Melissa Westwood

The IGF axis and placental function. a mini review.

It is well known that the insulin-like growth factor (IGF) axis is an important regulator of foetal growth and in recent years, it has been suggested that the ligands IGF-I and IGF-II may, in part, mediate this effect by promoting proper placental development and function. In other tissues, IGF effects on metabolism, proliferation and differentiation are primarily mediated via IGF binding protein-regulated interaction of IGFs with the type 1 IGF receptor and therefore here, we review the placental expression and postulated role, of each of the IGF axis components and discuss the cellular mechanisms through which these effects are exerted.

The SNAT4 isoform of the system A amino acid transporter is functional in human placental microvillous plasma membrane

Placental system A activity is important for the supply of neutral amino acids needed for fetal growth. There are three system A isoforms: SNAT1, SNAT2 and SNAT4, but the contribution of each to system A‐mediated transport is unknown. Here, we have used immunohistochemistry to demonstrate that all three isoforms are present in the syncytiotrophoblast suggesting each plays a role in amino acid transport across the placenta. We next tested the hypothesis that the SNAT4 isoform is functional in microvillous plasma membrane vesicles (MVM) from normal human placenta using a method which exploits the unique property of SNAT4 to transport both cationic amino acids as well as the system A‐specific substrate MeAIB. The data show that SNAT4 contribution to system A‐specific amino acid transport across MVM is higher in first trimester placenta compared to term (approx. 70% and 33%, respectively, P < 0.01). Further experiments performed under more physiological conditions using intact placental villous fragments suggest a contribution of SNAT4 to system A activity in first trimester placenta but minimal contribution at term. In agreement, Western blotting revealed that SNAT4 protein expression is higher in first trimester MVM compared to term (P < 0.05). This study provides the first evidence of SNAT4 activity in human placenta and demonstrates the contribution of SNAT4 to system A‐mediated transport decreases between first trimester and term: our data lead us to speculate that at later stages of gestation SNAT1 and/or SNAT2 are more important for the supply of amino acids required for normal fetal growth.

Methods for siRNA-mediated Reduction of mRNA and Protein Expression in Human Placental Explants, Isolated Primary Cells and Cell Lines

The use of RNA interference (RNAi) to deplete individual proteins from cells or tissue has revolutionised our ability to characterise gene function. The placenta is an attractive target for studies in which the role of specific proteins can be compared with cell culture models and explanted villous tissue where physiological function can be maintained ex vivo. In this study, we compared a variety of commercially available reagents and approaches to define methods for efficient delivery of siRNA to placental cells. Protocols optimised using fluorescently-labelled siRNA were subsequently tested using siRNA sequences that target placental alkaline phosphatase (PLAP), chosen because of its high abundance in trophoblast. mRNA abundance was assayed using qRT-PCR, and the effect on protein was examined using immunolocalisation. We report that different protocols are required for BeWo choriocarcinoma cells (nucleofection), primary cytotrophoblast cells (lipid-based transfection) and villous tissue explants (nucleofection). The results provide guidelines for optimal siRNA-mediated knockdown in these three models of the human placenta.

State-trait anxiety inventory (STAI) scores during pregnancy following intervention with complementary therapies

OBJECTIVEnWe review intervention trials that have used the State-Trait Anxiety inventory (STAI) as a measure of maternal anxiety in pregnancy in order to provide ranges in scores before and after participation in complementary therapy-based interventions to highlight the expected ranges of scores in pregnancy and determine whether anxiety in pregnancy is amenable to change when measured by the STAI.nnnMETHODSnCombinations of the key words STAI, state anxiety, pregnancy, anxiety, maternal, stress, outcome and intervention were used to search publications between January 1970 and January 2011. Studies eligible for inclusion recruited low risk, adult women to a non-pharmacological intervention or a comparator group, and measured anxiety at baseline and post-intervention.nnnRESULTSnTen studies were eligible. Scores were routinely high compared to expected ranges in non-pregnant populations. Studies examining the immediate effects of an intervention consistently reported significantly lowered STAI scores after a single session. Likewise, studies examining the effect of interventions consisting of multiple sessions over the course of pregnancy found that those in the intervention group were more likely to show an improvement in STAI scores.nnnLIMITATIONSnHeterogeneity in type and duration of intervention prevent drawing conclusions on which were most effective in reducing anxiety.nnnCONCLUSIONnScores on the STAI appear amenable to change during pregnancy, both after a single session and multiple sessions of interventions designed to reduce maternal anxiety. This review offers a guideline for the expected range of scores for future studies examining the efficacy of interventions in pregnancy when using the STAI.

