of rodent diets, and tissues were harvested on embryonic day (E) 10.5, 12.5, 15.5 or 18.5 for gene expression, protein abundance and immunohistochemical analyses., Results: The choline-induced changes in the inflammatory and angiogenic markers were a function of fetal sex. Specifically, 4X (versus 1X) choline reduced the transcript (P ≤ 0.05) and protein (P ≤ 0.06) expression of TNF-a and IL-1β in the male placentas at E10.5 and E18.5, respectively. In the female placentas, 4X (versus 1X) choline modulated the transcript expression of Il1b in a biphasic pattern with reduced Il1b at E12.5 (P = 0.045) and E18.5 (P = 0.067) but increased Il1b at E15.5 (P = 0.031). MCS also induced an upregulation of Vegfa expression in the female placentas at E15.5 (P = 0.034; 4X versus 2X) and E18.5 (P = 0.026; 4X versus 1X). MCS decreased (P = 0.011; 4X versus 1X) placental apoptosis at E10.5. Additionally, the luminal area of the maternal spiral arteries was larger (P ≤ 0.05; 4X versus 1X) in response to extra choline throughout gestation., Discussion: MCS during murine pregnancy has fetal sex-specific effects on placental inflammation and angiogenesis, with possible consequences on placental vascular development., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Fetal growth is dependent on placental nutrient supply, which is influenced by placental perfusion and transporter abundance. Previous research indicates that adequate choline nutrition during pregnancy improves placental vascular development, supporting the hypothesis that choline may affect placental nutrient transport.Objective: The present study sought to determine the impact of maternal choline supplementation (MCS) on placental nutrient transporter abundance and nutrient metabolism during late gestation.Methods: Female non-Swiss albino mice were randomly assigned to the 1×, 2×, or 4× choline diet (1.4, 2.8, and 5.6 g choline chloride/kg diet, respectively) 5 d before mating (n = 16 dams/group). The placentas and fetuses were harvested on gestational day (E) 15.5 and E18.5. The placental abundance of macronutrient, choline, and acetylcholine transporters and glycogen metabolic enzymes, and the placental concentration of glycogen were quantified. Choline metabolites and docosahexaenoic acid (DHA) concentrations were measured in the placentas and/or fetal brains. Data were stratified by gestational day and fetal sex and were analyzed by using mixed linear models.Results: At E15.5, MCS downregulated the placental transcript and protein abundance of glucose transporter 1 (GLUT1) (-40% to -73%, P < 0.05) and the placental transcript abundance of glycogen-synthesizing enzymes (-24% to -50%, P ≤ 0.05). At E18.5, MCS upregulated GLUT3 protein abundance (+55%, P = 0.016) and the transcript abundance of glycogen-synthesizing enzymes only in the female placentas (+36% to +60%, P < 0.05), resulting in a doubling (P = 0.01) of the glycogen concentration. A higher placental transcript abundance of the transporters for DHA, choline, and acetylcholine was also detected in response to MCS, consequently altering their concentrations in the placentas or fetal brains (P ≤ 0.05).Conclusions: These data suggest that MCS modulates placental nutrient transporter abundance and nutrient metabolism in late gestation of mouse pregnancy, with subsequent effects on nutrient supply for the developing fetus. [ABSTRACT FROM AUTHOR]