Molecular regulators of defective placental and cardiovascular development in fetal growth restriction.

Placental insufficiency is one of the major causes of fetal growth restriction (FGR), a significant pregnancy disorder in which the fetus fails to achieve its full growth potential in utero. As well as the acute consequences of being born too small, affected offspring are at increased risk of cardiovascular disease, diabetes and other chronic diseases in later life. The placenta and heart develop concurrently, therefore placental maldevelopment and function in FGR may have profound effect on the growth and differentiation of many organ systems, including the heart. Hence, understanding the key molecular players that are synergistically linked in the development of the placenta and heart is critical. This review highlights the key growth factors, angiogenic molecules and transcription factors that are common causes of defective placental and cardiovascular development.

Decreased Expression of Placental Proteins in Recurrent Pregnancy Loss: Functional Relevance and Diagnostic Value.

Miscarriages affect 50-70% of all conceptions and 15-20% of clinically recognized pregnancies. Recurrent pregnancy loss (RPL, ≥2 miscarriages) affects 1-5% of recognized pregnancies. Nevertheless, our knowledge about the etiologies and pathophysiology of RPL is incomplete, and thus, reliable diagnostic/preventive tools are not yet available. Here, we aimed to define the diagnostic value of three placental proteins for RPL: human chorionic gonadotropin free beta-subunit (free-ß-hCG), pregnancy-associated plasma protein-A (PAPP-A), and placental growth factor (PlGF). Blood samples were collected from women with RPL (n = 14) and controls undergoing elective termination of pregnancy (n = 30) at the time of surgery. Maternal serum protein concentrations were measured by BRAHMS KRYPTOR Analyzer. Daily multiple of median (dMoM) values were calculated for gestational age-specific normalization. To obtain classifiers, logistic regression analysis was performed, and ROC curves were calculated. There were differences in changes of maternal serum protein concentrations with advancing healthy gestation. Between 6 and 13 weeks, women with RPL had lower concentrations and dMoMs of free ß-hCG, PAPP-A, and PlGF than controls. PAPP-A dMoM had the best discriminative properties (AUC = 0.880). Between 9 and 13 weeks, discriminative properties of all protein dMoMs were excellent (free ß-hCG: AUC = 0.975; PAPP-A: AUC = 0.998; PlGF: AUC = 0.924). In conclusion, free-ß-hCG and PAPP-A are valuable biomarkers for RPL, especially between 9 and 13 weeks. Their decreased concentrations indicate the deterioration of placental functions, while lower PlGF levels indicate problems with placental angiogenesis after 9 weeks.

Association of Vascular Endothelial Growth Factors (VEGFs) with Recurrent Miscarriage: A Systematic Review of the Literature.

Recurrent miscarriage (RM) can be defined as two or more consecutive miscarriages before 20 weeks' gestation. Vascular endothelial growth factors (VEGFs) play an important role in endometrial angiogenesis and decidualization, prerequisites for successful pregnancy outcomes. We conducted a systematic review of the published literature investigating the role of VEGFs in RM. In particular, we explored the methodological inconsistencies between the published reports on this topic. To our knowledge, this is the first systematic literature review to examine the role of VEGFs in RM. Our systematic search followed PRISMA guidelines. Three databases, Medline (Ovid), PubMed, and Embase, were searched. Assessment-bias analyses were conducted using the Joanna Bigger Institute critical appraisal method for case-control studies. Thirteen papers were included in the final analyses. These studies included 677 cases with RM and 724 controls. Endometrial levels of VEGFs were consistently lower in RM cases compared to controls. There were no consistent significant findings with respect to VEGFs levels in decidua, fetoplacental tissues, and serum when RM cases were compared to controls. The interpretation of studies that explored the relationship between VEGFs and RM is hampered by inconsistencies in defining clinical, sampling, and analytical variables. To clarify the association between VEGF and RM in future studies, researchers ideally should use similarly defined clinical groups, similar samples collected in the same way, and laboratory analyses undertaken using the same methods.

Proteoglycans: Systems-Level Insight into Their Expression in Healthy and Diseased Placentas.

