Demands Of Fetal Growth Research Articles

Antepartum Care for Pregnant People with Overt Hypothyroidism, Subclinical Hypothyroidism, and Positive Thyroid Autoantibodies.

Maternal thyroid hormones are critical for the growth and development of a healthy fetus. During pregnancy, maternal thyroid hormone production is increased to meet the demands of fetal growth. The purpose of this review is to provide clinicians with current evidence about possible adverse maternal and fetal effects of hypothyroidism in pregnancy, including the role of hypothyroidism in recurrent pregnancy loss. In addition, clinical presentation, evidence-based screening guidelines, and evidence-based treatments are discussed.

Male and Female, Prenatal Brain Development Differs in Response to the Maternal Omega 3 and 6 Nutritional Status

ObjectivesTo establish why prenatal brain growth in male fetuses responded to an omega-3-rich supplement but the females did not. MethodsIn a study of maternal lipid status during pregnancy in relation to regional fetal brain development a supplement containing long chain polyenoic fatty acids (300 mg of docosahexaenoic acid DHA, 42 mg eicosapentaenoic (EPA), 8.4 mg arachidonic and placebo 721 mg of oleic acid). Magnetic resonance images were obtained of newborn brains. Quantitative analysis of regions of the brain showed the supplement enhanced brain volume, with and without CSF, cortex, whole grey matter, and corpus callosum but only boys. ResultsWe wish to report correlations for arachidonic and stearic acids with several regions of brain growth in girls but not boys: for example, maternal RBC stearic acid at recruitment correlated with whole cortex (0.85 p < 0.0002), grey matter (0.847 p < 0.0002), corpus callosum (0.699 p < 0.008), whole brain (0.792 p < 0.001), brain plus CSF (0.733 p < 0.004, n = 13). Correlations with arachidonic acid at delivery reflected its index of arachidonic biomagnification and linoleic bioreduction. This measure of placental efficiency for arachidonic was for cortex (0.748 p < 0. 0034), deep grey matter (0.659 p < 0.014), whole grey matter (0.753 p < 0.003), hippocampus (0.611 p < 0.03), lentiform (0.774 p < 0.002), thalami (0.654 p < 0.015), corpus callosum (0.640 p < 0.018), brain (0.685 p < 0.0098), brain with CSF (0.774 p < 0.0019 n = 12). None were seen with the placebo boys (n = 22). Following embryogenesis, the placenta develops ahead of the demands of fetal growth. The fetal cardiovascular system is required to develop to support organogenesis and the brain growth thrust. The fetal immune system is required to help maintain pregnancy and for birth. All these systems are rich in arachidonic acid with little omega 3. The placenta biomagnifies arachidonic acid for the fetus: (typically maternal plasma lecithin 8.76% ± 1.49 CFD fetal cord 17.5% ± 3.22 p < 0.0001 (n = 44), whereas DHA in the same mothers was 4.13% ± 0.98 vs 5.79% ± 1.69 p < 0.0001. ConclusionsWe conclude that arachidonic acid is playing an as yet, unseen role, and female physiology is more focused on arachidonic acid to serve the basics of reproduction. Funding SourcesThe Mother and Child Foundation, Waterloo Foundation and the BORNE Charity.

Persistent DNA Damage and Senescence in the Placenta Impacts Developmental Outcomes of Embryos.

Cohesin is an evolutionarily conserved chromosome-associated protein complex essential for chromosome segregation, gene expression, and repair of DNA damage. Mutations that affect this complex cause the human developmental disorder Cornelia de Lange syndrome (CdLS), thought to arise from defective embryonic transcription. We establish a significant role for placental defects in the development of CdLS mouse embryos (Nipbl and Hdac8). Placenta is a naturally senescent tissue; we demonstrate that persistent DNA damage potentiates senescence and activates cytokine signaling. Mutant embryo developmental outcomes are significantly improved in the context of a wild-type placenta or by genetically restricting cytokine signaling. Our study highlights that cohesin is required for maintaining ploidy and the repair of spontaneous DNA damage in placental cells, suggesting that genotoxic stress and ensuing placental senescence and cytokine production could represent a broad theme in embryo health and viability.

