Complex, coordinated and highly regulated changes in placental signaling and nutrient transport capacity in IUGR

Abstract

The most common cause of intrauterine growth restriction (IUGR) in the developed world is placental insufficiency, a concept often used synonymously with reduced utero-placental and umbilical blood flows. However, placental insufficiency and IUGR are associated with complex, coordinated and highly regulated changes in placental signaling and nutrient transport including inhibition of insulin and mTOR signaling and down-regulation of specific amino acid transporters, Na⁺/K⁺-ATPase, the Na^+/H⁺-exchanger, folate and lactate transporters. In contrast, placental glucose transport capacity is unaltered and Ca²⁺-ATPase activity and the expression of proteins involved in placental lipid transport are increased in IUGR. These findings are not entirely consistent with the traditional view that the placenta is dysfunctional in IUGR, but rather suggest that the placenta adapts to reduce fetal growth in response to an inability of the mother to allocate resources to the fetus. This new model has implications for the understanding of the mechanisms underpinning IUGR and for the development of intervention strategies.

Introduction

Intrauterine growth restriction (IUGR) is often defined as the failure of the fetus to reach its genetic growth potential in utero and affects 3–7% of all newborns [1]. IUGR increases the risk of perinatal complications [2, 3], including stillbirth [4, 5], and predisposes the infant to the development of diabetes and cardiovascular disease in childhood and adult age [6–8]. However, the pathophysiology underlying IUGR remains poorly understood, no specific treatment is available and biomarkers for early detection are lacking [9, 10].

An incomplete trophoblast invasion resulting in poor spiral artery remodeling, which restricts the normal increase in utero-placental blood flow in mid-pregnancy is believed to be the most common etiology of IUGR in developed countries. In the literature it is often assumed that placental insufficiency, i.e. impaired delivery of nutrients and oxygen to the fetus, is directly caused by the reduced placental blood flow. However, the placental blood flow reduction per se is unlikely to adequately explain the impaired placental transfer in IUGR. Oxygen, which is highly lipophilic, diffuses rapidly across the placental barrier and placental oxygen transfer is therefore primarily limited by the rate of blood flow on the two sides of the barrier, and to some degree the barrier thickness. Thus, it is likely that the reduction in utero-placental and umbilical blood flows contribute to fetal hypoxia that develops in some IUGR fetuses. In contrast, transplacental transport of nutrients such as glucose and amino acids are less affected by changes in blood flow because the transport across the barrier is the primary limiting factor for the transfer of these molecules. Thus, placental insufficiency may not be just a matter of reduced blood flow or a small placenta.

Herein, we will briefly review experimental evidence suggesting that complex regulation of multiple placental signaling pathways and down-regulation of placental nutrient transporters directly contributes to the development of IUGR. Although the focus will be on changes in placental function in human IUGR, we will discuss selected animal models to address mechanistic links and to illustrate that very different causes of IUGR appear to be associated with strikingly similar changes in placental signaling and function. Moreover, we will speculate on the evolutionary origins of placental responses to changes in utero-placental blood flow as well as maternal nutrition and metabolism. Subsequently, we will present a model that can help explain a range of fetal growth patterns in response to variation in the ability of the maternal supply line to allocate resources for fetal growth. Finally, we will discuss novel avenues to prevent or improve outcomes in IUGR targeting placental function based on this new understanding.

The placental barrier in the human

In the term human placenta, there are only two largely continuous cell layers between the maternal blood in the intervillous space and the fetal blood in the capillaries of the terminal villi: the syncytiotrophoblast and the capillary endothelial cells (Fig 1). The syncytiotrophoblast is the transporting epithelium of the human placenta and is a true syncytium generated by fusion of underlying cytotrophoblast cells. Because the fetal-placental capillary endothelial cells are of the so called “continuous” type, and therefore allow a relatively unrestricted transfer of molecules such as glucose and amino acids through intercellular junctions [11], the syncytiotrophoblast represents the primary barrier for movement of most solutes from the maternal to the fetal circulations (Fig 1). Specifically, transfer across the two polarized plasma membranes of the syncytiotrophoblast, the apical or microvillous plasma membrane (MVM) directed to maternal blood in the intervillous space and the basal plasma membrane (BM) facing the fetal capillaries constitute the limiting step. Maternal-fetal exchange of most nutrients and ions occurs by mediated transfer involving transporter proteins expressed in the syncytiotrophoblast plasma membranes.

Fig 1. The placental barrier at term and a model for placental transport of neutral amino acids.

The syncytiotrophoblast cell layer (ST) and in particular its two polarized plasma membranes, the microvilllous (MVM) and basal plasma membranes (BM) constitute the primary barrier for the transfer of molecules such as glucose, amino acids and folate. The uptake of neutral amino acids from the maternal circulation across the MVM represents the active step of amino acid transport and is largely mediated by the System A and System L amino acid transporters. System A is predominantly expressed in the MVM and transports non-essential neutral amino acids (NEAA) against a concentration gradient energized by the inwardly directed Na⁺-gradient. System L is an exchanger, which uses the steep outwardly directed concentration gradient of some NEAAs to drive uptake of essential amino acids (EAA) against their concentration gradients. Amino acids are transferred across BM by facilitated diffusion driven by the outwardly directed concentration gradient mediated by System L and efflux transporters. N,nucleus; ET, endothelial cells of the fetal capillary.

The description of the human placental barrier above is a simplification. First, a number of other cell types are present in the barrier even at term, including cytotrophoblasts, macrophages (Hofbauer cells), and fibroblasts. Albeit not forming a continuous layer and therefore not directly limiting transplacental transfer, these cell types may influence transport across the barrier by their substrate metabolism. For example, it has been reported that cytotrophoblast cells contribute significantly to glycolysis and oxidative phosphorylation in the term placenta [12] and changes in cytotrophoblast metabolism could therefore impact transplacental transport of, for example, glucose. Second, for larger molecules including immunoglobulins and lipids, the endothelial cell barrier becomes limiting for transplacental transport and for these molecules mechanisms accounting for the transport across the endothelial cells must be considered in order to model transfer across the entire barrier [11, 13]. Third, in early pregnancy cytotrophoblast cells are highly abundant, forming a largely continuous cell layer between the syncytium and the fetal capillary [14] which is therefore likely to directly contribute to the barrier properties of the human placenta in first trimester.

