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Endometrial cancer is the most diagnosed gynecological malignancy. Despite numerous scientific advances, the incidence and mortality rate of endometrial cancer continues to rise. Emerging evidence suggests a putative role of the (pro)renin receptor ((P)RR), in the ontogenesis of endometrial cancer. The (P)RR is implicated in breast cancer and pancreatic carcinoma pathophysiology by virtue of its role in proliferation, angiogenesis, fibrosis, migration and invasion. Thus, we aimed to investigate the functional role of the (P)RR in human endometrial cancer. We employed an siRNA-mediated knockdown approach to abrogate (P)RR expression in the endometrial epithelial cell lines; Ishikawa, AN3CA and HEC-1-A and examined cellular proliferation and viability. We also carried out a sophisticated proteomic screen to explore potential pathways via which the (P)RR is acting in endometrial cancer physiology. These data confirmed that the (P)RR is critical for endometrial cancer development, contributing to both its proliferative capacity and in the maintenance of cell viability. This is likely mediated through proteins such as MGA, SLC4A7, SLC7A11 or DHRS2, which were reduced following (P)RR knockdown. These putative protein interactions/pathways, which rely on the presence of the (P)RR, are likely to contribute to endometrial cancer progression and could therefore, represent several novel therapeutic targets for endometrial cancer.

Background We carried out a study to determine the impact of prematurity on kidney development in the first 2 years of life. Methods In this prospective study, extremely preterm neonates (gestation Results Fifty-three premature and 31 term neonates (control) were recruited. At the age of 24 months (corrected age), infants born preterm had significantly smaller TKV (56.1 (9.4) vs. 64.8 (10.2) mL; P = 0.006). There was no difference in eGFR. These preterm infants were smaller (11.25 (1.53) vs. 12.9 (1.8) kg; P = 0.002) and shorter (83.8 (3.0) vs. 86.3 (3.4) cm; P = 0.02) when compared with the control group. At 6, 12, and 18 months respectively, preterm infants had, relative to their height, significantly smaller kidney volumes (0.54 (0.1) vs. 0.59 (0.1) mL/cm, P = 0.05; 0.61 (0.1) vs.0.71 (0.1) mL/cm, P = 0.003; and 0.67 (0.1) vs.0.76 (0.1) mL/cm, P = 0.006). Conclusions Relative to body length, TKV in premature infants is smaller. Since length reflects adult body proportions more accurately than BSA, TKV to height ratio may be a more important measure in the child. Despite smaller TKV (and therefore fewer nephrons), infants born prematurely achieve similar eGFRs in the first 24 months of life, probably due to single-nephron hyperfiltration.

Angiotensin-converting enzyme 2 (ACE2) is a membrane-bound protein containing 805 amino acids. ACE2 shows approximately 42% sequence similarity to somatic ACE but has different biochemical activities. The key role of ACE2 is to catalyze the vasoconstrictor peptide angiotensin (ANG) II to Ang-(1–7), thus regulating the two major counterbalancing pathways of the renin-angiotensin system (RAS). In this way, ACE2 plays a protective role in end-organ damage by protecting tissues from the proinflammatory actions of ANG II. The circulating RAS is activated in normal pregnancy and is essential for maintaining fluid and electrolyte homeostasis and blood pressure. Renin-angiotensin systems are also found in the conceptus. In this review, we summarize the current knowledge on the regulation and function of circulating and uteroplacental ACE2 in uncomplicated and complicated pregnancies, including those affected by preeclampsia and fetal growth restriction. Since ACE2 is the receptor for SARS-CoV-2, and COVID-19 in pregnancy is associated with more severe disease and increased risk of abnormal pregnancy outcomes, we also discuss the role of ACE2 in mediating some of these adverse consequences. We propose that dysregulation of ACE2 plays a critical role in the development of preeclampsia, fetal growth restriction, and COVID-19-associated pregnancy pathologies and suggest that human recombinant soluble ACE2 could be a novel therapeutic to treat and/or prevent these pregnancy complications.

This study aimed to determine if the (pro)renin receptor (ATP6AP2) changes the cellular profile of choriocarcinomas from cytotrophoblast cells to terminally syncytialised cells and ascertain whether this impacts the invasive potential of choriocarcinoma cells. Additionally, we aimed to confirm that FURIN and/or site 1 protease (MBTPS1) cleave soluble ATP6AP2 (sATP6AP2) in BeWo choriocarcinoma cells and determine whether sATP6AP2 levels reflect the cellular profile of choriocarcinomas. BeWo choriocarcinoma cells were treated with ATP6AP2 siRNA, FURIN siRNA, DEC-RVKR-CMK (to inhibit FURIN activity), or PF 429242 (to inhibit MBTPS1 activity). Cells were also treated with forskolin, to induce syncytialisation, or vehicle and incubated for 48 h before collection of cells and supernatants. Syncytialisation was assessed by measuring hCG secretion (by ELISA) and E-cadherin protein levels (by immunoblot and immunocytochemistry). Cellular invasion was measured using the xCELLigence real-time cell analysis system and secreted sATP6AP2 levels measured by ELISA. Forskolin successfully induced syncytialisation and significantly increased both BeWo choriocarcinoma cell invasion (P < 0.0001) and sATP6AP2 levels (P = 0.02). Treatment with ATP6AP2 siRNA significantly inhibited syncytialisation (decreased hCG secretion (P = 0.005), the percent of nuclei in syncytia (P = 0.05)), forskolin-induced invasion (P = 0.046), and sATP6AP2 levels (P < 0.0001). FURIN siRNA and DEC-RVKR-CMK significantly decreased sATP6AP2 levels (both P < 0.0001). In conclusion, ATP6AP2 is important for syncytialisation of choriocarcinoma cells and thereby limits choriocarcinoma cell invasion. We postulate that sATP6AP2 could be used as a biomarker measuring the invasive potential of choriocarcinomas. Additionally, we confirmed that FURIN, not MBTPS1, cleaves sATP6AP2 in BeWo cells, but other proteases (inhibited by DEC-RVKR-CMK) may also be involved.