Circadian Clock Regulation of Developmental Time in the Kidney

We report the emergence of an endogenous circadian clock that regulates organogenesis in mouse fetal kidney. We detect circadian rhythms both invivo with transcriptional profiling and exvivo by bioluminescence. High-resolution structural analysis of embryonic explants reveals that global or local clock disruption results in defects that resemble human congenital abnormalities of the kidney. The onset of fetal rhythms strongly correlates with the timing of a distinct transition in branching and growth rates during a gestational window of high fetal growth demands. Defects in clock mutants typically have been attributed to accelerated aging; however, our study establishes a role for the fetal circadian clock as a developmental timer that regulates the pathways that control organogenesis, branching rate, and nephron number and thus plays a fundamental role in kidney development.

Nitrite mediated vasorelaxation in human chorionic plate vessels is enhanced by hypoxia and dependent on the NO-sGC-cGMP pathway

Adequate perfusion of the placental vasculature is essential to meet the metabolic demands of fetal growth and development. Lacking neural control, local tissue metabolites, circulating and physical factors contribute significantly to blood flow regulation. Nitric oxide (NO) is a key regulator of fetoplacental vascular tone. Nitrite, previously considered an inert end-product of NO oxidation, has been shown to provide an important source of NO. Reduction of nitrite to NO may be particularly relevant in tissue when the oxygen-dependent NO synthase (NOS) activity is compromised, e.g. in hypoxia. The contribution of this pathway in the placenta is currently unknown. We hypothesised that nitrite vasodilates human placental blood vessels, with enhanced efficacy under hypoxia.Placentas were collected from uncomplicated pregnancies and the vasorelaxant effect of nitrite (10−6–5x10−3 M) was assessed using wire myography on isolated pre-constricted chorionic plate arteries (CPAs) and veins (CPVs) under normoxic (pO2 ∼5%) and hypoxic (pO2 ∼1%) conditions. The dependency on the NO–sGC–cGMP pathway and known nitrite reductase (NiR) activities was also investigated. Nitrite caused concentration-dependent vasorelaxation in both arteries and veins, and this effect was enhanced by hypoxia, significantly in CPVs (P < 0.01) and with a trend in CPAs (P = 0.054). Pre-incubation with NO scavengers (cPTIO and oxyhemoglobin) attenuated (P < 0.01 and P < 0.0001, respectively), and the sGC inhibitor ODQ completely abolished nitrite-mediated vasorelaxation, confirming the involvement of NO and sGC. Inhibition of potential NiR enzymes xanthine oxidoreductase, mitochondrial aldehyde dehydrogenase and mitochondrial bc1 complex did not attenuate vasorelaxation. This data suggests that nitrite may provide an important reservoir of NO bioactivity within the placenta to enhance blood flow when fetoplacental oxygenation is impaired, as occurring in pregnancy diseases such as pre-eclampsia and fetal growth restriction.

The effects of Nrf2 deletion on placental morphology and exchange capacity in the mouse

Objectives: Intrauterine growth restriction (IUGR) is defined as a pathological decreased fetal growth. Oxidative stress has been connected to the restriction in the fetal growth. The transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) is a potent activator of the cellular antioxidant response. The effect Nrf2 on fetal–placental development has not yet been sufficiently investigated. Here, we evaluated the placental and fetal growth in Nrf2 knockout (Nrf2-KO) and Nrf2-wild type mice (Nrf2-WT) throughout pregnancy. Methods: Heterozygote Nrf2 (Nrf2+/−) mice were paired to get Nrf2-KO and Nrf2-WT in the litters. Placentae and embryos from both genotypes were collected and weighed on days 13.5, 15.5 and 18.5 post coitum. The absolute volumes of the labyrinth zone and the total volume of the placenta were determined using the Cavalieri principle. Results: On E 18.5 the fetal weight in Nrf2-KO was significantly reduced versus Nrf2-WT indicating a decrease in placental efficiency. A significant reduction in both total and labyrinth-volume in the placenta of Nrf2-KO mice was observed. Conclusion: This data points out the necessity of functional Nrf2 for fetal and placental growth. A deficiency in Nrf2 signaling may negatively affect nutrient transfer capacity which is then no longer able to meet fetal growth demands.

Pregnancy-induced changes in the circadian expression of hepatic clock genes: implications for maternal glucose homeostasis.