Changes in placental signaling in IUGR

The syncytiotrophoblast serves not only as the transporting epithelium but also as the primary endocrine cell of the human placenta, integrating a multitude of maternal and fetal signals to balance maternal supply and fetal demand. It does so by altering placental growth and function and by secreting signaling molecules, which directly alter maternal physiology and fetal development. Although complex signaling pathways with extensive cross-talk regulate this functional diversity, the discussion of placental signaling in IUGR will be limited to a few signaling pathways that have been studied in some detail and that have been linked to specific placental functions. This data is summarized in Fig 2.

Fig 2. Changes in placental signaling in IUGR.

Placental insulin/IGF-1 and mTORC1 and mTORC2 signaling is inhibited and the ER stress pathway is activated in IUGR. For details, see text. Abbreviations: IGF-1: insulin-like growth factor I; IGF1-R: insulin-like growth factor 1 receptor; AKT: protein kinase B; mTORC1: mechanistic target of rapamycin complex 1; mTORC2: mechanistic target of rapamycin complex 2; eIF2α: eukaryotic initiation factor 2α.

Insulin/IGF signaling

The insulin-like growth factor (IGF) family consists of polypeptide ligands (insulin, IGF-1, and IGF-2), tyrosine kinase receptors (in particular the IGF1 receptor and insulin receptors) and IGF binding proteins (IGFBPs). Insulin [15–17] and IGF-1 [17–19] positively regulate the system A amino acid transporter in the trophoblast and the placenta. The insulin receptor (INSR) gene undergoes differential splicing generating two isoforms, INSR-A and INSR-B. Insulin binds to both INSR-A and ISNR-B with high affinity. IGF-1 predominantly interacts with the IGF-1 receptor and the biological effects of IGF-2 are mediated by binding to INSR-A [20]. IGFs, in particular IGF-1, are key regulators of fetal growth [21, 22] and placental and maternal IGFs promote placental growth and function [23–25], such as amino acid transport [26]. The bioavailability of IGFs is tightly regulated by IGF binding proteins and IGFBP-1 is believed to be the predominant IGF binding protein in the fetus and in pregnant women. Phosphorylation of IGFBP-1 at three serine residues (Ser101, 119 and 169) is known to markedly increase its affinity for binding IGF-1 [27], thus affecting the ability of IGF-1 to interact with the IGF-1 receptor, resulting in inhibition of IGF-1 function [28, 29]. In addition, phosphorylation makes IGFBP-1 more resistant to proteolysis [28, 30]. Functionally, phosphorylation increases the capacity of IGFBP-1 to inhibit IGF-1-stimulated cell proliferation, DNA synthesis, amino acid transport and apoptosis [31–33].

Insulin [34–38] and IGF-1 receptors [39] are abundantly expressed in the syncytiotrophoblast microvillous plasma membrane, and the actions of both insulin and IGF-1 in the maternal circulation and the maternal-fetal interface serve as powerful positive regulators of placental function and growth. Therefore, alterations in the maternal insulin, IGF-1 and/or IGFBP1 levels in pregnancies complicated by IUGR may directly contribute to the restricted fetal growth. Locally in the placental barrier, IGFBP-1 produced by the decidua inhibits trophoblast invasion by sequestering IGF-1 [40, 41]. Furthermore, the decidua constitutes the major source for maternal circulating IGFBP-1 in pregnancy [42, 43]. Serum IGF-1 concentrations are decreased in mothers delivering IUGR babies [44] and most [44–54], but not all [55–58], studies show that IUGR or low birth weight is associated with increased maternal serum IGFBP-1 levels. Moreover, placental IGFBP-1 protein expression has been reported to be increased in IUGR [59, 60]. We demonstrated that first trimester maternal plasma IGFBP-1 is hyperphosphorylated in women who later delivered IUGR infants [61]. This suggests that hyperphosphorylation of maternal IGFBP-1 may represent a novel early biomarker of IUGR with implications for early intervention and ultimately prevention of perinatal complications associated with this pregnancy complication.

In addition to decreased maternal circulating levels of IGF-1 in IUGR [44], decreased IGF-1 secretion from decidual explants [62] and lower placental expression of IGF-1 has been reported in human IUGR [63]. After ligand binding, the INSR and IGF1-R activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and protein kinase B (AKT/PKB) pathways, which mediate many of the cellular effects of insulin/IGF-1. Overall, the activity of the placental insulin/IGF-1 signaling, as determined by the degree of phosphorylation of key kinases in the signaling pathway, is decreased in human IUGR [64–66] (Fig 2). The protein expression of INSR [67] and the gene [63] and protein expression [68] of IGF-1R have been reported to be decreased in the IUGR placenta. In contrast, other reports suggest that placental IGF-1R gene expression is increased in association with IUGR [69]. Moreover, Iniguez and co-workers reported that total protein expression of IGF-1R, IRS-1 and Akt was increased in the IUGR placenta [70], reminiscent to the increased total placental mTOR protein expression demonstrated in IUGR previously [71]. These changes may be compensatory to inhibition of basal insulin/IGF-1 activity in the IUGR placenta. However, other reports suggest that IGF-1R expression is decreased in the IUGR placenta [68]. Because the insulin/IGF-1 signaling regulates multiple trophoblast functions such as hormone synthesis including human placental lactogen, chorion gonadotrophin, and progesterone [72], nutrient transport [16, 73] and protein synthesis (mediated by mTOR signaling, see below), decreased placental insulin/IGF-1 signaling in IUGR is expected to inhibit these functions (Fig 2).

