CONTEXT:The regulation of pubertal timing and reproductive axis maturation is influenced by a myriad of physiologic and environmental inputs yet remains incompletely understood. OBJECTIVE:To contrast differences in bile acid isoform profiles across defined stages of reproductive maturity in humans and a rat model of puberty and to characterize the role of bile acid signaling via hypothalamic expression of bile acid receptor populations in the rodent model. METHODS:Secondary analysis and pilot studies of clinical cohorts, rodent models, ex vivo analyses of rodent hypothalamic tissues. Bile acid concentrations is the main outcome measure. RESULTS:Lower circulatory conjugated:deconjugated bile acid concentrations and higher total secondary bile acids were observed in postmenarcheal vs pre-/early pubertal adolescents, with similar shifts observed in infantile (postnatal day [PN]14) vs early juvenile (PN21) rats alongside increased tgr5 receptor mRNA expression within the mediobasal hypothalamus of female rats. 16S rRNA gene sequencing of the rodent gut microbiome across postnatal life revealed changes in the gut microbial composition predicted to have bile salt hydrolase activity, which was observed in parallel with the increased deconjugated and increased concentrations of secondary bile acids. We show that TGR5-stimulated GnRH release from hypothalamic explants is mediated through kisspeptin receptors and that early overexpression of human-TGR5 within the arcuate nucleus accelerates pubertal onset in female rats. CONCLUSION:Bile acid isoform shifts along stages of reproductive maturation are conserved across rodents and humans, with preclinical models providing mechanistic insight for the neuroendocrine-hepatic-gut microbiome axis as a potential moderator of pubertal timing in females.

The intricate changes in endocrine function during sexual maturation in the heifer are slowly being elucidated through basic research endeavors. Understanding the hypothalamic-pituitary-gonadal axis of the pre-, peri- and postpubertal bovine female will be of great benefit in developing management techniques that impact on age at which puberty occurs. Age at which heifers reach puberty is influenced by the breeds of the sire and dam used to produce replacement heifers. Because puberty is such a highly heritable trait the use of sires and dams which reached puberty earlier in life would result in an earlier age at puberty in replacement heifers. Brahman heifers exhibited a definite seasonal variation in sexual activity as measured by presence of corpora lutea and size and tone of the uterus. Sexual activity increased during the spring and was highest during the summer. Responsiveness of heifers to potential pheromonal cues from bulls appeared to be dependent upon body weight of heifers.

AbstractThe major facilitator superfamily domain 2a protein was identified recently as a lysophosphatidylcholine (LPC) symporter with high affinity for LPC species enriched with DHA (LPC-DHA). To test the hypothesis that reproductive state and choline intake influence plasma LPC-DHA, we performed a post hoc analysis of samples available through 10 weeks of a previously conducted feeding study, which provided two doses of choline (480 and 930 mg/d) to non-pregnant (n 21), third-trimester pregnant (n 26), and lactating (n 24) women; all participants consumed 200 mg of supplemental DHA and 22 % of their daily choline intake as 2H-labelled choline. The effects of reproductive state and choline intake on total LPC-DHA (expressed as a percentage of LPC) and plasma enrichments of labelled LPC and LPC-DHA were assessed using mixed and generalised linear models. Reproductive state interacted with time (P = 0·001) to influence total LPC-DHA, which significantly increased by week 10 in non-pregnant women, but not in pregnant or lactating women. Contrary to total LPC-DHA, patterns of labelled LPC-DHA enrichments were discordant between pregnant and lactating women (P < 0·05), suggestive of unique, reproductive state-specific mechanisms that result in reduced production and/or enhanced clearance of LPC-DHA during pregnancy and lactation. Regardless of the reproductive state, women consuming 930 v. 480 mg choline per d exhibited no change in total LPC-DHA but higher d3-LPC-DHA (P = 0·02), indicating that higher choline intakes favour the production of LPC-DHA from the phosphatidylethanolamine N-methyltransferase pathway of phosphatidylcholine biosynthesis. Our results warrant further investigation into the effect of reproductive state and dietary choline on LPC-DHA dynamics and its contribution to DHA status.

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.