Adaptations in maternal carbohydrate metabolism are particularly important in pregnancy because glucose is the principal energy substrate used by the fetus. As metabolic homeostasis is intricately linked to the circadian system via the rhythmic expression of clock genes, it is likely that metabolic adaptations during pregnancy also involve shifts in maternal circadian function. We hypothesized that maternal adaptation in pregnancy involves changes in the hepatic expression of clock genes, which drive downstream shifts in circadian expression of glucoregulatory genes. Maternal liver and plasma (n = 6-8/group) were collected across 24-h periods (0800, 1200, 1600, 2000, 0000, 0400) from C57Bl/6J mice under isoflurane-nitrous oxide anesthesia prior to and on days 6, 10, 14 and 18 of pregnancy (term = day 19). Hepatic expression of clock genes and glucoregulatory genes was determined by RT-qPCR. Hepatic clock gene expression was substantially altered across pregnancy, most notably in late gestation when the circadian rhythmicity of several clock genes was attenuated (≤64% reduced amplitude on day 18). These changes were associated with a similar decline in rhythmicity of the key glucoregulatory genes Pck1, G6Pase, and Gk, and by day 18, Pck1 was no longer rhythmic. Overall, our data show marked adaptations in the liver clock during mouse pregnancy, changes that may contribute to the altered circadian variation in glucoregulatory genes near term. We propose that the observed reduction of daily oscillations in glucose metabolism ensure a sustained supply of glucose to meet the high demands of fetal growth.

Preconception Counseling for Women with Congenital Heart Disease.

With advances that have been made over the recent decades in transcatheter and surgical interventions, most patients with congenital heart disease (CHD) can survive into adulthood. Overall, probably half of these surviving patients are female. When these female CHD patients reach childbearing age, however, pregnancy management will be a major issue. In order to meet the demands of fetal growth, the maternal cardiovascular system starts a series of adaptations beginning in early pregnancy. These adaptations include: decreased systemic and pulmonary vascular resistances, decreased blood pressure, expansion of the blood volume, increased heart rate and increased cardiac output. For women with CHD, this hemodynamic alteration may increase the risks of adverse cardiovascular events as well as the fetal and neonatal complications. Therefore, proper risk stratification and effective counseling for women with CHD who are planning their pregnancies is an important undertaking. Congenital heart disease; Pregnancy.

Peroxisome proliferator activated receptor gamma 2 modulates late pregnancy homeostatic metabolic adaptations.

Pregnancy requires the adaptation of maternal energy metabolism including expansion and functional modifications of adipose tissue. Insulin resistance (IR), predominantly during late gestation, is a physiological metabolic adaptation that serves to support the metabolic demands of fetal growth. The molecular mechanisms underlying these adaptations are not fully understood and may contribute to gestational diabetes mellitus. Peroxisome proliferator-activated receptor gamma (PPARγ) controls adipogenesis, glucose and lipid metabolism and insulin sensitivity. The PPARγ2 isoform is mainly expressed in adipocytes and is thus likely to contribute to adipose tissue adaptation during late pregnancy. In the present study, we investigated the contribution of PPARγ2 to the metabolic adaptations occurring during the late phase of pregnancy in the context of IR. Using a model of late pregnancy in PPARγ2 knockout (KO) mice, we found that deletion of PPARγ2 exacerbated IR in association with lower serum adiponectin levels, increased body weight and enhanced lipid accumulation in liver. Lack of PPARγ2 provoked changes in the distribution of fat mass and preferentially prevented the expansion of the perigonadal depot while at the same time exacerbating inflammation. PPARγ2KO pregnant mice presented adipose tissue depot-dependent decreased expression of genes involved in lipid metabolism. Collectively, these data indicate that PPARγ2 is essential to promote healthy adipose tissue expansion and immune and metabolic functionality during pregnancy, contributing to the physiological adaptations that lead gestation to term.

Maternal Factors Are Associated with the Expression of Placental Genes Involved in Amino Acid Metabolism and Transport.