mTOR signaling

Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase highly expressed in the syncytiotrophoblast [71]. mTOR regulates cellular metabolism, growth and proliferation [74–78]. mTOR is present in the cell in two complexes, mTOR complex (mTORC) 1 and 2, with the protein raptor associated to mTORC1 and the protein rictor constituting an integral part of mTORC2. mTORC1 promotes protein translation mediated by phosphorylation of S6K1 and 4EBP1 [74–79]. In contrast, activation of mTORC2 phosphorylates Akt, PKCα, and serum glucocorticoid-regulated kinase 1 (SGK1) and regulates the actin skeleton, cell-cycle progression, anabolism and cell survival [80–82]. Deptor is an endogenous inhibitor of both mTORC1 and 2 [83]. mTORC1 integrates a large number of metabolic signals, including hormones and growth factors, such as insulin, IGF-1 and EGF, cellular ATP levels, hypoxia, DNA damage, amino acids, glucose and fatty acids, to regulate cellular metabolism, growth and proliferation [84, 85]. In contrast to mTORC1, mTORC2 predominantly responds to insulin/PI3K signaling [84].

It was recently reported that mTORC1, but not mTORC2, is a positive regulator of oxidative phosphorylation mediated by influencing mitochondrial biogenesis [86]. Thus, by controlling syncytiotrophoblast ATP production, mTORC1 signaling indirectly regulates all energy-requiring processes including active transport and protein synthesis, In addition, using human placental villous explants and primary human trophoblast (PHT) cells, we have demonstrated that mTOR signaling directly regulates key trophoblast nutrient transporters [17, 71, 73, 87–89]. Specifically, inhibition of both mTORC1 and/or mTORC2 downregulates trophoblast System A and L amino acid transport activity by affecting the plasma membrane trafficking of specific System A (SNAT2, SLC38A2) and System L isoforms (LAT1, SLC7A5) [87]. Furthermore, it was demonstrated that Nedd4–2, an E3 ubiquitin ligase, is required for the regulation of plasma membrane trafficking of amino acid transporter isoforms by mTORC1, but not mTORC2 [88]. In contrast, mTORC2 regulates the plasma membrane trafficking of specific amino acid transporter isoforms activating the Rho GTPases Cdc42 and Rac1, which influence the actin skeleton (Rosario et al, unpublished observations). The powerful effects of mTOR signaling on nutrient transport are not limited to amino acids. For example, both mTORC1 and 2 are positive regulators of trophoblast folate uptake by modulating the cell surface expression of folate receptor-α (FR-α) and the reduced folate carrier (RFC) [89].

Using the phosphorylation of key downstream targets as functional readouts of mTOR signaling, we and others have reported that placental mTORC1 [90–94] and mTORC2 [92, 94] signaling activity is inhibited in human idiopathic IUGR as well in IUGR induced by placental malaria [94]. On the other hand, the total protein expression of mTOR has been reported to be up-regulated in the IUGR placenta in some studies [90, 95] but not all [91], possibly as a regulatory event in response to the inhibited activity.

ER stress signaling

Diverse signals may impair the ability of cells to properly execute folding and post-translational modifications of secretory and transmembrane proteins in the endoplasmic reticulum (ER) causing ER stress, which is associated with accumulation of misfolded proteins in the ER [96]. For survival, cells that undergo ER stress must rapidly restore protein-folding capacity to match protein-folding demand. In the presence of high levels of misfolded proteins in the ER, an intracellular signaling pathway called the unfolded protein response (UPR) initiates homeostatic mechanisms to prevent further damage but prolonged UPR activation can lead to cell death [96]. The UPR is comprised of three principal signaling pathways with involvement of PKR-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE-1) and activating transcription factor 6 (ATF6). ER stress and activation of the UPR leads to increased phosphorylation of eukaryotic initiation factor 2α (eIF2α), resulting in inhibition of global translation but to increased translation of activating transcription factor (ATF) 4. ATF4 increases the expression of a small group of target genes involved in transport, metabolism, and oxidative stress [97]. IUGR has been reported to be associated with placental ER stress and protein synthesis inhibition [91, 93, 98]. The reduced protein synthesis in the IUGR placenta in response to inhibition of mTOR signaling and activation of the ER stress pathways has been proposed to contribute to a smaller placental size, which is characteristic of the IUGR phenotype. UPR activation may also indirectly regulate trophoblast nutrient transporters. First, ATF4 has been proposed to increase the transcription of the two amino acid transporter isoforms SNAT2 (SLC38A2) and CAT-1 (SLC7A1) [97]. Second, eIF2α phosphorylation results in inhibition of mTORC1 and mTORC2, which are positive regulators of several specific amino acid transporter isoforms, and is mediated in part by increased expression of REDD1 (regulated in development and DNA damage response 1) [99, 100].

PPARγ signaling

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor that regulates gene expression. PPARγ is involved in an array of biological processes including glucose homeostasis, adipocyte differentiation and inflammatory responses [101]. PPARγ forms a heterodimer with RXR and promotes fatty acid uptake and lipid storage, typically resulting in increased insulin sensitivity [101, 102]. PPARγ regulates trophoblast invasion and differentiation [103, 104] and controls placental vascular proliferation [105] and is therefore critical for placental development. PPARγ stimulates fatty acid uptake [106] and increases L-type amino acid and taurine transporter protein expression [107] in primary human trophoblast cells. The literature on placental PPARγ expression in IUGR is conflicting. Some investigators have reported an increased placental PPARγ expression in IUGR, which has been suggested to represent a protection against hypoxia and/or nutrient deficiency caused by placental insufficiency [108, 109]. However, Rodie and coworkers reported that PPARγ is unchanged [110], whereas other investigators observed a decreased placental PPARγ protein [107] and gene expression [111] in small-for-gestational age infants. Therefore, though the important regulatory role of PPARγ in placental development is clear, the relationship between alterations in placental PPARγ expression and the development of IUGR remains to be elucidated.