Suboptimal nutrition during pregnancy is recognised as a significant modifiable determinant in the development of chronic disease in offspring in later life. The current study aimed: (i) to assess the dietary intakes of pregnant Indigenous Australian women against national recommendations and (ii) to investigate the associations between maternal nutrition during pregnancy and the growth of the offspring, including kidney development in late gestation in the Gomeroi gaaynggal cohort (n = 103). Maternal dietary intake in the third trimester was assessed using the Australian Eating Survey Food Frequency Questionnaire. Estimated fetal weight (EFW) and kidney size were obtained by ultrasound. Birth weight was retrieved from hospital birth records. Of the five key nutrients for optimal reproductive health (folate, iron, calcium, zinc and fibre), the nutrients with the highest percentage of pregnant women achieving the nutrient reference values (NRVs) were zinc (75.7%) and folate (57.3%), whereas iron was the lowest. Only four people achieved all NRVs (folate, iron, calcium, zinc and fibre) important in pregnancy. Sodium and saturated fat intake exceeded recommended levels and diet quality was low, with a median score of 28 out of 73 points. After adjusting for smoking and pre-pregnancy body mass index, only maternal intake of retinol equivalents and the proportion of energy from nutrient-dense or energy-dense, nutrient-poor (EDNP) foods were associated with fetal growth. EFW decreased by 0.13 g and birth weight decreased by 0.24 g for every µg increase in maternal dietary retinol intake. Interestingly, EFW, but not actual birth weight, was positively associated with percentage energy from nutrient dense foods and negatively associated with percentage energy from EDNP foods. Dietary supplement usage was associated with increased birthweight, most significantly iron and folate supplementation. Current dietary intakes of pregnant Australian women from this cohort do not align with national guidelines. Furthermore, current findings show that maternal retinol intake and diet composition during pregnancy can influence fetal growth, but not fetal kidney growth in late gestation. Strategies that aim to support and optimise nutrient intakes of Indigenous pregnant women are urgently needed. Future studies with long-term follow-up of the children in the current cohort to assess renal damage and blood pressure are imperative.

The (pro)renin receptor ((P)RR) is a multi-functional protein that can be proteolytically cleaved and released in a soluble form (s(P)RR). Recently, the (P)RR and s(P)RR have become of interest in pregnancy and its associated pathologies. This is because the (P)RR not only activates tissue renin angiotensin systems, but it is also an integral component of vacuolar-ATPase, activates the wingless/integrated (Wnt)/β-catenin and extracellular signal regulated kinases 1 and 2/mitogen-activated protein kinase signalling pathways, and stabilises the β subunit of pyruvate dehydrogenase. Additionally, s(P)RR is detected in plasma and urine, and maternal plasma levels are elevated in pregnancy complications including fetal growth restriction, preeclampsia and gestational diabetes mellitus. Therefore, s(P)RR has potential as a biomarker for these pregnancy pathologies. Preliminary functional findings suggest that s(P)RR may be important for regulating fluid balance, inflammation and blood pressure, all of which contribute to a successful pregnancy. The (P)RR and s(P)RR regulate pathways that are known to be important in maintaining pregnancy, however their role in the physiological context of pregnancy is poorly characterised. This review summarises the known and potential functions of the (P)RR and s(P)RR in pregnancy, and how their dysregulation may contribute to pregnancy complications. It also highlights the need for further research into the source and function of s(P)RR in pregnancy. Soluble (P)RR levels could be indicative of placental, kidney or liver dysfunction and therefore be a novel clinical biomarker, or therapeutic target, to improve the detection and treatment of pregnancy pathologies.

Human placental renin-angiotensin system (RAS) expression is highest in early gestation, at a time when placental oxygen tension is at its lowest (1-3%), and promotes placental development. Some miRNAs predicted to target RAS mRNAs are downregulated in early gestation. We tested the hypothesis that low oxygen suppresses expression of miRNAs that target placental RAS mRNAs, thus increasing concentrations of RAS mRNAs. HTR-8/SVneo cells were cultured in 1, 5 and 20% oxygen for 48 h. Differences in miRNA expression were measured on an Affymetrix miRNA microarray (n = 3/group). Those predicted to target RAS mRNAs, or that were decreased in early gestation, were confirmed by qPCR (n = 9/group). RAS protein levels were assessed by ELISAs or immuno-blotting. Microarray analysis identified four miRNAs predicted to target RAS mRNAs that were differentially expressed between 1 and 5% oxygen. Using qPCR, 15 miRNAs that target the RAS were measured in HTR-8/SVneo cells. Five miRNAs were downregulated in 1% compared with 5% oxygen. Expression of a number of RAS mRNAs (ATP6AP2, AGT, ACE and AGTR1) were increased in either, or both, 1 and 5% oxygen compared with 20% oxygen. AGT protein levels were increased in 1% oxygen compared with 5%. Further validation is needed to confirm that these miRNAs target RAS mRNAs directly and that placental development is partly regulated by oxygen-sensitive miRNAs that target RAS mRNAs. Since placental oxygen tension changes across gestation, changes in expression of these miRNAs may contribute to the transgestational changes in placental RAS expression and the resulting effects on placental development.