Proteoglycan macromolecules play key roles in several physiological processes (e.g., adhesion, proliferation, migration, invasion, angiogenesis, and apoptosis), all of which are important for placentation and healthy pregnancy. However, their precise roles in human reproduction have not been clarified. To fill this gap, herein, we provide an overview of the proteoglycans' expression and role in the placenta, in trophoblast development, and in pregnancy complications (pre-eclampsia, fetal growth restriction), highlighting one of the most important members of this family, syndecan-1 (SDC1). Microarray data analysis showed that of 34 placentally expressed proteoglycans, SDC1 production is markedly the highest in the placenta and that SDC1 is the most upregulated gene during trophoblast differentiation into the syncytiotrophoblast. Furthermore, placental transcriptomic data identified dysregulated proteoglycan genes in pre-eclampsia and in fetal growth restriction, including SDC1, which is supported by the lower concentration of syndecan-1 in maternal blood in these syndromes. Overall, our clinical and in vitro studies, data analyses, and literature search pointed out that proteoglycans, as important components of the placenta, may regulate various stages of placental development and participate in the maintenance of a healthy pregnancy. Moreover, syndecan-1 may serve as a useful marker of syncytialization and a prognostic marker of adverse pregnancy outcomes. Further studies are warranted to explore the role of proteoglycans in healthy and complicated pregnancies, which may help in diagnostic or therapeutic developments.

Inflammasomes-A Molecular Link for Altered Immunoregulation and Inflammation Mediated Vascular Dysfunction in Preeclampsia.

Preeclampsia (PE) is a pregnancy-specific multisystem disorder and is associated with maladaptation of the maternal cardiovascular system and abnormal placentation. One of the important characteristics in the pathophysiology of PE is a dysfunction of the placenta. Placental insufficiency is associated with poor trophoblast uterine invasion and impaired transformation of the uterine spiral arterioles to high capacity and low impedance vessels and/or abnormalities in the development of chorionic villi. Significant progress in identifying potential molecular targets in the pathophysiology of PE is underway. The human placenta is immunologically functional with the trophoblast able to generate specific and diverse innate immune-like responses through their expression of multimeric self-assembling protein complexes, termed inflammasomes. However, the type of response is highly dependent upon the stimuli, the receptor(s) expressed and activated, the downstream signaling pathways involved, and the timing of gestation. Recent findings highlight that inflammasomes can act as a molecular link for several components at the syncytiotrophoblast surface and also in maternal blood thereby directly influencing each other. Thus, the inflammasome molecular platform can promote adverse inflammatory effects when chronically activated. This review highlights current knowledge in placental inflammasome expression and activity in PE-affected pregnancies, and consequently, vascular dysfunction in PE that must be addressed as an interdependent interactive process.

Hydroxychloroquine Mitigates the Production of 8-Isoprostane and Improves Vascular Dysfunction: Implications for Treating Preeclampsia.

In preeclampsia, widespread maternal endothelial dysfunction is often secondary to excessive generation of placental-derived anti-angiogenic factors, including soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), along with proinflammatory cytokines such as tumour necrosis factor-α (TNF-α) and activin A, understanding of which offers potential opportunities for the development of novel therapies. The antimalarial hydroxychloroquine is an anti-inflammatory drug improving endothelial homeostasis in lupus. It has not been explored as to whether it can improve placental and endothelial function in preeclampsia. In this in vitro study, term placental explants were used to assess the effects of hydroxychloroquine on placental production of sFlt-1, sEng, TNF-α, activin A, and 8-isoprostane after exposure to hypoxic injury or oxidative stress. Similarly, human umbilical vein endothelial cells (HUVECs) were used to assess the effects of hydroxychloroquine on in vitro markers of endothelial dysfunction. Hydroxychloroquine had no effect on the release of sFlt-1, sEng, TNF-α, activin A, or 8-isoprostane from placental explants exposed to hypoxic injury or oxidative stress. However, hydroxychloroquine mitigated TNF-α-induced HUVEC production of 8-isoprostane and Nicotinanamide adenine dinucleotide phosphate (NADPH) oxidase expression. Hydroxychloroquine also mitigated TNF-α and preeclamptic serum-induced HUVEC monolayer permeability and rescued the loss of zona occludens protein zona occludens 1 (ZO-1). Although hydroxychloroquine had no apparent effects on trophoblast function, it may be a useful endothelial protectant in women presenting with preeclampsia.