Placental mitochondrial abundance and function in IUGR

Increased mitochondrial activity and biogenesis across gestation sustains the metabolic activity of the placenta during normal pregnancy. Mitochondrial dysfunction and excess generation of reactive oxygen species can affect placental function, signaling and nutrient transport which are vital for normal fetal growth. Studies exploring placental mitochondrial DNA content, electron transport chain complex expression and function in IUGR have generated conflicting results. Specifically, placental mitochondrial DNA content has been reported to be increased [112, 113] unaltered [114] or decreased [115, 116] in IUGR. Moreover, using enzyme activity measurements Lefebvre and coworkers concluded that placental mitochondrial function is unchanged in IUGR [114], whereas Mando and collaborators studied cultured primary human cytotrophoblast cells using high resolution respirometry and reported increased trophoblast mitochondrial function in IUGR [112]. The reasons for the divergent findings in the different studies remains to be established but may be related to small sample sizes in some studies and variable definitions of IUGR. We recently reported that placental protein expression of electron transport chain complexes is decreased and positively correlated to mTOR signaling activity in IUGR [86]. These observations, together with the demonstration that mTORC1 is a positive regulator of trophoblast function [86], are consistent with the possibility that reduced placental mTOR activity in IUGR may impair mitochondrial respiration and contribute to placental insufficiency in this pregnancy complication.

Placental nutrient transport in IUGR

Nutrient availability is a key determinant of fetal growth, and changes in fetal nutrient supply not only results in abnormal fetal growth but programs the fetus for diseases later in life [8, 117–120]. Fetal nutrient availability is critically dependent on placental nutrient transport and metabolism and common pregnancy complications are associated with specific changes in placental nutrient transporter expression and activity [121–125]. Thus, various aspects of placental nutrient transport in IUGR have been studied in some detail, as reviewed in the literature [121–123, 126–135].

As discussed above, it is the syncytiotrophoblast cell layer and in particular its two polarized plasma membranes, the microvillous (MVM) and basal plasma membranes (BM), that constitute the actual barrier for the transfer of molecules such as glucose, amino acids, and folate. This provides the rationale for isolating these plasma membranes to study their transport characteristics in vitro. Reported changes in MVM or BM nutrient transporter activity in IUGR placentas are summarized in Fig 3.

Figure 3. Alterations in Placental Transport in IUGR.

Increased (red), decreased (blue) and unchanged (grey) transporter activity in microvillous (MVM) and basal (BM) syncytiotrophoblast plasma membranes isolated from IUGR placentas, as compared to gestational age matched AGA controls. For details, see text.

Glucose

Transplacental glucose transport is mediated by facilitated diffusion through specific glucose transporters (GLUTs). In late pregnancy GLUT 1 (SLC2A1) is believed to constitute the primary transporter mediating transfer of glucose across the placental barrier in the human [136] (Fig 3). GLUT1 expression has been estimated to be three-fold higher in the MVM compared to BM [136], reflecting the higher glucose transport activity in the MVM [136]. Given the 6–7-fold larger area of the microvillous surface of the syncytiotrophoblast compared to the basal plasma membrane at term it has been estimated that the total glucose transport capacity of the MVM may be 20-fold higher than BM [136]. Based on these findings it was suggested that transport across the BM constitutes the rate-limiting step of transplacental glucose transport [136]. In addition to GLUT1, additional glucose transporters have been shown to be expressed in different cell types of the human term placenta, however their functional significance remain to be fully established. For example, the insulin-sensitive GLUT4 is reported to be expressed in human term villous stromal cells [137], GLUT9 protein is expressed in both MVM and BM [138, 139] and GLUT3 is reported to be localized in the BM [140], though other investigators have found that GLUT3 protein expression in the term human placenta is restricted to fetal capillary endothelial cells [141]. In the first trimester, at least four different GLUT isoforms are expressed in the syncytiotrophoblast, primarily in the MVM: GLUT1, 3, 4 and 12 [142, 143] of which GLUT4 and 12 are sensitive to regulation by insulin.

MVM and BM GLUT1 protein and glucose transport activity are unaffected by IUGR [136, 144]. However, BM GLUT1 protein expression has been reported to be decreased in women residing at high altitude who give birth to smaller infants, albeit not meeting the clinical definition of IUGR [145]. Using immunohistochemistry, Janzen and co-workers found that GLUT3 protein expression is increased in the IUGR placenta, in particular in cytotrophoblasts [146]. Thus, fetal hypoglycemia in IUGR is unlikely to be due to changes in placental glucose transporter expression or activity. It is possible that an increased placental consumption of glucose in IUGR could explain a decreased transplacental glucose transfer, even with maintained GLUT transporter expression and activity. Some studies [147, 148], but not all [149], have provided evidence in support of this hypothesis.

Amino Acids

More than twenty-five amino acid transporters with distinct but overlapping substrate specificity are expressed in the syncytiotrophoblast [150, 151]. System A is a sodium-dependent transporter mediating the uptake of non-essential neutral amino acids into the cell [152]. All three known isoforms of System A, SNAT 1 (SLC38A1), SNAT 2 (SLC38A2), and SNAT 4 (SLC38A4) are expressed in the human placenta [153]. System A activity establishes the high intracellular concentration of amino acids like glycine, which is used to exchange for extracellular essential amino acids via system L. Thus, System A activity is critical for placental transport of both non-essential and essential amino acids (Fig 1). System L is a sodium-independent exchanger mediating cellular uptake of essential amino acids including leucine [154]. The System L amino acid transporter is a heterodimer, consisting of a light chain, typically LAT1 (SLC7A5) or LAT2 (SLC7A8), and a heavy chain, 4F2hc/CD98 (SLC3A2). System β or TAUT (SLC6A6) mediates cellular uptake of taurine, an essential amino acid in fetal life.