The contribution of SNAT1 to system A amino acid transporter activity in human placental trophoblast

Research highlights ► mRNA levels for SNAT1 are higher than other system A subtype mRNAs in primary human cytotrophoblast. ► SNAT1 knockdown in cytotrophoblast cells significantly reduces system A activity. ► SNAT1 is a key contributor to system A-mediated amino acid transport in human placenta.

Taurine transport in human placental trophoblast is important for regulation of cell differentiation and survival

The outer epithelial cell layer of human placenta, the syncytiotrophoblast, is a specialised terminally differentiated multinucleate tissue. It is generated and renewed from underlying cytotrophoblast cells that undergo proliferation, differentiation and fusion with syncytiotrophoblast. Acquisition of fresh cellular components is thought to be balanced by apoptosis and shedding of aged nuclei. This process of trophoblast cell turnover maintains a functional syncytiotrophoblast, capable of sufficient nutrient transfer from mother to foetus. Foetal growth restriction (FGR) is a pregnancy complication associated with aberrant trophoblast turnover and reduced activity of certain amino acid transporters, including the taurine transporter (TauT). Taurine is the most abundant amino acid in human placenta implying an important physiological role within this tissue. Unlike other amino acids, taurine is not incorporated into proteins and in non-placental cell types represents an important osmolyte involved in cell volume regulation, and is also cytoprotective. Here, we investigated the role of taurine in trophoblast turnover using RNA interference to deplete primary human trophoblast cells of TauT and reduce intracellular taurine content. Trophoblast differentiation was compromised in TauT-deficient cells, and susceptibility of these cells to an inflammatory cytokine that is elevated in FGR was increased, evidenced by elevated levels of apoptosis. These data suggest an important role for taurine in trophoblast turnover and cytoprotection.

Dicer-dependent miRNAs provide an endogenous restraint on cytotrophoblast proliferation

Mature microRNAs (miRNAs) are processed from non-functional (pre)-miRNAs by the enzyme Dicer. In this study, manipulation of Dicer level was used to explore the influence of miRNAs on cytotrophoblast proliferation in human placenta. Immunohistochemistry (IHC) showed Dicer in cytotrophoblast, but not in terminally differentiated syncytiotrophoblast. siRNA-mediated knockdown of Dicer was used to effect a global reduction in miRNA in first trimester placental explants, as a result of which cytotrophoblast proliferation was significantly enhanced. QPCR and IHC analysis following Dicer knockdown revealed that the expression of two nodal pro-mitogenic signalling molecules expressed within cytotrophoblast, ERK and SHP-2, was significantly enhanced. Studies are now required to identify individual miRNAs involved in regulating trophoblast proliferation.

A limitation of the method for siRNA delivery into primary human cytotrophoblast cells

Transfection using cationic lipid reagents such as DharmaFECT2 is an efficient tool for introducing siRNA into primary human cytotrophoblast cells. However, we now report a limitation to the use of this method as the concentration of DharamFECT2 needed for effective siRNA delivery causes ligand-independent activation of the insulin/insulin-like growth factor (IGF) receptor and consequently, functional resistance to cytotrophoblast stimulation by these two hormones. We therefore advise researchers against the use of this method of transfection when investigating the mechanisms by which insulin/IGF, and potentially other hormones that exert their effects through kinase signalling molecules, influence cytotrophoblast cell function.