Extracellular-signal-regulated kinases (ERK) 1 and 2 regulate many aspects of the hypothalamic-pituitary-gonadal axis. We sought to understand the role of ERK1/2 signaling in cells expressing a Cre allele regulated by the endogenous GnRHR promoter (GRIC-ERKdko). Adult female GRIC-ERKdko mice were hypogonadotropic and anovulatory. Gonadotropin administration and mating led to pregnancy in one-third of the ERKdko females. Litters from ERKdko females and pup weights were reduced coincident with delayed parturition and 100% neonatal mortality. Based on this, we examined Cre expression in implantation sites as a potential mechanism. GnRHR mRNA levels at e10.5 and e12.5 were comparable to pituitary levels from adult female mice at proestrus and GnRHR mRNA in decidua was enriched compared to whole implantation site. In vivo studies confirmed recombination in decidua, and GRIC-ERKdko placentas showed reduced ERK2 expression. Histopathology revealed abnormalities in placental architecture in the GRIC-ERKdko animals. Regions of apoptosis at the decidual/uterine interface at e18.5 were observed in control animals but apoptotic tone in these regions was reduced in ERKdko animals. These studies support a potential model of ERK-dependent signaling within the implantation site leading to loss of placental architecture and mis-regulation of apoptotic events at parturition occurring coincident with prolonged gestation and neonatal mortality.

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.

Extracellular signal-regulated kinase (ERK) signaling regulates hormone action in the reproductive axis, but specific mechanisms have yet to be completely elucidated. In the current study, ERK1 null and ERK2 floxed mice were combined with a gonadotropin-releasing hormone receptor (GnRHR)-internal ribosomal entry site-Cre (GRIC) driver. Female ERK double-knockout (ERKdko) animals were hypogonadotropic, resulting in anovulation and complete infertility. Transcript levels of four gonadotrope-specific genes (GnRHR and the three gonadotropin subunits) were reduced in pituitaries at estrus in ERKdko females, and the postcastration response to endogenous GnRH hyperstimulation was blunted. As females aged, they exhibited abnormal ovarian histology, as well as increased body weight. ERKdko males were initially less affected, showing moderate subfertility, up to 6 months of age. Male ERKdko mice also displayed a blunted response to endogenous GnRH following castration. By 12 months of age, ERKdko males had reduced testicular weights and sperm production. By 18 months of age, the ERKdko males displayed reduced testis and seminal vesicle weights, marked seminiferous tubule degeneration, and a 77% reduction in sperm production relative to controls. As the GRIC is also active in the male germ line, we examined the specific role of ERK loss in the testes using the stimulated by retinoic acid 8 (Stra8)-Cre driver. Whereas ERK loss in GRIC and Stra8 males resulted in comparable losses in sperm production, seminiferous tubule histological degeneration was only observed in the GRIC-ERKdko animals. Our data suggest that loss of ERK signaling and hypogonadotropism within the reproductive axis impacts fertility and gonadal aging.

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; Punadjusted = 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.

The natriuretic peptides, Atrial-, B-type and C-type natriuretric peptides (ANP, BNP, CNP), are regulators of many endocrine tissues and exert their effects predominantly through the activation of their specific guanylyl cyclase receptors (GC-A and GC-B) to generate cGMP. Whereas cGMP-independent signalling has been reported in response to natriuretic peptides, this is mediated via either the clearance receptor (Npr-C) or a renal-specific NPR-Bi isoform, which both lack intrinsic guanylyl cyclase activity. Here, we report evidence of GC-B-dependent cGMP-independent signalling in pituitary GH3 cells. Stimulation of GH3 cells with CNP resulted in a rapid and sustained enhancement of ERK1/2 phosphorylation (P-ERK1/2), an effect that was not mimicked by dibutryl-cGMP. Furthermore, CNP-stimulated P-ERK1/2 occurred at concentrations below that required for cGMP accumulation. The effect of CNP on P-ERK1/2 was sensitive to pharmacological blockade of MEK (U0126) and Src kinases (PP2). Silencing of the GC-B1 and GC-B2 splice variants of the GC-B receptor by using targeted short interfering RNAs completely blocked the CNP effects on P-ERK1/2. CNP failed to alter GH3 cell proliferation or cell cycle distribution but caused a concentration-dependent increase in the activity of the human glycoprotein α-subunit promoter (αGSU) in a MEK-dependent manner. Finally, CNP also activated the p38 and JNK MAPK pathways in GH3 cells. These findings reveal an additional mechanism of GC-B signalling and suggest additional biological roles for CNP in its target tissues.