The Effects of Early-Onset Pre-Eclampsia on Placental Creatine Metabolism in the Third Trimester.

Creatine is a metabolite important for cellular energy homeostasis as it provides spatio-temporal adenosine triphosphate (ATP) buffering for cells with fluctuating energy demands. Here, we examined whether placental creatine metabolism was altered in cases of early-onset pre-eclampsia (PE), a condition known to cause placental metabolic dysfunction. We studied third trimester human placentae collected between 27-40 weeks' gestation from women with early-onset PE (n = 20) and gestation-matched normotensive control pregnancies (n = 20). Placental total creatine and creatine precursor guanidinoacetate (GAA) content were measured. mRNA expression of the creatine synthesizing enzymes arginine:glycine aminotransferase (GATM) and guanidinoacetate methyltransferase (GAMT), the creatine transporter (SLC6A8), and the creatine kinases (mitochondrial CKMT1A & cytosolic BBCK) was assessed. Placental protein levels of arginine:glycine aminotransferase (AGAT), GAMT, CKMT1A and BBCK were also determined. Key findings; total creatine content of PE placentae was 38% higher than controls (p < 0.01). mRNA expression of GATM (p < 0.001), GAMT (p < 0.001), SLC6A8 (p = 0.021) and BBCK (p < 0.001) was also elevated in PE placentae. No differences in GAA content, nor protein levels of AGAT, GAMT, BBCK or CKMT1A were observed between cohorts. Advancing gestation and birth weight were associated with a down-regulation in placental GATM mRNA expression, and a reduction in GAA content, in control placentae. These relationships were absent in PE cases. Our results suggest PE placentae may have an ongoing reliance on the creatine kinase circuit for maintenance of cellular energetics with increased total creatine content and transcriptional changes to creatine synthesizing enzymes and the creatine transporter. Understanding the functional consequences of these changes warrants further investigation.

The role of insulin-like growth factor 2 receptor-mediated homeobox gene expression in human placental apoptosis, and its implications in idiopathic fetal growth restriction.

Fetal growth restriction (FGR) is caused by poor placental development and function early in gestation. It is well known that placentas from women with FGR exhibit reduced cell growth, elevated levels of apoptosis and perturbed expression of the growth factors, cytokines and the homeobox gene family of transcription factors. Previous studies have reported that insulin-like growth factor-2 (IGF2) interacts with its receptor-2 (IGF2R) to regulate villous trophoblast survival and apoptosis. In this study, we hypothesized that human placental IGF2R-mediated homeobox gene expression is altered in FGR and contributes to abnormal trophoblast function. This study was designed to determine the association between IGF2R, homeobox gene expression and cell survival in pregnancies affected by FGR. Third trimester placentas were collected from FGR-affected pregnancies (n = 29) and gestation matched with control pregnancies (n = 30). Functional analyses were then performed in vitro using term placental explants (n = 4) and BeWo trophoblast cells. mRNA expression was determined by real-time PCR, while protein expression was examined by immunoblotting and immunohistochemistry. siRNA transfection was used to silence IGF2R expression in placental explants and the BeWo cell-line. cDNA arrays were used to screen for downstream targets of IGF2R, specifically homeobox gene transcription factors and apoptosis-related genes. Functional effects of silencing IGF2R were then verified by ß-hCG ELISA, caspase activity assays and a real-time electrical cell-impedance assay for differentiation, apoptosis and cell growth potential, respectively. IGF2R expression was significantly decreased in placentas from pregnancies complicated by idiopathic FGR (P < 0.05 versus control). siRNA-mediated IGF2R knockdown in term placental explants and the trophoblast cell line BeWo resulted in altered expression of homeobox gene transcription factors, including increased expression of distal-less homeobox gene 5 (DLX5), and decreased expression of H2.0-Like Homeobox 1 (HLX) (P < 0.05 versus control). Knockdown of IGF2R transcription increased the expression and activity of caspase-6 and caspase-8 in placental explants, decreased BeWo proliferation and increased BeWo differentiation (all P < 0.05 compared to respective controls). This is the first study linking IGF2R placental expression with changes in the expression of homeobox genes that control cellular signalling pathways responsible for increased trophoblast cell apoptosis, which is a characteristic feature of FGR.