Introduction Placental development occurs in a low oxygen environment, which stimulates angiogenesis by upregulating vascular endothelial growth factor A (VEGFA), plasminogen activator inhibitor-1 (SERPINE1) and the angiopoietin-2/-1 ratio (ANGPT2/1). At this time, Angiotensin II type 1 receptor (AT1R) is highly expressed. We postulated that the early gestation placental oxygen milieu, by stimulating the angiotensin (Ang) II/AT1R pathway, increases expression of proliferative/angiogenic factors. Methods HTR-8/SVneo cells were cultured in 1%, 5% or 20% O2 with the AT1R antagonist (losartan) for 48 h. mRNA and protein levels of angiogenic factors were determined by qPCR and ELISA. Angiogenesis and cell viability were assessed by HUVEC tube formation and resazurin assay. Results Culture in low oxygen (1%) increased angiogenic VEGFA, SERPINE1 and placental growth factor (PGF) mRNA and VEGFA and SERPINE1 protein levels, and reduced anti-angiogenic ANGPT1, endoglin (ENG) and soluble fms-like tyrosine kinase-e15a (sFlt-e15a) mRNA (all P = 0.0001). At 1% oxygen, losartan significantly reduced intracellular VEGFA and SERPINE1 levels and secreted VEGF levels (P = 0.008, 0.0001 and 0.0001). HUVEC tube formation was increased in cells grown in HTR-8/SVneo conditioned medium from 1 to 5% cultures (all P = 0.0001). HUVECs cultured in medium from losartan treated HTR-8/SVneo cells had a reduced number of meshes, branching points and total branching length (P = 0.004, 0.003 and 0.0002). At 1% oxygen, losartan partially inhibited the oxygen-induced increase in cell viability (P = 0.0001). Discussion Thus, AT1R blockade antagonised the low oxygen induced increase in pro-angiogenic factor expression and cell viability. Our findings highlight a role for an oxygen-sensitive Ang II/AT1R pathway during placentation.

The (pro)renin receptor (ATP6AP2) is cleaved and released as soluble ATP6AP2 (sATP6AP2). The sATP6AP2 is detected in plasma and urine and is elevated in women with gestational diabetes and preeclampsia. The source and cleavage pathway of sATP6AP2 in pregnancy is unknown. The syncytiotrophoblast is the major placental secretory layer and is in direct contact with maternal blood. Both FURIN and Site 1 protease (MBTPS1) cleave sATP6AP2 in non-placental cells. We postulated that ATP6AP2 was cleaved by FURIN and/or MBTPS1 and that sATP6AP2 is secreted by the placental syncytiotrophoblast.Term primary trophoblast cells were transfected with FURIN siRNA, negative control siRNA or vehicle. In a separate experiment, primary trophoblasts were treated with a pro-protein convertase inhibitor (DEC-RVKR-CMK), an MBTPS1 inhibitor (PF 429242) or vehicle. Trophoblasts were left to spontaneously syncytialise before cells and supernatants were collected and intracellular and extracellular sATP6AP2 levels analysed by immunoblot.sATP6AP2 is secreted by placental trophoblasts. Levels of intra and extra-cellular sATP6AP2 decrease with syncytialisation (P = 0.01 and P = 0.02, respectively), as do FURIN mRNA (P = 0.0003) and protein (P = 0.0007). FURIN siRNA decreased FURIN mRNA and protein levels (both P 0.0001). Neither FURIN siRNA or PF 429242 affected sATP6AP2 levels. DEC-RVKR-CMK significantly decreased extracellular sATP6AP2 protein levels (P = 0.02).Soluble ATP6AP2 is secreted by placental trophoblasts and levels decrease with syncytialisation. DEC-RVKR-CMK, a broad inhibitor of pro-protein convertases reduced extracellular sATP6AP2 levels, but FURIN siRNA and MBTPS1 inhibition had no effect. Hence, a convertase other than FURIN or MBTPS1 is most likely responsible for placental sATP6AP2 secretion.