IntroductionMaternal environment and lifestyle factors may modify placental function to match the mother’s capacity to support the demands of fetal growth. Much remains to be understood about maternal influences on placental metabolic and amino acid transporter gene expression. We investigated the influences of maternal lifestyle and body composition (e.g. fat and muscle content) on a selection of metabolic and amino acid transporter genes and their associations with fetal growth.MethodsRNA was extracted from 102 term Southampton Women’s Survey placental samples. Expression of nine metabolic, seven exchange, eight accumulative and three facilitated transporter genes was analyzed using quantitative real-time PCR.ResultsIncreased placental LAT2 (p = 0.01), y + LAT2 (p = 0.03), aspartate aminotransferase 2 (p = 0.02) and decreased aspartate aminotransferase 1 (p = 0.04) mRNA expression associated with pre-pregnancy maternal smoking. Placental mRNA expression of TAT1 (p = 0.01), ASCT1 (p = 0.03), mitochondrial branched chain aminotransferase (p = 0.02) and glutamine synthetase (p = 0.05) was positively associated with maternal strenuous exercise. Increased glutamine synthetase mRNA expression (r = 0.20, p = 0.05) associated with higher maternal diet quality (prudent dietary pattern) pre-pregnancy. Lower LAT4 (r = -0.25, p = 0.05) and aspartate aminotransferase 2 mRNA expression (r = -0.28, p = 0.01) associated with higher early pregnancy diet quality. Lower placental ASCT1 mRNA expression associated with measures of increased maternal fat mass, including pre-pregnancy BMI (r = -0.26, p = 0.01). Lower placental mRNA expression of alanine aminotransferase 2 associated with greater neonatal adiposity, for example neonatal subscapular skinfold thickness (r = -0.33, p = 0.001).ConclusionA number of maternal influences have been linked with outcomes in childhood, independently of neonatal size; our finding of associations between placental expression of transporter and metabolic genes and maternal smoking, physical activity and diet raises the possibility that their effects are mediated in part through alterations in placental function. The observed changes in placental gene expression in relation to modifiable maternal factors are important as they could form part of interventions aimed at maintaining a healthy lifestyle for the mother and for optimal fetal development.

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Objectives A well-developed placenta is essential for a healthy pregnancy. A widespread oxidative stress and free radical damage and the resultant damage of the maternal endothelial cells have been implicated in preeclampsia. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a potent transcription activator that regulates the expression a group of antioxidants and detoxication enzymes. Recent discussion about a link between Nrf2 and vascular angiogenic balance in preeclampsia has focused on the downstream target protein, heme oxygenase-1 (HO-1). The effect Nrf2 on placental development has not been yet discussed. In particular, we evaluated the placental and fetal growth, the feto-placental phenotype and oxidative stress status in Nrf2 knockout (Nrf2−/−) mice at different stages of pregnancy. Methods Placentae from Nrf2−/− and wild type Nrf2+/+ were collected on days 13.5, 15.5 and 18.5 post coitum. We performed H&E and Periodeic Acid Schiff on paraffin-embedded samples. Markers of oxidative stress, VEGF, Nrf2, 4-Hydroxynonenal (4-HNE) and HO-1 were assessed by immunohistochemical methods. Results At 18.5 days post coitum the fetal weight in Nrf2−/− was reduced (P In the placentas of Nrf2−/− the labyrinth cells showed increased levels of the lipid peroxidation product 4-hydroxynonenal (4-HNE). Furthermore, in Nrf2−/−-placentas there was a significant decrease in HO-1, an important endogenous antioxidant enzyme and a key regulator of trophoblast invasion and placental function. The labyrinth zones of these placentas were smaller when compared to Nrf2+/+-ones. Conclusions Our results indicated that deficiency in Nrf2 signaling may reduce the placental functions, increase the levels of oxidative stress, which may negatively affect the nutrient transfer capacity to meet fetal growth demands. Disclosures N. Kweider: None. J. Lambertz: None. T. Pufe: None. C.J. Wruck: None. W. Rath: None.

Review: Adaptation in placental nutrient supply to meet fetal growth demand: Implications for programming

This review considers the hypothesis that adaptations in blood flow, exchange surface area and transporter activity enable placental supply capacity to meet fetal demand and cause alterations in fetal composition which result in life-long programming of homeostatic set points. We consider the components of placental supply capacity and describe the predominant changes each of these could impose on solute and water exchange across the placenta. We next consider the evidence that adaptations in placental nutrient supply to meet the demands of fetal growth and development do occur. Evidence from human and mouse studies suggests that adaptations occur in regulation of blood flow through the fetoplacental circulation, in exchange barrier surface area and in transporter-mediated processes for amino acids and calcium. Crucially there appear to be differences in the gestational timing of these adaptations. Finally we suggest that each of these adaptations could have separate effects on the composition of the fetus. These could affect physiological set points in different ways and so programme the lifetime responses of the individual.