Placental creatine metabolism in cases of placental insufficiency and reduced fetal growth.

Creatine is a metabolite involved in cellular energy homeostasis. In this study, we examined placental creatine content, and expression of the enzymes required for creatine synthesis, transport and the creatine kinase reaction, in pregnancies complicated by low birthweight. We studied first trimester chorionic villus biopsies (CVBs) of small for gestational age (SGA) and appropriately grown infants (AGA), along with third trimester placental samples from fetal growth restricted (FGR) and healthy gestation-matched controls. Placental creatine and creatine precursor (guanidinoacetate-GAA) levels were measured. Maternal and cord serum from control and FGR pregnancies were also analyzed for creatine concentration. mRNA expression of the creatine transporter (SLC6A8); synthesizing enzymes arginine:glycine aminotransferase (GATM) and guanidinoacetate methyltransferase (GAMT); mitochondrial (mtCK) and cytosolic (BBCK) creatine kinases; and amino acid transporters (SLC7A1 & SLC7A2) was assessed in both CVBs and placental samples. Protein levels of AGAT (arginine:glycine aminotransferase), GAMT, mtCK and BBCK were also measured in placental samples. Key findings; total creatine content of the third trimester FGR placentae was 43% higher than controls. The increased creatine content of placental tissue was not reflected in maternal or fetal serum from FGR pregnancies. Tissue concentrations of GAA were lower in the third trimester FGR placentae compared to controls, with lower GATM and GAMT mRNA expression also observed. No differences in the mRNA expression of GATM, GAMT or SLC6A8 were observed between CVBs from SGA and AGA pregnancies. These results suggest placental creatine metabolism in FGR pregnancies is altered in late gestation. The relevance of these changes on placental bioenergetics should be the focus of future investigations.

Dysregulation of the placental renin-angiotensin system in human fetal growth restriction.

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.

Formyl peptide receptor-2 is decreased in foetal growth restriction and contributes to placental dysfunction.

STUDY QUESTION: What is the association between placental formyl peptide receptor 2 (FPR2) and trophoblast and endothelial functions in pregnancies affected by foetal growth restriction (FGR)? SUMMARY ANSWER: Reduced FPR2 placental expression in idiopathic FGR results in significantly altered trophoblast differentiation and endothelial function in vitro. WHAT IS KNOWN ALREADY: FGR is associated with placental insufficiency, where defective trophoblast and endothelial functions contribute to reduced feto-placental growth. STUDY DESIGN, SIZE, DURATION: The expression of FPR2 in placental tissues from human pregnancies complicated with FGR was compared to that in gestation-matched uncomplicated control pregnancies (n = 25 from each group). Fpr2 expression was also determined in placental tissues obtained from a murine model of FGR (n = 4). The functional role of FPR2 in primary trophoblasts and endothelial cells in vitro was assessed in diverse assays in a time-dependent manner. PARTICIPANTS/MATERIALS, SETTING, METHODS: Placentae from third-trimester pregnancies complicated by idiopathic FGR (n = 25) and those from gestation-matched pregnancies with appropriately grown infants as controls (n = 25) were collected at gestation 27-40 weeks. Placental tissues were also collected from a spontaneous CBA/CaH × DBA/2 J murine model of FGR. Placental FPR2/Fpr2 mRNA expression was determined by real-time PCR, while protein expression was examined by immunoblotting and immunohistochemistry. siRNA transfection was used to silence FPR2 expression in primary trophoblasts and in human umbilical vein endothelial cells (HUVEC), and the quantitation of cytokines, chemokines and apoptosis was performed following a cDNA array analyses. Functional effects of trophoblast differentiation were measured using HCGB/ß-hCG and syncytin-2 expression as well as markers of apoptosis, tumour protein 53 (TP53), caspase 8, B cell lymphoma 2 (BCL2) and BCL associated X (BAX). Endothelial function was assessed by proliferation, network formation and permeability assays. MAIN RESULTS AND THE ROLE OF CHANCE: Placental FPR2/Fpr2 expression was significantly decreased in FGR placentae (n = 25, P < 0.05) as well as in murine FGR placentae compared to controls (n = 4, P < 0.05). FPR2 siRNA (siFPR2) in term trophoblasts significantly increased differentiation markers, HCGB and syncytin-2; cytokines, interleukin (IL)-6, CXCL8; and apoptotic markers, TP53, caspase 8 and BAX, but significantly reduced the expression of the chemokines CXCL12 and its receptors CXCR4 and CXCR7; CXCL16 and its receptor, CXCR6; and cytokine, IL-10, compared with control siRNA (siCONT). Treatment of HUVECs with siFPR2 significantly reduced proliferation and endothelial tube formation, but significantly increased permeability of HUVECs. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Reduced expression of placental FPR2/Fpr2 was observed in the third trimester at delivery after development of FGR, suggesting that FPR2 is associated with FGR pregnancies. However, there is a possibility that the decreased placental FPR2 observed in FGR may be a consequence rather than a cause of FGR, although our in vitro functional analyses using primary trophoblasts and endothelial cells suggest that FPR2 may have a direct or indirect regulatory role on trophoblast differentiation and endothelial function in FGR. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study linking placental FPR2 expression with changes in the trophoblast and endothelial functions that may explain the placental insufficiency observed in FGR. STUDY FUNDING/COMPETING INTERESTS: P.M. and P.R.E. received funding from the Australian Institute of Musculoskeletal Science, Western Health, St. Albans, Victoria 3021, Australia. M.L. is supported by a Career Development Fellowship from the National Health and Medical Research Council (NHMRC; Grant no. 1047025). Monash Health is supported by the Victorian Government's Operational Infrastructure Support Programme. The authors declare that there is no conflict of interest in publishing this work.