Introduction In the placenta, the (pro)renin receptor (ATP6AP2) is localised to the syncytiotrophoblast. ATP6AP2 can activate the placental renin-angiotensin system (RAS), producing Angiotensin II (Ang II) which, acting via the angiotensin II type 1 receptor (AGTR1), is important for placental development and function. ATP6AP2 can also independently stimulate intracellular signalling pathways known to regulate trophoblast syncytialisation. We proposed that ATP6AP2 plays a role in trophoblast syncytialisation. Methods Primary trophoblast cells were isolated from human placentae and transfected with an ATP6AP2 siRNA, a negative control siRNA or vehicle and allowed to spontaneously syncytialise. Syncytialisation was determined by secretion of human chorionic gonadotrophin (hCG) and by decreased CDH1 (E-cadherin) levels. Expression of RAS mRNAs and proteins were measured by qPCR and immunoblotting, respectively. Results Primary trophoblast cells spontaneously syncytialised in culture. Syncytialisation did not affect ATP6AP2 mRNA or protein levels. However, the expression of REN, AGT and AGTR1 mRNAs were increased (P = 0.02, P = 0.01 and P = 0.03, respectively). ATP6AP2 siRNA had no effect on syncytialisation. Discussion In primary trophoblasts, syncytialisation was associated with increased expression of the RAS. hCG was increased during syncytialisation and is known to stimulate REN and possibly AGT, however further experiments are needed to confirm that this was the mechanism via which the RAS was activated. Therefore, syncytialisation of primary trophoblasts may involve hCG-induced RAS activation and downstream activation of signalling pathways and growth factors, which can be stimulated via the interaction of Ang II with AGTR1. Nevertheless, it appears that the (pro)renin receptor is not involved.

Placental renin-angiotensin system (RAS) components; prorenin, angiotensinogen, and angiotensin (Ang) II type 1 receptor (AT

KEY POINTS Preterm infants often have poor cardiovascular function that is associated with adverse neurodevelopmental outcomes. Preterm infants may be vulnerable to hypovolaemia due to excessive vasodilatation and leaky capillaries. Following reduction in blood volume, cardiac output and mean arterial pressure were reduced to the same extent in term and preterm piglets. Cerebral blood flow was maintained following blood volume reduction in term but not in preterm piglets. Effective detection and treatment of functional hypovolaemia may reduce the risk of brain injury in preterm infants. ABSTRACT Preterm infants often have impaired cardiovascular function that may contribute to poor neurodevelopmental outcomes. The study aimed to determine the effects of reduced blood volume on cardiovascular function, including cerebral blood flow, in preterm and term piglets. In preterm (97/115 days) and term piglets, up to 10% of the estimated blood volume was removed. Removal of blood was stopped if MAP dropped below 20 mmHg. Heart rate, cardiac contractility and relaxation, cardiac output, mean arterial pressure (MAP), and cerebral blood flow were measured at baseline and again after blood volume reduction. The volume of blood removed was less in preterm piglets than in term piglets (5.1 ± 1.8 vs. 7.7 ± 0.9 mL kg-1 , mean ± SD, P

The intrarenal renin-angiotensin system (iRAS) is implicated in the pathogenesis of hypertension, chronic kidney disease and diabetic nephropathy. Urinary angiotensinogen (uAGT) levels reflect the activity of the iRAS and are altered in women with preeclampsia. Since Indigenous Australians suffer high rates and early onset of renal disease, we hypothesised that Indigenous Australian pregnant women, like non-Indigenous women with pregnancy complications, would have altered uAGT levels. The excretion of RAS proteins was measured in non-Indigenous and Indigenous Australian women with uncomplicated or complicated pregnancies (preeclampsia, diabetes/gestational diabetes, proteinuria/albuminuria, hypertension, small/large for gestational age, preterm birth), and in non-pregnant non-Indigenous women.Non-Indigenous pregnant women with uncomplicated pregnancies, had higher uAGT/creatinine levels than non-Indigenous non-pregnant women (P < 0.01), and levels increased as pregnancy progressed (P < 0.001). In non-Indigenous pregnant women with pregnancy complications, uAGT/creatinine was suppressed in the third trimester (P < 0.01). In Indigenous pregnant women with uncomplicated pregnancies, there was no change in uAGT/creatinine with gestational age and uAGT/creatinine was lower in the 2nd and 3rd trimesters than in nonIndigenous pregnant women with uncomplicated pregnancies (P < 0.03, P < 0.007, respectively). The uAGT/creatinine ratios of Indigenous women with uncomplicated or complicated pregnancies were the same.A decrease in uAGT/creatinine with advancing gestational age was associated with increased urinary albumin/creatinine, as is seen in preeclampsia, but it was not specific for this disorder. The reduced uAGT/creatinine in Indigenous pregnant women may reflect subclinical renal dysfunction which limits the ability of the kidney to maintain sodium balance and could indicate an increased risk of pregnancy complications and/or future renal disease.

No other physiological state is characterized by such high levels of renin, angiotensinogen, angiotensin II and aldosterone as occurs in pregnancy. Increased activity of the circulating renin-angiotensin-aldosterone system (cRAAS) and possibly the intrarenal renin-angiotensin system (RAS) counteract the effects of pregnancy on maternal cardiovascular and renal function caused by ovarian hormones. Of particular importance is the early activation of the cRAAS by estrogen-stimulated production of angiotensinogen, freeing it from feedback control of active renin release. This leads to increased salt and water retention that fills the maternal cardiovascular system, which is expanding under the influence of relaxin and other factors. The ovarian RAS plays key roles in ovulation and steroidogenesis. Placental and decidual RASs may play important roles in placentation and in regulating the timing of parturition. Thus, increased activity of tissue and circulating renin-angiotensin systems in pregnancy and their coordinated control are required to maintain pregnancy and ensure a healthy outcome.