The hypothalamic-pituitary-gonadal axis controls reproduction via a series of hormones regulating gonadal function through interconnected feedback loops. Secretion of hypothalamic-derived gonadotropin-releasing hormone (GnRH) integrates inputs from higher brain centers to coordinate the activity of the pituitary gonadotrope and the biosynthesis and secretion of the gonadotropins which ultimately regulate gonadal function. Failure of GnRH to serve as the central integrator of this system has been associated with hypogonadotropic-hypogonadism and clinical infertility, while pharmacological application of GnRH analogs and gonadotropins have important implications of the treatment of such infertility. Furthermore, the GnRH-GnRH receptor system has been characterized in several types of cancer and may offer therapeutic possibilities in their treatment. Given the central role of GnRH action in the control of fertility, it is of paramount importance to understand the molecular basis of control of GnRH action in the pituitary gonadotrope, including new and novel alternate ways to modulate GnRH action and gonadotropin secretion. The goal of this review is to discuss several new findings in this field focusing on novel regulators of GnRH action.

The placental transcription factors Distal-less 3 (DLX3) and Glial cell missing-1 (GCM1) have been shown to coordinate the specific regulation of PGF in human trophoblast cell lines. While both factors independently have a positive effect on PGF gene expression, when combined, DLX3 acts as an antagonist to GCM. Despite this understanding, potential mechanisms accounting for this regulatory interaction remain unexplored. We identify physical and functional interactions between specific domains of DLX3 and GCM1 in human trophoblast-derived cells by performing immunoprecipitation and mammalian one hybrid assays. Studies revealed that DLX3 binding reduced the transcriptional activity of GCM1, providing a mechanistic explanation of their functional antagonism in regulating PGF promoter activity. The DLX3 homeodomain (HD) was essential for DLX3-GCM1 interaction, and that the HD together with the DLX3 amino- or carboxyl-terminal domains was required for maximal inhibition of GCM1. Interestingly, a naturally occurring DLX3 mutant that disrupts the carboxyl-terminal domain leading to tricho-dento-osseous syndrome in humans displayed activities indistinguishable from wild type DLX3 in this system. Collectively, our studies demonstrate that DLX3 physically interacts with GCM1 and inhibits its transactivation activity, suggesting that DLX3 and GCM1 may form a complex to functionally regulate placental cell function through modulation of target gene expression.

Choline is an essential micronutrient that provides methyl groups for cellular DNA methylation, an epigenetic modification with downstream effects on gene expression. Maternal choline supplementation (MCS) during pregnancy has many beneficial effects on placental and fetal development, which may be mediated in part by an altered placental methylation profile. In addition to DNA methylation, other epigenetic mechanisms such as genomic imprinting and microRNA (miRNA) regulation are also important to normal placental and fetal development, but whether these markers are modulated by choline remains largely unknown. We sought to investigate the impact of MCS on these epigenetic markers using a subset of placental samples collected at gestational day 15.5 from dams consuming 1× or 4× the recommended choline level. Placental mRNA and miRNA were extracted and sequenced, and the RNAs with differential expression were determined using the edgeR package developed in R. In response to MCS, three imprinted genes (Dcn, Qpct and Tnfrsf23) were downregulated in the male placentas (Padj≤0.01) whereas four imprinted genes (Ampd3, Tfpi2, Gatm, and Aqp1) were upregulated in the female placentas (Padj≤0.05). Furthermore, five miRNAs (miR‐712‐5p, miR‐3470a, miR‐6538, miR‐6240 and miR‐5126) were significantly downregulated in the female placentas from the 4× choline group (Padj≤0.033). Consistent with the downregulation of these miRNAs, some of their predicted mRNA targets showed a higher expression (P<0.05). These target genes are involved in processes such as cell signaling, immune response and macronutrient transport, all of which are crucial to placental development and fetal health. In contrast, none of the miRNAs in the male placentas remained significant after correction for false discovery rate (Padj>0.05). Overall, these data indicate that the placental imprintome and miRNA profile are responsive to MCS, but the effects may differ between the male and the female placentas.Support or Funding InformationUSDA, NIH, Graduate Women in Science, Egg Nutrition Center

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.