Variable DAXX gene methylation is a common feature of placental trophoblast differentiation, preeclampsia, and response to hypoxia.

Placental functioning relies on the appropriate differentiation of progenitor villous cytotrophoblasts (CTBs) into extravillous cytotrophoblasts (EVCTs), including invasive EVCTs, and the multinucleated syncytiotrophoblast (ST) layer. This is accompanied by a general move away from a proliferative, immature phenotype. Genome-scale expression studies have provided valuable insight into genes that are associated with the shift to both an invasive EVCT and ST phenotype, whereas genome-scale DNA methylation analysis has shown that differentiation to ST involves widespread methylation shifts, which are counteracted by low oxygen. In the current study, we sought to identify DNA methylation variation that is associated with transition from CTB to ST in vitro and from a noninvasive to invasive EVCT phenotype after culture on Matrigel. Of the several hundred differentially methylated regions that were identified in each comparison, the majority showed a loss of methylation with differentiation. This included a large differentially methylated region (DMR) in the gene body of death domain-associated protein 6 (DAXX ), which lost methylation during both CTB syncytialization to ST and EVCT differentiation to invasive EVCT. Comparison to publicly available methylation array data identified the same DMR as among the most consistently differentially methylated genes in placental samples from preeclampsia pregnancies. Of interest, in vitro culture of CTB or ST in low oxygen increases methylation in the same region, which correlates with delayed differentiation. Analysis of combined epigenomics signatures confirmed DAXX DMR as a likely regulatory element, and direct gene expression analysis identified a positive association between methylation at this site and DAXX expression levels. The widespread dynamic nature of DAXX methylation in association with trophoblast differentiation and placenta-associated pathologies is consistent with an important role for this gene in proper placental development and function.-Novakovic, B., Evain-Brion, D., Murthi, P., Fournier, T., Saffery, R. Variable DAXX gene methylation is a common feature of placental trophoblast differentiation, preeclampsia, and response to hypoxia.

Expression of Biglycan in First Trimester Chorionic Villous Sampling Placental Samples and Altered Function in Telomerase-Immortalized Microvascular Endothelial Cells.