Maternal obesity during pregnancy has a detrimental impact on offspring renal development and function. This is pertinent to Indigenous Australians as they are twice as likely as non‐Indigenous Australians to develop chronic kidney disease (CKD). The aim of this study was to examine whether there was an association between maternal adiposity and fetal kidney growth in late gestation (>28 weeks) and kidney function in infants, 28 weeks gestation by bioelectrical impedance analysis. Fetal kidney structure was assessed by ultrasound. Renal function indicators (urinary albumin:creatinine and protein:creatinine) were measured in infants from a spot urine collection from nappies. Multiple linear regression and multi‐level mixed effects linear regression models with clustering were used to account for repeated measures of urine. 147 mother–child pairs were examined. Estimated fetal weight (EFW), but not fetal kidney size, was positively associated with maternal adiposity and pre‐pregnancy BMI. When adjusted for smoking, combined kidney volume relative to EFW was negatively associated with maternal percentage body fat. Infant kidney function was not influenced by maternal adiposity and pre‐pregnancy BMI (n = 84 observations). Current findings show that Indigenous babies born to obese mothers have reduced kidney size relative to EFW. We suggest that these babies are experiencing a degree of glomerular hyperfiltration in utero, and therefore are at risk of developing CKD in later life, especially if their propensity for obesity is maintained. Although no impact on renal function was observed at

The preterm newborn is at high risk of developing cardiovascular compromise during the first day of life and this is associated with increased risk of brain injury. Standard treatments are volume expansion and administration of inotropes, typically dopamine and/or dobutamine, but there is limited evidence that inotropes improve clinical outcomes. This study investigated the efficacy of dopamine and dobutamine for the treatment of cardiovascular compromise in the preterm newborn using a piglet model. Preterm and term piglets were assigned to either dopamine, dobutamine or control infusions. Heart rate, left ventricular contractility, cardiac output, blood pressure, and cerebral and regional blood flows were measured during baseline, low (10 µg/kg/h), and high (20 µg/kg/h) dose infusions. At baseline, preterm piglets had lower cardiac contractility, cardiac output, blood pressure, and cerebral blood flow compared to term piglets. The response of preterm piglets to either dopamine or dobutamine administration was less than in term piglets. In both preterm and term piglets, cardiac output and cerebral blood flow were unaltered by either inotrope. In order to provide better cardiovascular support, it may be necessary to develop treatments that target receptors with a more mature profile than adrenoceptors in the preterm newborn.

Endometrial cancer (EC) is the most common gynaecological malignancy and its incidence is increasing. Dysregulation of the endometrial renin–angiotensin system (RAS) could predispose to EC; therefore, we studied the prevalence of RAS single nucleotide polymorphisms (SNPs) in Australian women with EC. SNPs assessed were AGT M235T (rs699); AGTR1 A1166C (rs5186); ACE A240T and T93C (rs4291, rs4292) and ATP6AP2 (rs2968915). They were identified using TaqMan SNP Genotyping Assays. The C allele of the AGTR1 SNP (rs5186) was more prevalent in women with EC (odds ratio (OR) 1.7, 95% confidence interval (CI) (1.2–2.3), P=0.002). The CC genotype of this SNP is associated with upregulation of the angiotensin II type 1 receptor (AGTR1). The G allele of AGT rs699, which is associated with higher angiotensinogen (AGT) levels, was less prevalent in women with EC (OR 0.54, 95% CI (0.39–0.74), PAGTR1 SNP (rs5186) and the ACE SNPs (rs4291 and rs4292) was greater than in women who had no record of HRT; SNP rs4291 is associated with increased plasma ACE activity. These data suggest there is an interaction between genotype, oestrogen replacement therapy and EC. In conclusion, the prevalence of two SNPs that enhance RAS activity was different in women with EC compared with healthy controls. These genetic factors may interact with obesity and hyperoestrogenism, predisposing ageing, obese women to EC.

Adverse pregnancy outcomes including prematurity and low birth weight (LBW) have been associated with life-long chronic disease risk for the infant. Stress during pregnancy increases the risk of adverse pregnancy outcomes. Many studies have reported the incidence of adverse pregnancy outcomes in Indigenous populations and a smaller number of studies have measured rates of stress and depression in these populations. This study sought to examine the potential association between stress during pregnancy and the rate of adverse pregnancy outcomes in Australian Indigenous women residing in rural and remote communities in New South Wales. This study found a higher rate of post-traumatic stress disorder, depression and anxiety symptoms during pregnancy than the general population. There was also a higher incidence of prematurity and LBW deliveries. Unfortunately, missing post-traumatic stress disorder and depressive symptomatology data impeded the examination of associations of interest. This was largely due to the highly sensitive nature of the issues under investigation, and the need to ensure adequate levels of trust between Indigenous women and research staff before disclosure and recording of sensitive research data. We were unable to demonstrate a significant association between the level of stress and the incidence of adverse pregnancy outcomes at this stage. We recommend this longitudinal study continue until complete data sets are available. Future research in this area should ensure prioritization of building trust in participants and overestimating sample size to ensure no undue pressure is placed upon an already stressed participant.

