Hannah E.J. Yong

Increased decidual mRNA expression levels of candidate maternal pre-eclampsia susceptibility genes are associated with clinical severity

INTRODUCTION Pre-eclampsia (PE) has a familial association, with daughters of women who had PE during pregnancy having more than twice the risk of developing PE themselves. Through genome-wide linkage and genetic association studies in PE-affected families and large population samples, we previously identified the following as positional candidate maternal susceptibility genes for PE; ACVR1, INHA, INHBB, ERAP1, ERAP2, LNPEP, COL4A1 and COL4A2. The aims of this study were to determine mRNA expression levels of previously identified candidate maternal pre-eclampsia susceptibility genes from normotensive and severe PE (SPE) pregnancies and correlate mRNA expression levels with the clinical severity of SPE. METHODS Third trimester decidual tissues were collected from both normotensive (n = 21) and SPE pregnancies (n = 24) and mRNA expression levels were determined by real-time PCR. Gene expression was then correlated with several parameters of clinical severity in SPE. Statistical significance was determined by Mann-Whitney U test and Spearmans Correlation. RESULTS The data demonstrate significantly increased decidual mRNA expression levels of ACVR1, INHBB, ERAP1, ERAP2, LNPEP, COL4A1 and COL4A2 in SPE (p < 0.05). Increased mRNA expression levels of several genes - INHA, INHBB, COL4A1 and COL4A2 were correlated with earlier onset of PE and earlier delivery of the fetus (p < 0.05). CONCLUSION These results suggest altered expression of maternal susceptibility genes may play roles in PE development and the course of disease severity.

Role of the Placental Vitamin D Receptor in Modulating Feto-Placental Growth in Fetal Growth Restriction and Preeclampsia-Affected Pregnancies.

Fetal growth restriction (FGR) is a common pregnancy complication that affects up to 5% of pregnancies worldwide. Recent studies demonstrate that Vitamin D deficiency is implicated in reduced fetal growth, which may be rescued by supplementation of Vitamin D. Despite this, the pathway(s) by which Vitamin D modulate fetal growth remains to be investigated. Our own studies demonstrate that the Vitamin D receptor (VDR) is significantly decreased in placentae from human pregnancies complicated by FGR and contributes to abnormal placental trophoblast apoptosis and differentiation and regulation of cell-cycle genes in vitro. Thus, Vitamin D signaling is important for normal placental function and fetal growth. This review discusses the association of Vitamin D with fetal growth, the function of Vitamin D and its receptor in pregnancy, as well as the functional significance of a placental source of Vitamin D in FGR. Additionally, we propose that for Vitamin D to be clinically effective to prevent and manage FGR, the molecular mechanisms of Vitamin D and its receptor in modulating fetal growth requires further investigation.

Anti-angiogenic collagen fragment arresten is increased from 16 weeks' gestation in pre-eclamptic plasma.

INTRODUCTION Arresten and canstatin are endogenous anti-angiogenic factors derived from type IV collagen α-chains COL4A1 and COL4A2 respectively. While their functions are explored in cancer studies, little is known about their role in pregnancy. Pre-eclampsia (PE) is a common, serious hypertensive disorder of pregnancy that is characterised by systemic endothelial dysfunction. COL4A1 and COL4A2 are maternal PE susceptibility genes that have increased mRNA expression in PE decidua. Our study aim was to determine the levels of arresten and canstatin in plasma and decidua from PE and gestational age matched normotensive patients. METHODS Plasma was collected from normotensive (n = 44) and PE (n = 39) women during the second and third trimesters of pregnancy. Third trimester decidua was collected at delivery from normotensive and PE women (n = 4 each). Levels of arresten and canstatin were determined by Western immunoblotting. RESULTS Arresten levels were significantly increased in second and third trimester PE plasma, and in third trimester PE decidua (p < 0.05). Third trimester PE plasma arresten levels also significantly correlated with the need for MgSO4 treatment, where a 1.7 fold increase was observed in patients requiring MgSO4 treatment (p < 0.05). No significant change in canstatin levels was observed between normotensive and PE patients. DISCUSSION This is the first study to report significant increases in the levels of collagen fragment arresten in PE plasma and decidua. Given its significant increase before the onset of clinical disease and associations with clinical severity in the third trimester, arresten may be a useful biomarker for predicting PE and monitoring its severity.