OBJECTIVE: Biglycan (BGN) has reduced expression in placentae from pregnancies complicated by fetal growth restriction (FGR). We used first trimester placental samples from pregnancies with later small for gestational age (SGA) infants as a surrogate for FGR. The functional consequences of reduced BGN and the downstream targets of BGN were determined. Furthermore, the expression of targets was validated in primary placental endothelial cells isolated from FGR or control pregnancies. APPROACH AND RESULTS: BGN expression was determined using real-time polymerase chain reaction in placental tissues collected during chorionic villous sampling performed at 10 to 12 weeks' gestation from pregnancies that had known clinical outcomes, including SGA. Short-interference RNA reduced BGN expression in telomerase-immortalized microvascular endothelial cells, and the effect on proliferation, angiogenesis, and thrombin generation was determined. An angiogenesis array identified downstream targets of BGN, and their expression in control and FGR primary placental endothelial cells was validated using real-time polymerase chain reaction. Reduced BGN expression was observed in SGA placental tissues. BGN reduction decreased network formation of telomerase-immortalized microvascular endothelial cells but did not affect thrombin generation or cellular proliferation. The array identified target genes, which were further validated: angiopoetin 4 (ANGPT4), platelet-derived growth factor receptor α (PDGFRA), tumor necrosis factor superfamily member 15 (TNFSF15), angiogenin (ANG), serpin family C member 1 (SERPIN1), angiopoietin 2 (ANGPT2), and CXC motif chemokine 12 (CXCL12) in telomerase-immortalized microvascular endothelial cells and primary placental endothelial cells obtained from control and FGR pregnancies. CONCLUSIONS: This study reports a temporal relationship between altered placental BGN expression and subsequent development of SGA. Reduction of BGN in vascular endothelial cells leads to disrupted network formation and alterations in the expression of genes involved in angiogenesis. Therefore, differential expression of these may contribute to aberrant angiogenesis in SGA pregnancies.

Expression of Homeobox Gene HLX and its Downstream Target Genes are Altered in Placentae From Discordant Twin Pregnancies.

A discordant twin gestation, in which one fetus is significantly growth restricted, compared to the other normal twin, is a unique model that can be used to elucidate the mechanism(s) by which the intrauterine environment affects fetal growth. In many model systems, placental transcription factor genes regulate fetal growth. Transcription factors regulate growth through their activation or repression of downstream target genes that mediate important cell functions. The objective of this study was to determine the expression of the placental HLX homeobox gene transcription factor and its downstream target genes in dizygotic twins with growth discordance. In this cross-sectional study, HLX and its downstream target genes' retinoblastoma 1 (RB1) and cyclin kinase D (CDKN1C) expression levels were determined in placentae obtained from dichorionic diamniotic twin pregnancies (n = 23) where one of the twins was growth restricted. Fetal growth restriction (FGR) was defined as small for gestational age with abnormal umbilical artery Doppler indices when compared with the normal control co-twin. Homeobox gene HLX expression was significantly decreased at both the mRNA and protein levels in FGR twin placentae compared with the normal control co-twin placentae (p < .05). Downstream target genes CDKN1C and RB1 were also significantly decreased and increased, respectively, at both the mRNA and protein levels in FGR twin placentae compared with normal control co-twin placentae (p < .05). Together, these observations suggest an important association between HLX transcription factor expression and abnormal human placental development in discordant twin pregnancies.

Low-Dose Acetylsalicylic Acid Treatment Modulates the Production of Cytokines and Improves Trophoblast Function in an in Vitro Model of Early-Onset Preeclampsia.

Preeclampsia (PE), a serious hypertensive disorder of pregnancy, remains a leading cause of perinatal morbidity and mortality worldwide. Perturbed trophoblast function and impaired placental development early in pregnancy are key features. Low-dose acetylsalicylic acid (LDA) administered before 16 weeks' gestation significantly reduces the risk for PE. However, the exact mechanisms of action of LDA, particularly on trophoblast function, are unclear. We hypothesized that LDA influences placental trophoblast function and reverses PE-associated abnormalities. This study aimed to determine the effects of serum from normotensive women and from those with PE with or without LDA treatment on a model of placental syncytium. On cytokine profiling, LDA increased placental growth factor production and selectively restored PE serum-induced alterations in levels of cytokines [activated leukocyte cell adhesion molecule, CXCL-16, and ErbB3] to those in normotensive serum-treated cells. PE serum-induced increases in the apoptotic markers P53 mRNA expression, IKBKE mRNA expression, caspase 3 activity, and decreased BIRC8 mRNA expression, were attenuated by LDA treatment. LDA treatment also reduced abnormal differentiation caused by PE serum administration. Possible mechanisms by which LDA influences PE-affected trophoblast cells in vitro are by modulating cytokine secretion, reducing apoptosis to levels seen in normotensive serum-treated cells, and preventing the premature trophoblast differentiation commonly observed in PE.