Adaptations in placental nutrient transfer capacity to meet fetal growth demands depend on placental size in mice

Experimental reduction in placental growth often leads to increased placental efficiency measured as grams of fetus produced per gram of placenta, although little is known about the mechanisms involved. This study tested the hypothesis that the smallest placenta within a litter is the most efficient at supporting fetal growth by examining the natural intra-litter variation in placental nutrient transfer capacity in normal pregnant mice. The morphology, nutrient transfer and expression of key growth and nutrient supply genes (Igf2P0, Grb10, Slc2a1, Slc2a3, Slc38a1, Slc38a2 and Slc38a4) were compared in the lightest and heaviest placentas of a litter at days 16 and 19 of pregnancy, when mouse fetuses are growing most rapidly in absolute terms. The data show that there are morphological and functional adaptations in the lightest placenta within a litter, which increase active transport of amino acids per gram of placenta and maintain normal fetal growth close to term, despite the reduced placental mass. The specific placental adaptations differ with age. At E16, they are primarily morphological with an increase in the volume fraction of the labyrinthine zone responsible for nutrient exchange, whereas at E19 they are more functional with up-regulated placental expression of the glucose transporter gene, Slc2a1/GLUT1 and one isoform the System A family of amino acid transporters, Slc38a2/SNAT2. Thus, this adaptability in placental phenotype provides a functional reserve capacity for maximizing fetal growth during late gestation when placental growth is compromised.

Fasting-induced adipose factor identified as a key adipokine that is up-regulated in white adipose tissue during pregnancy and lactation in the rat

Adipokines, which are expressed and secreted from white adipose tissue (WAT), are potential factors that could contribute to the changes in energy homeostasis that occurs in pregnancy and lactation to meet the nutrient demands of fetal growth and milk production. The aim was to identify adipokines that could be involved by measuring the pattern of their mRNA expression in adipose tissue. Adipokine mRNAs were measured by quantitative RT-PCR in RNA isolated from white and brown adipose tissue (BAT) of rats at days 7, 14 and 21 of pregnancy, day 7 of lactation and virgin at dioestrus phase. The results for leptin, adiponectin and resistin expression in WAT essentially confirmed previous studies and it is unlikely that they are directly involved in the metabolic adaptations. The relative amounts of the mRNAs of the adipokines in BAT were comparable with those in WAT, but the patterns of expression did not follow those in WAT, except for apelin. Visfatin mRNA in WAT was elevated 2.5-fold only at day 21 of pregnancy. Apelin mRNA in WAT was increased 2.2-fold at day 7 of pregnancy. Retinol-binding protein 4 mRNA in WAT decreased to 46% of control at day 14 of pregnancy. Fasting-induced adipose factor (FIAF) mRNA in WAT was 2.2- to 2.5-fold higher throughout pregnancy and lactation. The marked induction of FIAF identifies this adipokine as a potential regulator of the metabolic adaptations that occur during pregnancy and lactation.

Effect of nutrient intake during pregnancy on fetal and placental growth and vascular development

Remarkable diversity of size and health of offspring exists after normal pregnancies. When pregnancies are complicated by an extrinsic variable such as inappropriate maternal nutrition, birth weight and health of the neonate are substantially affected. The placenta is the organ through which respiratory gases, nutrients, and wastes are exchanged between the maternal and fetal systems. Thus, transplacental exchange provides for all the metabolic demands of fetal growth. Transplacental exchange is dependent upon uterine and umbilical blood flow, and blood flow rates are in turn dependent in large part upon vascularization of the placenta. Therefore, factors that influence placental vascular development will have a dramatic impact on fetal growth and development, and thereby on neonatal mortality and morbidity. Recent work from our laboratories has focused on the effects of nutrient intake during pregnancy on placental growth and vascular development. Both nutrient restriction of the adult dam and overnourishment of the adolescent dam during pregnancy suppress placental cell proliferation and vascularity. Furthermore, placental expression of angiogenic factors and their receptors, factors that are known to affect vascular growth, are perturbed by level of nutrition. Studies in this area will lead to improved methods to manage nutritionally-compromised pregnancies.