The activity of several placental amino acid transporters is reduced in IUGR pregnancies. The activity of system A has been consistently shown to be lower in MVM isolated from IUGR placentas [92, 94, 144, 155] due to a decreased MVM protein expression of SNAT 1 (SLC38A1) and SNAT 2 (SLC38A2) [92]. Moreover, MVM system A activity is related to the level of fetal compromise in IUGR. Term IUGR fetuses are less affected than preterm IUGR [144] and the most severe cases of IUGR, as defined by abnormal pulsatility index in the umbilical artery and abnormal fetal heart rate tracings, are associated with the largest decreases in MVM System A activity [156]. However, it is unclear whether the reduction in placental amino acid transport in women occurs prior to fetal growth restriction or as a consequence of reduced fetal needs. We have reported that down-regulation of key placental amino acid transport systems precedes the development of IUGR in rodents [157, 158] and non-human primates [159–161], supporting the concept that down-regulation of placental nutrient transport is a primary event, which directly contributes to IUGR, rather than a response to a changing fetal demand.

The MVM and/or BM activity of transporters of essential amino acids, including system β (taurine) and system L (lysine and leucine), is also reduced in IUGR [162, 163]. Importantly, these in vitro findings are in agreement with a study using stable isotope techniques in pregnant women demonstrating that placental transfer of the essential amino acids leucine and phenylalanine is reduced in IUGR term pregnancies [164]. The deceased activity of amino acid transporters in the placental barrier in association with IUGR is believed to result in reduced placental amino acid transport, which may account for the low plasma levels of certain amino acids in growth restricted fetuses [165, 166].

Lipids

Numerous proteins involved in fatty acid transfer are expressed in the placenta [134]. For fatty acids in maternal lipoproteins to be accessible for placental transport, triglycerides must be hydrolyzed into non-esterified fatty acids (NEFAs). This is accomplished by lipases associated with the MVM. The placenta expresses several triglyceride lipases [167], two of which function in the extracellular compartment: lipoprotein lipase (LPL) and endothelial lipase (EL). Upon entering the cell FAs are believed to be esterified with coenzyme A and transferred by cytosolic fatty acid binding proteins (FABPs) to either (1) mitochondria for oxidation, (2) endoplasmic reticulum for re-esterification and lipid droplet formation, or 3) the fetal-facing syncytiotrophoblast basal plasma membrane (BM) for transport to the fetal circulation. The membrane-bound fatty acid transport proteins (FATPs) are important for cellular uptake of long-chain fatty acids [168]. The mammalian placenta expresses mRNA for five of the six known FATP isoforms: FATP1–4 and FATP6 [169]. FATP1 and FATP4, as well as CD36/FAT, have been demonstrated in both the MVM and BM of human placenta [170–172]. BM FATP2 protein expression has been reported to be up-regulated in maternal obesity [173].

The activity of lipoprotein lipase (LPL), an enzyme responsible for hydrolysis of lipoproteins, is reduced in MVM and the gene expression of EL is down-regulated in IUGR placentas [174]. In contrast, placental LPL mRNA expression is increased in IUGR [175, 176], possibly representing a compensatory mechanism or reflecting that MVM LPL activity is not regulated on the transcriptional level [176]. IUGR is associated with a reduced placental expression of lipoprotein receptors, low-density lipoprotein (LDL) and scavenger receptor class B type-I (SRBI), the key receptors for cholesterol uptake from maternal LDL and/or HDL [176]. In contrast, MVM FATP6 and CD36 protein expression is increased and long chain polyunsaturated fatty acids (LCPUFA) are preferentially routed towards cellular storage in triglyceride in the IUGR placenta [177], possibly to protect against oxidative stress associated with cellular fatty acid accumulation. We speculate that these changes may be caused by impaired efflux of fatty acids across the fetal-facing syncytiotrophoblast basal plasma membrane in IUGR placenta. Collectively, these findings suggest that placental lipid handling is abnormal in the IUGR placenta. However, further studies are required to obtain a more complete understanding of placental lipid transport in normal pregnancy and in pregnancy complications, such as IUGR.

Transport of solutes involved in acid-base balance

The fetus is dependent on the placenta for regulation of its acid-base balance, which involves the exchange of carbon dioxide, bicarbonate, protons, and lactate across the placental barrier. The sodium-proton exchanger (NHE) is the most important placental transporter involved in acid-base regulation. There are at least nine distinct human NHE genes (NHE1/SLC9A1-NHE9/SLC9A9) and this family of transporters is involved in a host of physiological processes in addition to removal of intracellular protons in exchange for sodium, including regulation of cell volume, proliferation and apoptosis [178]. NHEs are highly active in the MVM [179, 180], mediating the export of protons from the cytosol of the syncytium into maternal blood. Studies in villous fragments have confirmed that NHE is critical for maintenance of intracellular pH in the syncytiotrophoblast [181]. Pharmacological [182–184] and protein expression data [184, 185] suggest that NHE1 is the major isoform expressed in the MVM, however NHE2 and NHE3 are also present [184, 185]. Both the activity and expression of the Na⁺/H⁺ exchanger are reduced in IUGR [184, 185] and this finding could explain the development of acidosis in some of these infants due to decreased capacity to eliminate metabolically produced protons in the fetus to the maternal circulation.

Lactate is an important energy source in the fetal heart, brain and skeletal muscle. The placenta may be a source of lactate even when oxygen supplies are adequate [186]. However the placenta is also an important site for clearance of lactate produced in the fetus, especially in cases of fetal hypoxia [187]. Lactate is transported across cell membranes by members of the monocarboxylate transporter (MCT) family (SLC16), which mediates H⁺/lactate co-transport [188]. Both MVM [189, 190] and BM [190, 191] efficiently transport lactate in the presence of a proton gradient and the predominant MCT isoform in the MVM is MCT4, whereas MCT1 is highly expressed in the BM [190]. Decreased BM lactate transporter activity [192] may contribute to the increased fetal lactate levels associated with IUGR and impair the ability of the IUGR fetus to tolerate stress.