Fetal growth restriction (FGR) is a pregnancy complication wherein the foetus fails to reach its growth potential. The renin–angiotensin system (RAS) is a critical regulator of placental function, controlling trophoblast proliferation, angiogenesis and blood flow. The RAS significantly influences uteroplacental blood flow through the balance of its vasoconstrictive and vasodilatory pathways. Although the RAS is known to be dysregulated in placentae from women with preeclampsia, the expression of the RAS has not yet been studied in pregnancies compromised by FGR alone. This study investigated the mRNA expression and protein levels of RAS components in placentae from pregnancies compromised by FGR. Angiotensin II type 1 receptor (AGTR1) and angiotensin-converting enzyme 2 (ACE2) mRNA levels were reduced in FGR placentae compared with control (P = 0.012 and 0.018 respectively). Neprilysin (NEP) mRNA expression was lower in FGR placentae compared with control (P = 0.004). mRNA levels of angiotensinogen (AGT) tended to be higher in FGR placentae compared with control (P = 0.090). Expression of prorenin, AGT, angiotensin-converting enzyme (ACE) or ACE2 proteins were similar in control and FGR placentae. The renin-AGT reaction is a first order reaction so levels of expression of placental AGT determine levels of Ang II. Decreasing levels of ACE2 and/or NEP by limiting the production of Ang-(1-7), which is a vasodilator, and increasing placental Ang II levels (vasoconstrictor) may result in an imbalance between the vasoconstrictor and vasodilator arms of the placental RAS. Ultimately this dysregulation of the placental RAS could lead to reduced placental perfusion that is evident in FGR.

In early gestation, the human placental renin-angiotensin system (RAS) is upregulated and plays a role in placental development. Among other functions, signalling through the angiotensin II type 1 receptor (AT1R) initiates proliferation. Many microRNAs (miRNAs) targeting placental RAS mRNAs are downregulated at this time. We propose that in early gestation miRNAs that target the placental RAS are downregulated, allowing for the increased RAS expression and proliferation required for adequate placentation. HTR-8/SVneo cells (an immortalized human trophoblast cell line) were used to assess the effect of nine miRNA mimics (at 0.08, 0.16, 0.32 and 0.64 ng/μL) on trophoblast cell proliferation and predicted RAS target mRNAs. The effect of the miRNA mimics on the rate of cell proliferation was assessed using the xCELLigence real-time cell analysis system over 48 h. Levels of miRNAs and predicted RAS target mRNAs were determined by RT-PCR (qPCR, n = 9/group). Statistically different levels of expression were determined (P < 0.05). All nine miRNA mimics significantly affected the proliferation rates of HTR-8/SVneo cells. Five of the miRNA mimics (miR-181a-5p (predicted to target: renin (REN), angiotensin converting enzyme (ACE)), miR-378 (REN, ACE), miR-663 (REN), miR-483-3p (ACE, ACE2, angiotensinogen (AGT), angiotensin II type 1 receptor (AGTR1)) and miR-514 (AGT)) were associated with a dose-dependent reduction in cell proliferation. Seven of the mimics significantly decreased expression of at least one of their predicted target RAS mRNAs. Our study shows that miRNAs targeting placental RAS mRNAs play a role in controlling trophoblast proliferation. As placentation is largely a process of proliferation, changes in expression of these miRNAs may be partly responsible for the expression of the placental RAS, proliferation and placentation.