Human placental growth hormone increases expression of the p85 regulatory unit of phosphatidylinositol 3-kinase and triggers severe insulin resistance in skeletal muscle.

The insulin resistance of normal pregnancy is necessary to divert fuels to the fetus to meet fetal growth demands and is mediated by placental hormones. We recently demonstrated that human placental GH (hPGH) can trigger severe insulin resistance in transgenic (TG) mice. In this study we sought to elucidate the cellular mechanisms by which hPGH interferes with insulin signaling in muscle in TG mice. Insulin-stimulated GLUT-4 translocation to the plasma membrane (PM) was reduced in the TG compared with wild-type (WT) mice (P = 0.05). Insulin receptor (IR) levels were modestly reduced by 19% (P < 0.01) in TG mice, but there were no changes in phosphorylation of IR or IR substrate-1 (IRS-1) between WT and TG mice. A singular finding was a highly significant increase in the p85 alpha regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase; P < 0.001), yet a reduced ability of insulin to stimulate IRS-1-associated PI 3-kinase activity (P < 0.05). Although the levels of the p110 catalytic subunit protein of PI 3-kinase and IRS-1 were unchanged in the TG mice, insulin's ability to stimulate p110 association with IRS-1 was markedly reduced (P < 0.0001). We demonstrate a unique mechanism of insulin resistance and suggest that hPGH may contribute to the insulin resistance of normal pregnancy by increasing the expression of the p85 alpha monomer, which competes in a dominant negative fashion with the p85-p110 heterodimer for binding to IRS-1 protein.

Angiogenesis in the placenta.

The mammalian placenta is the organ through which respiratory gases, nutrients, and wastes are exchanged between the maternal and fetal systems. Thus, transplacental exchange provides for all the metabolic demands of fetal growth and development. The rate of transplacental exchange depends primarily on the rates of uterine (maternal placental) and umbilical (fetal placental) blood flows. In fact, increased uterine vascular resistance and reduced uterine blood flow can be used as predictors of high risk pregnancies and are associated with fetal growth retardation. The rates of placental blood flow, in turn, are dependent on placental vascularization, and placental angiogenesis is therefore critical for the successful development of viable, healthy offspring. Recent studies, including gene knockouts in mice, indicate that the vascular endothelial growth factors represent a major class of placental angiogenic factors. Other angiogenic factors, such as the fibroblast growth factors or perhaps the angiopoietins, also may play important roles in placental vascularization. In addition, recent observations suggest that these angiogenic factors interact with the local vasodilator nitric oxide to coordinate placental angiogenesis and blood flow. In the future, regulators of angiogenesis that are currently being developed may provide novel and powerful methods to ensure positive outcomes for most pregnancies.

GROWTH OF THE FETAL GUINEA PIG: PHYSICAL AND CHEMICAL CHARACTERISTICS

Achievement of the intrauterine growth rate is a goal frequently used in nutrition of preterm infants. Data on the chemical composition of the human are limited and there is curiously little similar data in experimental animals. We studied physical and chemical macronutrient accretion in the fetal guinea pig, a species in which the high fat content and rapid growth rate emphasize the demands of fetal growth upon maternal nutrition. We studied 32 fetuses in 12 litters ranging from 39 days to term (67 d), analyzing the fetuses for wet weight, dry weight, carbon, nitrogen, fat content, caloric content (bomb calorimeter) and amino acid composition. Body weight increased exponentially with gestational age at 7.1%/d. Dry weight concentration tripled from 10% to 30% by term and total dry weight increased at 10.1%/d. Fat concentration rose from 2% at 45 days to 12% at term and fat content increased 13.5%/d. The total caloric accretion rate was 100 kcal/kg/d at 45 days, increasing to 220 kcal/kg/d at term. Over 60% of the caloric accretion was due to fat accretion. Caloric values of fat and non-fat dry weight were 9.5 and 4.5 kcal/g respectively. Amino acids represented 80% of the total body nitrogen and 46% of the total body carbon. These data represent the first systematic study of chemical growth during fetal life in any experimental animal, provide accretion measurements unavailable in human studies and demonstrate the importance of fat accretion in caloric demands of fetal growth.