The placenta is responsible for delivering nutrients such as glucose to the fetus to support its growth and development, which is especially important during late gestation when fetal growth is most rapid. The ability of the placenta to efficiently provide nutrients is dependent on its vascular development and the abundance of nutrient transporters at the maternal fetal interface. Maternal choline supplementation (MCS) was shown to improve placental vasculature, but it is still unknown if MCS affects placental transporter abundance and fetal nutrient delivery. To test the hypothesis that MCS beneficially modulates placental glucose supply, pregnant mice were fed a diet containing 1, 2 or 4 times the recommended choline level. The placentas were collected at gestational day (E) 15.5 and 18.5 for evaluating the expression of key glucose transporters and enzymes involved in glycogen metabolism as well as the glycogen concentration. Data were analyzed using a mixed linear model. At E15.5, MCS reduced the membrane protein abundance of Glut1 transporter as well as the mRNA expression of the glycogen synthesizing enzyme Gys1 (p<0.05). The mRNA expression of the glycogen catabolic enzyme Pygm in the female placentas also tended to increase (p=0.08). Consistent with these findings, histological staining of placental glycogen tended to be lower (p=0.08). At E18.5, MCS had no effect on Glut1 transporter expression in either male or female placentas. However, MCS increased the membrane protein abundance of Glut3 transporter and the mRNA abundance of Gys1 in the female placentas (p<0.05). These changes resulted in a 2‐fold increase in glycogen concentration in the female placentas, as shown by histological staining and biochemical measurement (p<0.05). No such changes were seen in the male placentas at this time point. Overall, these data indicate that MCS modulates placental glucose transporter expression and glycogen metabolism in a manner highly dependent on fetal sex. We suggest that these choline‐mediated changes in placental glucose metabolism differentially influence glucose delivery to the fetus, with potential consequences on offspring development and disease risk in later life.Support or Funding InformationUSDA and Egg Nutrition Center

This chapter focuses on sunflower lecithin. Sunflower lecithin is not produced in considerable amounts worldwide. This fact is mainly because of the low lecithin content of crude sunflower oil as compared with 2.9% for soybean, 1.9% for rapeseed, 2.4% for cottonseed, and 2.0–2.7% for corn oil (normalized at 70% of insolubles in acetone). In Argentina, the production of sunflower oil is of utmost importance from an economic point of view. In this country, sunflower lecithin could represent an alternative to soybean lecithin because it is considered a non-Genetically Modified Organism (GMO) product, which is currently preferred by certain consumers. The chapter presents the phosphatide composition of vegetable lecithins obtained from different oils. Distribution of the main phospholipid components of sunflower lecithin appears to be rather similar to that of soybean lecithin. Moreover, the fatty acid composition of the phosphatides reflects the fatty acid composition of the oil in which these phosphatides occur, but it tends to have higher palmitic acid content and lower oleic acid content than the oil, as illustrated by the chapter. Sunflower lecithin is a promising alternative to soybean lecithin because it is the product of a non-GMO. Lecithin modification under industrial conditions with adequate techniques of analysis may be useful for evaluating the potential applications of these sunflower byproducts to the production of new emulsifiers.

Placental growth factor (PGF) is abundantly expressed by trophoblast cells within human placentae and is important for trophoblast development and placental vascularization. Circulating maternal serum levels of PGF are dynamically upregulated across gestation in normal pregnancies, whereas low circulating levels and placental production of PGF have been implicated in the pathogenesis of preeclampsia and other gestational diseases. However, the underlying molecular mechanism of regulating PGF expression in the human placenta remains poorly understood. In this study, we demonstrated that transcription factors Distal-less 3 (DLX3) and Glial cell missing-1 (GCM1) were both sufficient and required for PGF expression in human trophoblast-derived cells by overexpression and knockdown approaches. Surprisingly, while DLX3 and GCM1 were both positive regulators of PGF, co-overexpression of DLX3andGCM1 led to an antagonist effect on PGF expression on the endogenous gene and a luciferase reporter. Further, deletion and site-directed mutagenesis studies identified a novel regulatory element on the PGF promoter mediating both DLX3-and GCM1-dependent PGF expression. This regulatory region was also found to be essential for the basal activity of the PGF promoter. Finally, Chromatin-immunoprecipitation (ChIP) assays revealed colocalization of DLX3 and GCM1 at the identified regulatory region on the PGF promoter. Taken together, our studies provide important insights into intrinsic regulation of human placental PGF expression through the functional coordination of DLX3 and GCM1, and are likely to further the understanding of pathogenesis of PGF dysregulation in preeclampsia and other disease conditions.