Decidual ACVR2A regulates extravillous trophoblast functions of adhesion, proliferation, migration and invasion in vitro

Decidual stromal cells form the largest proportion of maternal cells at the maternal-fetal interface. Our aim was to investigate the role of the pre-eclampsia associated decidual activin receptor, ACVR2A, in regulating trophoblast functions at this interface. St-T1b and HTR-8/SVneo cell lines were used to model decidual stromal and trophoblast cells respectively. St-T1b conditioned medium inhibited HTR-8/SVneo adhesion, proliferation, migration and invasion; all effects that were attenuated by decidual ACVR2A siRNA transfection. These findings suggest that altered decidual ACVR2A expression perturbs the maternal-fetal crosstalk involved in regulating trophoblast function at the interface, which may affect placentation and lead to pre-eclampsia.

Placental Vitamin D-Binding Protein Expression in Human Idiopathic Fetal Growth Restriction

Vitamin D-binding protein is a multifunctional serum protein with multiple actions related to normal health. Vitamin D-binding protein transports vitamin D and influences the metabolism of this key hormone but it also has additional immunomodulatory and actin-clearing properties. We investigated whether vitamin D-binding protein expression is altered in fetal growth restriction-associated placental dysfunction. Protein was extracted from 35 placentae derived from 17 healthy control subjects and 18 gestation-matched subjects with fetal growth restriction (FGR). FGR subjects were further subdivided as idiopathic (n = 9) and nonidiopathic (n = 9). Vitamin D-binding protein and 25(OH) vitamin D were measured by ELISA and normalized to protein concentration. The results showed significantly reduced levels of placental vitamin D-binding protein (control versus FGR, p < 0.05, Students t-test) that were strongly associated with idiopathic fetal growth restriction (p < 0.01, Kruskal-Wallis), whereas levels of vitamin D-binding protein were not associated with placental 25(OH) vitamin D stores (p = 0.295, Pearsons correlation). As such, vitamin D-binding protein may be a factor in unexplained placental dysfunction associated with idiopathic fetal growth restriction and may potentially serve as a biomarker of this disease.

Genetic Approaches in Preeclampsia

Preeclampsia (PE) is a serious hypertensive disorder that affects up to 8% of all pregnancies annually. An established risk factor for PE is family history, clearly demonstrating an underlying genetic component to the disorder. To date, numerous genetic studies, using both the candidate gene and genome-wide approach, have been undertaken to tease out the genetic basis of PE and understand its origins. Such studies have identified some promising candidate genes such as STOX1 and ACVR2A. Nevertheless, researchers face ongoing challenges of replicating these genetic associations in different populations and performing the functional validation of identified genetic variants to determine their causality in the disorder. This chapter will review the genetic approaches used in the study of PE, discuss their limitations and possible confounders, and describe current strategies.

An Electrical Impedance-Based Assay to Examine Functions of Various Placental Cell Types In Vitro

In vitro functional analyses of cells are widely used to investigate the molecular mechanisms involved in preeclampsia. Common cellular functions studied include adhesion, apoptosis, proliferation, migration, and invasion. At present, most researchers will use endpoint experimental assays that only allow the determination of cell function at a single time point, with the need to repeat the experiment for an alternate time point. Here, we describe an electrical impedance-based tool that allows real-time monitoring of cells, which enables the efficient assessment of multiple time points over the duration of a single experiment.

Altered downstream target gene expression of the placental Vitamin D receptor in human idiopathic fetal growth restriction

ABSTRACT Fetal growth restriction (FGR) affects up to 5% of pregnancies and is associated with significant perinatal complications. Maternal deficiency of vitamin D, a secosteroid hormone, is common in FGR-affected pregnancies. We recently demonstrated that decreased expression of the vitamin D receptor (VDR) in idiopathic FGR placentae could impair trophoblast growth. As strict regulation of cell-cycle genes in trophoblast cells is critical for optimal feto-placental growth, we hypothesised that pathologically decreased placental VDR contributes to aberrant regulation of cell-cycle genes. The study aims were to (i) identify the downstream cell-cycle regulatory genes of VDR in trophoblast cells, and (ii) determine if expression was changed in cases of FGR. Targeted cell-cycle gene cDNA arrays were used to screen for downstream targets of VDR in VDR siRNA-transfected BeWo and HTR-8/SVneo trophoblast-derived cell lines, and in third trimester placentae from FGR and gestation-matched control pregnancies (n = 25 each). The six candidate genes identified were CDKN2A, CDKN2D, HDAC4, HDAC6, TGFB2 and TGFB3. TGFB3 was prioritised for further validation, as its expression is largely unknown in FGR. Significantly reduced mRNA and protein expression of TGFB3 was verified in FGR placentae and the BeWo and HTR-8/SVneo trophoblast cell lines, using real-time PCR and immunoblotting respectively. In summary, decreased placental VDR expression alters the expression of regulatory cell-cycle genes in FGR placentae. Aberrant regulation of cell-cycle genes in the placental trophoblast cells may constitute a mechanistic pathway by which decreased placental VDR reduces feto-placental growth.