Study question Are any microRNAs (miRNAs) that target the placental renin-angiotensin system (RAS) in the human placenta suppressed in early gestation? Summary answer Overall, 21 miRNAs with predicted RAS mRNA targets were less abundant in early versus term placentae and nine were more highly expressed. What is known already Regulation of human placental RAS expression could alter placental development and therefore normal pregnancy outcome. The expression of genes encoding prorenin (REN), angiotensinogen, (pro)renin receptor, angiotensin converting enzyme 2, and the angiotensin II type 1 receptor are highest in early gestation, at a time when oxygen tension is at its lowest. Studies have shown that the human placental RAS is sensitive to oxygen, as are some miRNAs that regulate RAS mRNAs. We propose that in early pregnancy, the prevailing low O2 tension, by suppression of levels of miRNAs that target RAS mRNAs, results in increased expression of RAS mRNAs and encoded proteins. As gestation proceeds and the prevailing oxygen tension rises, abundance of these miRNAs increases, and placental RAS mRNA expression is suppressed. Study design, size, duration The expression of miRNAs was compared in human placentae collected in early (10-11 weeks; n = 7) and mid-gestation (14-18 weeks; n = 8) with placenta collected at term (38-40 weeks; n = 8). Expression of placental miRNAs in women with early (29-35.1 weeks; n = 8) or late-onset pre-eclampsia (PE) (>34-weeks gestation; n = 8) and gestational age matched preterm (31.6-35.1 weeks; n = 8) and term normotensive controls were also compared. Participants/materials, setting, methods Agilent Human miRNA microarray v19 was used to detect up to 2006 miRNAs in four placentae from each group. Statistically different levels of expression were determined and refined using predictive modelling. Placental miRNAs predicted to target RAS mRNAs were identified in three databases. Differences detected on the array were confirmed for some miRNAs by semi-quantitative RT-PCR (qPCR, n = 7-8 for all groups). Two differentially expressed miRNAs that were known to target human renal REN mRNA (miR-181a-5p and miR-663) were transfected into human HTR-8/SVneo trophoblast cells to examine their effect on placental REN expression and prorenin levels. Main results and the role of chance In early gestation placentae, 186 miRNAs were differentially expressed compared with term placentae (109 increased, 77 decreased). Thirty of the differentially expressed miRNAs were predicted to target RAS components. In mid-gestation placentae, 117 miRNAs were differentially expressed compared with term placentae (69 increased, 48 decreased). Of these, 19 had RAS mRNAs as predicted targets. Eight miRNAs that were lower in early gestation and predicted to target RAS mRNAs were confirmed by qPCR. All showed an increase during gestation and could influence the transgestational profile of the human placental RAS. Additionally, on the array, three miRNAs predicted to target RAS mRNAs (miR-892c-3p, miR-378c and miR-514b-3p) were overexpressed in placentae from women with late-onset PE (P = 3.6E-10, P = 1.8E-05, P = 5.3E-06; respectively). miR-663, which suppresses renal REN mRNA expression, was overexpressed in early-onset PE placentae as determined by qRT-PCR analysis (P = 0.014). Transfection of miR-181a-5p and miR-663 into HTR-8/SVneo trophoblast cells suppressed REN mRNA expression (P = 0.05) and prorenin protein production (P = 0.001). Large scale data Data can be found via GEO accession number GSE109832. Limitations, reasons for caution Further validation that the differentially expressed miRNAs do indeed directly target RAS mRNAs and affect placental development and function is required. This study is limited by the small sample size. Therefore independent validation in a larger cohort is required. Wider implications of the findings We propose that suppression of miRNAs that target the placental RAS in early gestation is partly responsible for the increase in RAS expression at this time, in order to promote placental development. Later in pregnancy, we have detected overexpression of several miRNAs in placentae from women with PE. These may prove to be biomarkers for early detection of women at risk of developing PE. Since the placenta produces at least two miRNAs that were found in the kidney to target REN mRNA, and that also target placental REN mRNA, the escape of these miRNAs into the maternal circulation in excess amounts could affect maternal renal REN mRNA production and thereby disturb maternal fluid and electrolyte homoeostasis. Study funding/competing interest(s) This work was supported by the National Health and Medical Research Council, Australia (APP1043537). K.G.P. is supported by an Australian Research Council Future Fellowship (FT150100179). C.T.R. is supported by a Lloyd Cox Professorial Research Fellowship from the University of Adelaide. F.Z.M. is supported by a National Heart Foundation Future Leader Fellowship and Baker Heart and Diabetes Institute Fellowship. The authors declare that they have no competing interests.

Childhood obesity rates are higher among Indigenous compared with non-Indigenous Australian children. It has been hypothesized that early-life influences beginning with the intrauterine environment predict the development of obesity in the offspring. The aim of this paper was to assess, in 227 mother–child dyads from the Gomeroi gaaynggal cohort, associations between prematurity, Gestation Related-Optimal Weight (GROW) centiles, maternal adiposity (percentage body fat, visceral fat area), maternal non-fasting plasma glucose levels (measured at mean gestational age of 23.1 weeks) and offspring BMI and adiposity (abdominal circumference, subscapular skinfold thickness) in early childhood (mean age 23.4 months). Maternal non-fasting plasma glucose concentrations were positively associated with infant birth weight (P=0.005) and GROW customized birth weight centiles (P=0.008). There was a significant association between maternal percentage body fat (P=0.02) and visceral fat area (P=0.00) with infant body weight in early childhood. Body mass index (BMI) in early childhood was significantly higher in offspring born preterm compared with those born at term (P=0.03). GROW customized birth weight centiles was significantly associated with body weight (P=0.01), BMI (P=0.007) and abdominal circumference (P=0.039) at early childhood. Our findings suggest that being born preterm, large for gestational age or exposed to an obesogenic intrauterine environment and higher maternal non-fasting plasma glucose concentrations are associated with increased obesity risk in early childhood. Future strategies should aim to reduce the prevalence of overweight/obesity in women of child-bearing age and emphasize the importance of optimal glycemia during pregnancy, particularly in Indigenous women.

Preterm infants often have poor cardiovascular function that is associated with adverse neurodevelopmental outcomes. Preterm infants may be vulnerable to hypovolaemia due to excessive vasodilatation and leaky capillaries. Following reduction in blood volume, cardiac output and mean arterial pressure were reduced to the same extent in term and preterm piglets. Cerebral blood flow was maintained following blood volume reduction in term but not in preterm piglets. Effective detection and treatment of functional hypovolaemia may reduce the risk of brain injury in preterm infants.Preterm infants often have impaired cardiovascular function that may contribute to poor neurodevelopmental outcomes. The study aimed to determine the effects of reduced blood volume on cardiovascular function, including cerebral blood flow, in preterm and term piglets. In preterm (97/115 days) and term piglets, up to 10% of the estimated blood volume was removed. Removal of blood was stopped if MAP dropped below 20 mmHg. Heart rate, cardiac contractility and relaxation, cardiac output, mean arterial pressure (MAP), and cerebral blood flow were measured at baseline and again after blood volume reduction. The volume of blood removed was less in preterm piglets than in term piglets (5.1 ± 1.8 vs. 7.7 ± 0.9 mL kg