Sodium and calcium transport

A large number of Na⁺-dependent co-transporters have been shown to be active in MVM. These transporters utilize the inwardly directed sodium gradient across MVM to energize the uptake of, for example, amino acids, phosphate [193], biotin [194] and succinate [195] by co-transport or the efflux of molecules such as protons [179, 180] via exchange mechanisms. As in all other cells, low intracellular sodium concentrations in the syncytiotrophoblast are maintained by Na⁺K⁺-ATPase. However, in contrast to classical epithelia in which the sodium pump is expressed exclusively in the basolateral plasma membrane, Na⁺K⁺-ATPase is expressed and active in both BM [196, 197] and MVM [197] of the human syncytiotrophoblast. MVM Na⁺/K⁺ ATPase activity is decreased in IUGR [198], which may impair the function of all transporters dependent on the Na⁺ gradient for energy.

The fetus accumulates approximately 30g of calcium over nine months, predominantly in the third trimester when fetal bone mineralization occurs. As in other cells, the intracellular calcium concentration in the syncytiotrophoblast is four magnitudes lower than extracellular concentrations, which is critical for the role of intracellular calcium as a signal transducer. In principle, the transfer of calcium across the syncytiotrophoblast occurs in three distinct steps [reviewed in [199]] similar to calcium transport in the intestinal epithelium [reviewed in [200]]. The first step involves calcium entry into the cell across MVM. Subsequently, Ca²⁺ moves through the cytosol associated with calcium binding proteins and finally it is transported out of the cell mediated by Ca²⁺ ATPase. Interestingly, the BM Ca²⁺ ATPase is up-regulated in IUGR [201] possibly due to elevated fetal concentrations of PTHrp 38–94, a key regulator of the placental calcium pump [202]. In summary, the activity of syncytiotrophoblast Na⁺K⁺-ATPase and Ca²⁺ ATPase is altered in opposite directions, suggesting that functional changes in the IUGR placenta are due to homeostatic regulation rather than the result of injury.

Animals models of IUGR

The overall premise in the field is that a better understanding of the molecular and cellular mechanisms underpinning the development of IUGR will allow us to develop early biomarkers and novel intervention strategies to alleviate or prevent this serious pregnancy complication and its long-term adverse consequences. For example, prenatal identification of babies who are SGA significantly improves their perinatal outcomes as compared to diagnosis at birth [203]. Thus, identifying biomarkers for IUGR in early pregnancy could improve the clinical management of these patients by allowing early intervention. It is difficult, if not impossible, to perform mechanistic studies in women, and placental function can only be explored following delivery, typically in late gestation. These limitations can, at least in part, be circumvented by studies in carefully validated animal models, which have provided important insights into changes in placental function common to an array of different IUGR models in a range of species, changes in placental function across gestation and cause-and-effect links.

Changes in placental function in IUGR are remarkably similar in diverse animal models and are in general agreement with human IUGR due to placental insufficiency. For example, maternal calorie restriction from gestational day 30 to 165 (term 180–184) in the baboon was associated with an inhibition of placental insulin/IGF-1 and mTOR and signaling pathways, decreased expression of key placental amino acid and glucose transporter isoforms and lower fetal levels of essential amino acids and IUGR [161]. In pregnant rats subjected to protein restriction placental mTOR signaling is inhibited [158] and amino acid transport, as measured in vitro and in vivo, was reported to be decreased [157, 158, 204]. Maternal hyperthermia is one of the most common models of IUGR in the sheep. By increasing ambient temperature, maternal body temperature is elevated, which interferes with placental development, causing decreased placental oxygen diffusion and nutrient transport capacity [205]. Specifically, placental mTOR signaling is inhibited [206] and placental capacity to transport glucose [207], leucine [208], threonine [209] and ACP (a branched-chain amino acid analog) [210] is reduced in this IUGR model. In guinea-pigs, IUGR induced by uterine artery ligation in mid-pregnancy (GD 35) is associated with decreased placental capacity to transport amino acids, as measured in the unstressed chronically instrumented animal, in late pregnancy [211]. Similarly, uterine artery ligation in the rat resulted in a decreased in vivo transplacental glucose and amino acid transport and IUGR [212]. Furthermore, administration of corticosteroids to pregnant mice – a model of maternal stress - inhibits placental System A activity [213]. Chronic infusion of adiponectin in pregnant mice to mimic the high circulating adiponectin levels of very lean women, causes modest IUGR by inhibition of placental insulin and mTOR signaling and down regulation of placental nutrient transporters [214, 215]. Maternal folate deficiency inhibits placental mTOR signaling and placental amino acid transport and results in IUGR [216].

Importantly, animal IUGR models are not always associated with changes in placental function in agreement with this general model [217]. In particular, a series of studies in mice have provided evidence for compensatory increases in placental transport capacity in response to maternal under nutrition [218–220]. The possible reasons for the distinct placental responses to maternal under nutrition in mice as compared to the human, rat, guinea-pig and baboon have been discussed in some detail elsewhere [217]. Nevertheless, widely different causes of IUGR, such as maternal under nutrition, maternal stress, and reduced utero-placental blood flow following suboptimal trophoblast invasion, appear to result in strikingly similar changes in placental signaling and function, suggesting a common placental response to an inability of the maternal supply line to deliver nutrients and oxygen to the placenta.

In the absence of non-invasive approaches to study placental transport across gestation in women, gestational changes in placental signaling and function in normal and IUGR pregnancies can be explored in animal models. Studies in mice, baboons and rats strongly suggest that changes in placental nutrient transporters are a cause of, rather than a secondary consequence to, altered fetal growth [157–159, 161, 221]. Specifically, down-regulation of placental transport of amino acids precedes the development of intrauterine growth restriction in rats fed a low protein diet [157] and in baboons fed a calorie-restricted diet [161, 222], suggesting that these placental transport changes are a cause, rather than a consequence, of IUGR.