Fertility in mammals requires appropriate communication within the hypothalamic-pituitary-gonadal axis and the GnRH receptor (GnRHR) is a central conduit for this communication. The GnRHR resides in discrete membrane rafts and raft occupancy is required for signaling by GnRH. The present studies use immunoprecipitation and mass spectrometry to define peptides present within the raft associated with the GnRHR and flotillin-1, a key raft marker. These studies revealed peptides from the F0F1 ATP synthase complex. The catalytic subunits of the F1 domain were validated by immunoprecipitation, flow cytometry, and cell surface biotinylation studies demonstrating that this complex was present at the plasma membrane associated with the GnRHR. The F1 catalytic domain faces the extracellular space and catalyzes ATP synthesis when presented with ADP in normal mouse pituitary explants and a gonadotrope cell line. Steady-state extracellular ATP accumulation was blunted by coadministration of inhibitory factor 1, limiting inorganic phosphate in the media, and by chronic stimulation of the GnRHR. Steady-state extracellular ATP accumulation was enhanced by pharmacological inhibition of ecto-nucleoside triphosphate diphosphohydrolases. Kisspeptin administration induced coincident GnRH and ATP release from the median eminence into the hypophyseal-portal vasculature in ovariectomized sheep. Elevated levels of extracellular ATP augmented GnRH-induced secretion of LH from pituitary cells in primary culture, which was blocked in media containing low inorganic phosphate supporting the importance of extracellular ATP levels to gonadotrope cell function. These studies indicate that gonadotropes have intrinsic ability to metabolize ATP in the extracellular space and extracellular ATP may serve as a modulator of GnRH-induced LH secretion.

A properly functioning placenta allows efficient transfer of nutrients from mother to fetus and promotes fetal growth. Decreased placental efficiency is commonly associated with intrauterine growth restriction, preeclampsia, and miscarriage. Choline is an essential nutrient that is a precursor for several molecules with crucial roles in fetal development: betaine, an osmolyte and methyl donor; acetylcholine, a neurotransmitter and signaling molecule; and phosphatidylcholine (PC), the most prevalent membrane phospholipid. We previously showed that maternal choline supplementation (MCS) in the Dlx3+/ − mouse, a model of placental insufficiency, increases fetal growth in early gestation and lowers expression of pro‐angiogenic genes implicated in preeclampsia. To explore the mechanisms contributing to these effects, we measured choline metabolites in the maternal liver and placentas of Dlx3+/ − female mice consuming 1X (control), 2X or 4X the recommended intake levels of choline during gestation. Additionally, we measured expression of inflammatory genes that are elevated in preeclampsia and indicative of placental dysfunction. Dams were sacrificed at embryonic days E10.5, E12.5, E15.5, and E18.5. Data were analyzed using linear mixed models with dam as a random factor. MCS increased betaine concentrations in the maternal liver at all four time points (p<.01) and prevented a decline in maternal hepatic betaine stores in late pregnancy. 4X choline also increased phosphocholine, a precursor of PC, at E15.5 (p=.014), and decreased glycerophosphocholine, a product of PC breakdown, at E12.5 (p=.025). In the placenta, MCS increased betaine concentrations at E10.5 and 12.5 (p=.005 and .041, respectively). These data suggest that effects of MCS may be primarily driven by betaine, potentially through increased supply of methyl groups for one‐carbon reactions including DNA methylation. This is supported by data demonstrating widespread effects of choline on gene expression. In Dlx3+/ − placentas, choline decreased (p<0.05) expression of pro‐inflammatory genes Il1b (at E12.5, 15.5 and 18.5), Tnf‐α (at E12.5) and NfκB (at E15.5). Further studies are ongoing to identify downstream effects of increased betaine on placental DNA methylation and growth factor signaling pathways. Support or Funding Information Funding provided by USDA‐NIFA 2012‐67017‐30176