Genetic differences between ethnic populations affect susceptibility to disease and efficacy of drugs. This study examined and compared the prevalence of single nucleotide polymorphisms (SNPs) in genes of the renin-angiotensin system (RAS) in a desert community of Indigenous Australians and in non-Indigenous Australians. The polymorphisms were angiotensinogen, AGT G-217A (rs5049); AGT G+174A (rs4762); Angiotensin II type 1 receptor, AGTR1 A+1166C (rs5186); angiotensin converting enzyme, ACE A-240T (rs4291), ACE T-93C (rs4292); renin, REN T+1142C (rs5706). They were measured using allelic discrimination assays. The prevalence of REN T+1142C SNP was similar in the two populations; 99% were homozygous for the T allele. All other SNPs were differently distributed between the two populations (P < 0.0001). In non-Indigenous Australians, the A allele at position 204 of ACE rs4291 was prevalent (61.8%) whereas in the Indigenous Australians the A allele was less prevalent (28%). For rs4292, the C allele had a prevalence of 37.9% in non-Indigenous Australians but in Indigenous Australians the prevalence was only 1%. No Indigenous individuals were homozygous for the C allele of AGTR1 (rs5186). Thus the prevalence of RAS SNPs in this Indigenous Australian desert community was different from non-Indigenous Australians as was the prevalence of cytokine SNPs (as shown in a previous study). These differences may affect susceptibility to chronic renal and cardiovascular disease and may alter the efficacy of drugs used to inhibit the RAS. These studies highlight the need to study the pharmacogenetics of drug absorption, distribution, metabolism and excretion in Indigenous Australians for safe prescribing guidelines.

Pregnancy outcome is influenced, in part, by the sex of the fetus. Decidual renin messenger RNA (REN) abundance is greater in women carrying a female fetus than a male fetus. Here, we explore whether the sex of the fetus also influences the regulation of decidual RAS expression with a known stimulator of renal renin and cyclic adenosine monophosphate (cAMP). Cyclic adenosine monophosphate had no affect on decidual REN expression, since REN abundance was still greater in decidual explants from women carrying a female fetus than a male fetus after cAMP treatment. Cyclic adenosine monophosphate decreased prorenin levels in the supernatant if the fetus was female (ie, prorenin levels were no longer sexually dimorphic) and altered the fetal sex-specific differences in other RAS genes seen in vitro. Therefore, fetal sex influences the decidual renin-angiotensin system response to cAMP. This may be related to the presence of fetal cells in the maternal decidua.

Objectives:The newborn circulating, cardiac and renal renin–angiotensin systems (RASs) are essential for blood pressure control, and for cardiac and renal development. If cardiac and renal RASs are immature this may contribute to cardiovascular compromise in preterm infants. This study measured mRNA expression of cardiac and renal RAS components in preterm, glucocorticoid (GC) exposed preterm, and term piglets.Methods:Renal and cardiac RAS mRNA levels were measured using real-time polymerase chain reaction (PCR). Genes studied were: (pro)renin receptor, renin, angiotensinogen, angiotensin converting enzyme (ACE), ACE2, angiotensin type 1 receptor (AT1R) and angiotensin type 2 receptor (AT2R).Results:All the genes studied were expressed in the kidney; neither renin nor AT2R mRNA were detected in the heart. There were no gestational changes in (pro)renin receptor, renin, ACE or AT1R mRNA levels. Right ventricular angiotensinogen mRNA levels in females were lower in preterm animals than at term, and GC exposure increased levels in male piglets. Renal angiotensinogen mRNA levels in female term piglets were lower than females from both preterm groups, and lower than male term piglets. Left ventricular ACE2 mRNA expression was lower in GC treated preterm piglets. Renal AT2R mRNA abundance was highest in GC treated preterm piglets, and the AT1R/AT2R ratio was increased at term.Conclusions:Preterm cardiac and renal RAS mRNA levels were similar to term piglets, suggesting that immaturity of these RASs does not contribute to preterm cardiovascular compromise. Since preterm expression of both renal and cardiac angiotensin II-AT1R is similar to term animals, cardiovascular dysfunction in the sick preterm human neonate might be effectively treated by agents acting on their RASs.

Uterine arteries from pregnant animals are one of only a few adult blood vessels which express predominantly AT2 receptors. Fetal systemic arteries also contain a high density of AT2 receptors. To assess whether fetal systemic arteries, like uterine arteries from pregnant animals, were relatively insensitive to angiotensin II (Ang II), we compared the contractile responses of fetal carotid arteries, as well as the receptor binding properties of fetal aortae, with those of uterine arteries from pregnant ewes. Ang II receptor binding properties were measured in arterial membrane preparations using 125I [Sar1Ile8] Ang II. Proportions of AT1 and AT2 receptors were determined by inhibiting 125I [Sar1Ile8] Ang II with losartan (AT1 antagonist) or PD 123319 (AT2 antagonist). Both fetal aortae and uterine arteries contained predominantly AT2 receptors. However, the AT2 receptor in fetal aortae had a higher affinity than that of the AT2 receptor in uterine arteries (P