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.
Dams were sacrificed at embryonic days E Higher concentrations of hepatic and placental betaine were detected in 4X versus 1X choline, placental betaine was positively associated with embryo weight. No differences in fetal growth parameters were detected at E Placental betaine may be responsible for the growth-promoting effects of choline. The placenta mediates the delivery of nutrients to the fetus and is thus a major determinant of fetal growth [ 1 ].
Impairments in placental development can lead to placental insufficiency, an inability of the placenta to provide adequate nutrients for the fetus, and contribute to adverse pregnancy outcomes, including intrauterine growth restriction IUGRmiscarriage, and preeclampsia [ 23 ].
At present, very few treatment options are available for placental-induced pregnancy complications that adversely affect the health of the mother and child.
Nutritional Choline supplements and sexual growth represent a promising area of research for improving maternal and child health [ 4 ], especially since some pharmacological treatments may be unsafe during pregnancy. Choline is a water-soluble essential nutrient that is necessary for the synthesis of acetylcholine a neurotransmitterphosphatidylcholine a membrane phospholipidand betaine a methyl donor and osmolyte [ 5 ].
During pregnancy, these molecules support rapid cell division, govern genomic methylation patterns, and modulate placental and fetal development [ 6 ]. Choline has previously been shown to reduce a risk factor of Choline supplements and sexual growth, sFLT1, in a randomized, controlled feeding trial of maternal choline supplementation MCS during the third trimester of pregnancy in healthy women [ 7 ]. In addition, choline has been shown to modulate inflammation, oxidative stress, and apoptosis of immortalized human placental trophoblast cells in culture [ 8 ] and in normal murine pregnancy [ 9 ].
Therefore, we hypothesized that choline supplementation would be beneficial for pregnancies complicated by placental insufficiency. During pregnancy, the homeodomain-containing transcription factor distal-less homeobox 3 Dlx3 plays a crucial role in the development of the fetal-maternal interface [ 10 ]. Mice heterozygous for this gene have been shown to Choline supplements and sexual growth inadequate vascularization and development of the placental labyrinth, which is the location of nutrient exchange between
Choline supplements and sexual growth and fetus [ 11 ].
Mice that are homozygous null for Dlx3 die between embryonic days 9. Heterozygous embryos are viable but have been reported to display placental abnormalities, including a reduced invasion of trophoblasts and impaired remodeling of maternal spiral arteries as well as increased placental oxidative stress and apoptosis within the labyrinth and maternal decidua [ 11 ].
The expression of IGF genes as well as the epidermal growth factor receptor EGFRwhich interacts with IGF1R [ 15 ], has been shown to be modulated by choline supply in various tissues and gestational time points of pregnancy [ 161718 ].
The present study was undertaken to investigate the effects of MCS on the determinants and parameters of fetal growth in the Dlx3 murine model of placental insufficiency. We hypothesized that a maternal choline supplementation would support fetal growth in placental insufficiency and b this effect may occur through the IGF axis. All animal protocols and procedures used in this study were approved by the Institutional Animal Care and Use Committees at Cornell University and conducted in accordance with the Guide for the Care and Use of Laboratory Animals protocol number The primers were designed to amplify wildtype or knockout allele amplicons or both indicating a heterozygote Supplementary Table S1.
Breeding animals were given ad libitum access to commercial rodent chow Teklad and water. The 2X treatment was chosen to correspond with the previous human feeding study conducted by our research group [ 19 ].
A 4X treatment is widely used to investigate cognitive effects in rodent models. The presence of a vaginal plug was used to designate embryonic E day 0. Pregnant mice were euthanized at four different gestational time points E The number of implantations and resorptions in the gravid uterus were recorded. Embryos and placentas were carefully dissected to minimize decidual contamination and weighed.
Due to the smaller tissue size, E The primers are listed in Supplementary Table S1. Fetal body composition analyses were determined on fetuses acquired from E Fetuses were decapitated at "Choline supplements and sexual growth" to allow for analyses of the brain in a separate study; all body composition data is reported on the remaining tissue.
Briefly, the fetuses were thawed and weighed, followed by desiccation to determine water weight. Total lipid extractions were successively performed until no change in weight occurred. The protein content was determined colorimetrically, following the digestion of pulverized fetal tissue in radioimmunoprecipitation assay RIPA buffer.
Choline supplements and sexual growth
Choline, betaine, and methionine in the placenta were measured using the method of Holm et al. Two or three placentas per dam were randomly selected for extraction. The reaction conditions were as follows: A melting curve analysis was included at the end of the amplification Choline supplements and sexual growth to ensure the specificity of the PCR product. Tbp has been shown to be stable in response to various choline treatments [ 24 ]. For maternal measurements, data were analyzed using general linear models with treatment and litter size as independent variables.
For placental and fetal measurements, data were analyzed using a linear mixed model.
The model included choline treatment, fetal sex, and litter size as fixed effects and maternal identifier as a random effect. Data was either stratified by fetal Dlx3 genotype or, when all genotypes were combined, was adjusted for fetal genotype as a fixed effect. Data were assessed for normality and log-transformed if the residuals were not normally distributed. Corrections for multiple analyses were not performed due to the hypothesis-driven nature of the study and the "Choline supplements and sexual growth" limited sample size.
To determine whether choline treatment during pregnancy could improve viability, we examined indicators of embryo survival. We also assessed whether MCS could increase or prolong the survival of homozygous null fetuses at E Choline treatment at 2X or 4X did not significantly alter embryo genotype distributions at any gestational time point.
Fetal and placental growth characteristics by choline treatment 1X control, 2X, and 4X and Dlx3 genotype. Embryo weight at A E Placental weight at C E Crown-rump length at E E Data were analyzed using mixed linear models controlling for maternal identifier, fetal sex, and litter size.
Placental weight was significantly higher at E Placental weight was not influenced by choline treatment at E However, when comparing 2X to 4X, heterozygotes in the 4X group had slightly lower embryo weights at E We also assessed the impact of altered growth patterns on fetal body composition at E Choline treatment at 2X or 4X did not alter the percentage of body water, fat, or protein compared to 1X choline Supplementary Table S2.
Since the maternal liver is the source of choline metabolites, which are used by the placenta to support fetal growth and development, we measured the concentration of maternal hepatic choline metabolites Table 1.
Maternal liver of choline metabolites per gram tissue at E Data were analyzed using general linear models with treatment and litter size as fixed factors. No significant differences in GPC at E No significant differences in hepatic choline, methionine, phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin were detected at any gestational time point Table 1.
Placental concentrations of betaine were also higher in the 4X group at E However, betaine concentration did not differ by treatment in late pregnancy E Placental concentrations of free choline and methionine were not significantly affected by choline treatment at any time point; however, methionine tended to be lower at E Data were analyzed using mixed linear models with treatment, genotype, sex, and litter size as fixed effects and maternal ID as random effect.
Since changes in fetal and placental growth were seen with choline treatment at E Choline supplements and sexual growth placental expression of Igf2 did not significantly differ at either time point Figure 2 B. Neither Igf1r nor Igf2r was significantly affected by choline treatment at E Fold changes are expressed relative to the housekeeping gene Tbpwith the 1X control group normalized to 1.