Because of the available advanced gene tools, mouse models provide unique opportunities for rigorous cause-and-effect experiments. Traditional transgenesis using a short fragment of the human Cyp19 promoter, Cyp19I.1 as a trophoblast specific promoter [223–225] and lentivirus transfection of mouse blastocysts [226–228] have been used as approaches to achieve trophoblast-specific gene targeting. However, despite the promise of these approaches, they have not been entirely reproducible between labs and studies systematically targeting placental nutrient transporters have yet to be published.

Evolution and placental nutrient sensing

Imprinted genes are characterized by parental-specific monoallelic expression and many of the approximately ~100 imprinted genes currently known in humans are highly expressed in the placenta [229]. The “parental conflict theory” proposes that paternally expressed (i.e. maternally imprinted) genes in the placenta, such as IGF2, are designed to stimulate fetal growth. In contrast, maternally expressed (paternally imprinted) placental genes, including the IGF2 receptor (IGF2R), which functions as a IGF-2 clearance receptor, tend to limit nutrient supply to the embryo in order to preserve maternal reproductive resources [230]. Given the intimate relationship between placental nutrient transport and fetal growth, it is not surprising that some genes that encode transporter proteins (SLC38A4) or proteins that are involved in the regulation of nutrient transport (IGF2) are imprinted in the placenta. The expression of imprinted genes in the placenta may set the general trajectory for placental growth/function and fetal growth, whereas other regulatory mechanisms are believed to respond to changes in the ability of the maternal supply line to deliver oxygen and nutrients to the placenta.

The down-regulation of a multitude of placental nutrient transporters in IUGR in women and a range of animal models appears, at first glance, counterintuitive given the paradigm of the fetus as the ultimate parasite and the fact that classic physiology would predict a homeostatic up-regulation of placental nutrient transport in response to decreased fetal nutrient delivery. There are two fundamentally distinct and opposing, but not mutually exclusive, models for how placental function in general, and placental nutrient transporters in particular, are regulated by changes in the ability of the maternal circulation to supply oxygen and nutrients to the placenta. In the regulation by maternal supply model [123–125, 231, 232], the placenta responds to maternal nutritional cues, resulting in down-regulation of placental nutrient transporters in response to, for example, maternal under-nutrition, stress, high altitude hypoxia or restricted utero-placental blood flow (Fig 4). As a result, fetal nutrient availability is decreased and IUGR may develop. The regulation by maternal supply model therefore constitutes a mechanism by which fetal growth is matched to the ability of the maternal supply line to allocate resources to the fetus. In this model, changes in placental growth and nutrient transport directly contribute to, or cause, alterations in fetal growth. Maternal nutritional signaling molecules include maternal metabolic hormones such as insulin, cortisol, leptin, and adiponectin (Fig 4), which are known to be influenced by maternal nutrition and regulate placental transport. Other signals proposed to be sensed by the placenta are oxygen and nutrient levels. It has been suggested that placental mTOR signaling plays an important role in integrating all these signals and regulating fetal nutrient availability by modulating placental growth and nutrient transport [123–125, 231, 232].

Fig 4. Placental nutrient sensing.

The placenta integrates signals conveying information on the ability of the maternal supply line to deliver nutrients and oxygen to the placenta (maternal supply) and on fetal demand through intrinsic nutrient sensors, such as mechanistic target of rapamycin (mTOR) signaling. These signals then regulate placental growth, mitochondrial respiration, nutrient transport and hormone secretion to balance fetal demand with the ability of the mother to support pregnancy. Thus, the placenta plays a critical role in modulating maternal-fetal resource allocation, thereby affecting fetal growth and the long-term health of the offspring. See text for detailed explanation. IGF-2, insulin-like growth factor 2; PTHrp, parathyroid hormone related peptide.

Predominantly based on elegant mouse studies, placental nutrient transport capacity has been proposed to be largely regulated by fetal demand signals [233–235]. According to this model, in response to limitations in fetal nutrient supply due to, for example, maternal under-nutrition or a lack of normal gestational increase in utero-placental blood flow, the fetus signals to the placenta to up-regulate placental growth and nutrient transport. This model represents a classical homeostatic mechanism by which the fetus compensates for changes in nutrient availability by regulating nutrient supply (i.e., placental transport) in the opposite direction.

As discussed above, most studies of placental transport functions in human IUGR show a down-regulation of key placental transporters for amino acids, lipids and ions (Fig 3), which is inconsistent with a homeostatic or fetal demand model for regulation of placenta transport. Indeed, there is a paucity of data demonstrating the presence of fetal demand signals in other species than mice. For example, when placental nutrient transport capacity and fetal growth were studied across the last third of pregnancy in rats exposed to protein restriction [157, 204] and baboons subjected to a 30% reduction in calorie intake [160, 161, 222] inhibition of placental mTOR signaling and down-regulation of placental nutrient transport occurred prior to slowing of fetal growth and without signs of compensatory up-regulation of placental function. However, fetal demand signals are difficult to identify unequivocally and it is possible that these signals exist in all species. Moreover, human studies of placental function in IUGR have been performed at term, or in a few cases using tissue obtained from preterm deliveries in third trimester [144, 174, 184]. It is therefore possible that compensatory changes consistent with fetal demand signals may be present earlier in pregnancy, as is evident in mouse models of IUGR. Finally, the distinct up-regulation of BM Ca²⁺-ATPase activity in IUGR placentas [236] may represent a compensatory activation of the placental calcium transport system stimulated by an increased fetal demand.

We have proposed a model (placental nutrient sensing) in which the placenta integrates a multitude of maternal (regulation by maternal supply) and fetal nutritional cues (regulation by fetal demand) with information from intrinsic nutrient sensing signaling pathways to balance fetal demand with the ability of the mother to support the pregnancy by regulating maternal physiology, placental growth and nutrient transport (Figure 4). These regulatory loops may also function in the “reverse” direction in response to maternal ‘over-nutrition’, represented by maternal obesity and gestational diabetes. In support of this concept, placental capacity to transport amino acids [237, 238], glucose [138] [239] and fatty acids [167, 173, 174] may be increased in obese and/or diabetic women, particularly in cases of fetal overgrowth.