Maternal 25-hydroxyvitamin D is inversely correlated with foetal serotonin.

OBJECTIVE: Maternal vitamin D deficiency during pregnancy has been linked to impaired neurocognitive development in childhood. The mechanism by which vitamin D affects childhood neurocognition is unclear but may be via interactions with serotonin, a neurotransmitter involved in foetal brain development. In this study, we aimed to explore associations between maternal and foetal vitamin D concentrations, and foetal serotonin concentrations at term. STUDY DESIGN AND MEASUREMENTS: Serum 25-hydroxyvitamin D (25(OH)D, nmol/l) and serotonin (5-HT, nmol/l) concentrations were measured in maternal and umbilical cord blood from mother-infant pairs (n = 64). Association between maternal 25(OH)D, cord 25(OH)D and cord serotonin was explored using linear regression, before and after adjusting for maternal serotonin levels. We also assessed the effects of siRNA knockdown of the vitamin D receptor (VDR) and administration of 10 nm 1,25-dihydroxyvitamin D3 on serotonin secretion in human umbilical vein endothelial cells (HUVECs) in vitro. RESULTS: We observed an inverse relationship between both maternal and cord 25(OH)D concentrations with cord serotonin concentrations. The treatment of HUVECs with 1,25-dihydroxyvitamin D3 in vitro decreased the release of serotonin (193·9 ±14·8 nmol/l vs 458·9 ± 317·5 nmol/l, control, P < 0·05). Conversely, inactivation of VDR increased serotonin release in cultured HUVECs. CONCLUSIONS: These observations provide the first evidence of an inverse relationship between maternal 25(OH)D and foetal serotonin concentrations. We propose that maternal vitamin D deficiency increases foetal serotonin concentrations and thereby contributes to longer-term neurocognitive impairment in infants and children.

Homeobox gene TGIF-1 is increased in placental endothelial cells of human fetal growth restriction.

Aberrant placental angiogenesis is associated with fetal growth restriction (FGR). In mice, targeted disruption of the homeobox gene, transforming growth ß-induced factor (Tgif-1), which is also a transcription factor, causes defective placental vascularisation. Nevertheless, the role of TGIF-1 in human placental angiogenesis is unclear. We have previously reported increased TGIF-1 expression in human FGR placentae and demonstrated localisation of TGIF-1 protein in placental endothelial cells (ECs). However, its functional role remains to be investigated. In this study, we aimed to specifically compare TGIF-1 mRNA expression in placental ECs isolated from human FGR-affected pregnancies with gestation-matched control pregnancies in two independent cohorts from Australia and Canada and to identify the functional role of TGIF-1 in placental angiogenesis using the human umbilical vein endothelial cell-derived cell line, SGHEC-7, and primary human umbilical vein ECs. Real-time PCR revealed that TGIF-1 mRNA expression was significantly increased in ECs isolated from FGR-affected placentae compared with that of controls. The functional roles of TGIF-1 were determined in ECs after TGIF-1 siRNA transfection. TGIF-1 inactivation in ECs significantly reduced TGIF-1 at both the mRNA and protein levels, as well as the proliferative and invasive potential, but significantly increased the angiogenic potential. Using angiogenesis PCR screening arrays, we identified ITGAV, NRP-1, ANPGT-1 and ANPGT-2 as novel downstream targets of TGIF-1, after TGIF-1 inactivation in ECs. Collectively, these results show that TGIF-1 regulates EC function and the expression of angiogenic molecules; and when abnormally expressed, may contribute to the aberrant placental angiogenesis observed in FGR.

Placenta-derived angiogenic proteins and their contribution to the pathogenesis of preeclampsia.

Placental angiogenesis is critical to the success of human pregnancy. Angiogenesis is defined as the formation of new blood vessels from existing vasculature. Angiogenesis is necessary for the establishment of adequate placental perfusion, which is important for providing the optimum in utero environment to support fetal development. Defective placental angiogenesis is associated with several pregnancy complications, the most clinically important of which is preeclampsia; the multisystem disorder is characterized by maternal hypertension, proteinuria, and endothelial dysfunction. Here, we review our current understanding of several key angiogenic factors that are associated with placental angiogenesis. We also discuss their importance with respect to preeclampsia, where aberrant expression and release of these factors into the maternal circulation is thought to contribute to the pathogenesis and pathophysiology of preeclampsia.