The link between changes in placental function and restricted fetal growth may be modulated by fetal sex because pregnancies with a male fetus are more likely to have a poor outcome [240–242], an association that is present also in IUGR or small-for-gestational age infants [243, 244]. The underlying mechanisms are elusive, however sexual dimorphism in placenta gene expression [245–247] and function [248] may play a role. Emerging evidence suggest that differences in polyamine metabolism underpin sex differences in placental function in normal pregnancies and in pregnancy complications such as IUGR [249].

We propose that placental nutrient sensing has evolved due to the evolutionary pressures of maternal under-nutrition or starvation, which likely occurred frequently across evolutionary time. Matching fetal growth to maternal resources in response to maternal under-nutrition will produce an offspring that is smaller in size but who, in most instances, will survive and reproduce. Reduced fetal growth may be a better alternative than the fetus extracting all the nutrients needed for normal growth from an already deprived mother, thereby potentially jeopardizing both maternal and fetal survival. It may be speculated that the relative importance of placental nutrient sensing and fetal demand signals for the regulation of placental function may differ between species and depend on the type, duration and severity of the nutritional perturbation. For example, it is plausible that regulation by fetal demand signals dominates when the nutritional challenge is moderate and brief whereas regulation by maternal supply may override fetal demand if the nutritional challenge is more severe or prolonged.

Avenues to target placental function to improve outcomes in IUGR

In terms of preventing the development of IUGR two interventions have received significant interest; aspirin and low molecular weight heparin. Low dose aspirin therapy has an array of actions that could offer benefits in preventing IUGR, including the well-established irreversible inhibition of the synthesis of platelet thromboxane A2, a powerful vasoconstrictor and prothrombotic agent, increased acetylation of endothelial nitric oxide synthase promoting nitric oxide release [250] and increased activity of heme oxygenase-1 which catabolizes heme leading to decreased oxidative stress and inflammation [251]. However, recent systematic meta-analyses [252, 253] suggest that aspirin only has a modest effect on decreasing the risk of a SGA infant in high-risk pregnancies. Low molecular weight heparin has been safely used in pregnancy on the indications thromboprophylaxis and venous thromboembolism and initial randomized clinical trials suggested that low molecular weight heparin may reduce the risk of developing preeclampsia and/or IUGR [254, 255]. However more recent meta-analyses suggest no clear benefit [256, 257].

A lack of normal increase in utero-placental blood flow across gestation is believed to underpin the development of IUGR, providing a strong premise for the efforts of the past 40 years to develop prevention and treatment strategies aiming at increasing utero-placental blood flow in IUGR [258, 259]. Unfortunately, systemic maternal administration of vasodilators has in many cases been reported to be associated with poor fetal outcomes. For example, maternal administration of prostacyclin, a potent vasodilator, in severe IUGR resulted in fetal demise [260] and a recent large trial of sildenafil, a phosphodiesterase type 5 inhibitor potentiating the action of nitric oxide, had to be halted after multiple fetal deaths [261]. There are several reasons for the lack of efficacy of systemically administered vasodilators in improving utero-placental blood flow and fetal outcomes in IUGR. One explanation is likely to be that for many vasodilators, the systemic circulation is more sensitive to the vasodilatory effects of the agent than the utero-placental circulation resulting in a decrease of placental perfusion due to lowered mean arterial blood pressure. Animal experiments [262] and pilot studies in women [263] have suggested that atrial natriuretic peptide may represent a vasodilator for which the opposite is true, i.e., the uteroplacental circulation is more sensitive for the vasodilatory effect of ANP. Collectively, there are currently no specific treatment of IUGR available but some innovative approaches targeting the uteroplacental vasculature, including maternal VEGF gene therapy to increase VEGF levels locally in the uterine artery [264, 265], are currently being developed.

As discussed in this review, placental insufficiency is much more than just a matter of reduced blood flow because IUGR is associated with complex, coordinated and highly regulated changes in placental signaling and nutrient transport capacity that directly contributes to poor outcomes. Thus, approaches targeting the trophoblast to improve placental function may improve fetal growth and alleviate adverse outcomes in IUGR. Albeit in very early stages of development, IGF-2 or the antioxidant MitoQ have been specifically targeted to trophoblast cells by using nanoparticles coated with tumor-homing peptide sequences (which is also recognized by the trophoblast) [266, 267]. Given that mTOR signaling regulates a wide range of key placental functions including amino acid and folate transport, mitochondrial biogenesis and protein synthesis and that IUGR is associated with inhibition of placental mTOR signaling, specific approaches to activate trophoblast mTOR signaling in IUGR hold promise for improving fetal growth and preventing poor outcomes in this important pregnancy complication.

In conclusion, the development of IUGR involves regulation of multiple placental signaling pathways and down-regulation of placental nutrient transporters, contributing to placental insufficiency and restricted fetal growth. Our proposed ‘placental nutrient sensing’ model provides a framework for considering the complex regulatory pathways by which the placenta balances maternal supply with fetal demand to optimize fetal growth. Further investigation into placental function and potential mechanistic pathways are needed to identify the time points at which intervention may help prevent the development of IUGR and to improve the clinical management of the already affected IUGR fetus.

Highlights.

• IUGR involves complex regulation of multiple placental signaling pathways

• An array of placental nutrient transporters are down-regulated in IUGR

• These changes contribute to placental insufficiency and restricted fetal growth

• Placental insufficiency is much more than just a matter of reduced blood flow

• Altered placental function should be considered when IUGR therapies are developed

Abbreviations:

GLUT1

glucose transporter 1

A

system A

L

system L

β

system β

NHE1

sodium/hydrogen exchanger 1

LPL

lipoprotein lipase

CD36

cluster of differentiation 36, fatty acid translocase

FATP6

fatty acid transporter 6

MCT1

monocarboxylate transporter 1

Ala

alanine

Ser

serine

Gln

glutamine

Gly

glycine

Leu

leucine

Tau

taurine

TG

triglyceride

FA

fatty acid