An EG-VEGF-Dependent Decrease in Homeobox Gene NKX3.1 Contributes to Cytotrophoblast Dysfunction: A Possible Mechanism in Human Fetal Growth Restriction.

Idiopathic fetal growth restriction (FGR) is frequently associated with placental insufficiency. Previous reports have provided evidence that endocrine gland-derived vascular endothelial growth factor (EG-VEGF), a placental secreted protein, is expressed during the first trimester of pregnancy, controls both trophoblast proliferation and invasion, and its increased expression is associated with human FGR. In this study, we hypothesize that EG-VEGF-dependent changes in placental homeobox gene expressions contribute to trophoblast dysfunction in idiopathic FGR. The changes in EG-VEGF-dependent homeobox gene expressions were determined using a homeobox gene cDNA array on placental explants of 8-12 wks gestation after stimulation with EG-VEGF in vitro for 24 h. The homeobox gene array identified a greater-than-five-fold increase in HOXA9, HOXC8, HOXC10, HOXD1, HOXD8, HOXD9 and HOXD11, while NKX 3.1 showed a greater-than-two-fold decrease in mRNA expression compared with untreated controls. Homeobox gene NKX3.1 was selected as a candidate because it is a downstream target of EG-VEGF and its expression and functional roles are largely unknown in control and idiopathic FGR-affected placentae. Real-time PCR and immunoblotting showed a significant decrease in NKX3.1 mRNA and protein levels, respectively, in placentae from FGR compared with control pregnancies. Gene inactivation in vitro using short-interference RNA specific for NKX3.1 demonstrated an increase in BeWo cell differentiation and a decrease in HTR-8/SVneo proliferation. We conclude that the decreased expression of homeobox gene NKX3.1 downstream of EG-VEGF may contribute to the trophoblast dysfunction associated with idiopathic FGR pregnancies.

Human amniotic epithelial cells suppress relapse of corticosteroid-remitted experimental autoimmune disease.

BACKGROUND AIMS: Multiple sclerosis (MS) is considered to be a T-cell-mediated disease. Although MS remits with corticosteroid treatment, the disease relapses on discontinuation of therapy. Human amniotic epithelial cells (hAEC) from the placenta are readily accessible in large quantities and have anti-inflammatory properties. Previously we reported that hAEC given near disease onset ameliorated clinical signs and decreased myelin oligodendrocyte glycoprotein (MOG)-specific immune responses in MOG-induced experimental autoimmune encephalomyelitis (EAE), an experimental MS model. METHODS: To examine the therapeutic effect of hAEC in a clinically relevant setting, we first treated MOG peptide-induced EAE mice with a corticosteroid, prednisolone, in drinking water to induce remission. hAEC were then infused intravenously into the remitted mice. Anti-MOG antibodies in serum were detected by enzyme-linked immunoassay. Splenocyte proliferation was assessed by (3)H-thymidine incorporation. Immune cell subpopulations in spleens and lymph nodes and secreted cytokines in splenocyte culture were quantified by flow cytometry. Central nervous system histology was examined with the use of hematoxylin and eosin, Luxol fast blue and immunostaining. RESULTS: With cessation of prednisolone treatment, hAEC delayed EAE relapse for 7 days, and, after another 7 days, largely remitted disease in six of eight responder mice. Splenocyte proliferation was suppressed, anti-MOG35-55 antibodies in serum were decreased and interleukin-2 and interleukin-5 production by splenocytes were elevated after hAEC treatment. In the central nervous system, hAEC-treated mice had decreased demyelination and fewer macrophages in the inflammatory infiltrates. hAEC treatment also increased CD4(+)CD25(+)FoxP3(+) regulatory T cells in inguinal lymph nodes. CONCLUSIONS: These data demonstrate that the therapeutic effects of hAEC after corticosteroid treatment in an MS model probably are the consequence of peripheral immunoregulation. We suggest that hAEC may have potential as a cell therapy for remitted MS.