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Fetal growth restriction (FGR) describes a fetus which has not achieved its genetic growth potential; it is closely linked to placental dysfunction and uteroplacental hypoxia. Estrogen-related receptor gamma (ESRRG) is regulated by hypoxia and is highly expressed in the placenta. We hypothesized ESRRG is a regulator of hypoxia-mediated placental dysfunction in FGR pregnancies. Placentas were collected from women delivering appropriate for gestational age (AGA; n = 14) or FGR (n = 14) infants. Placental explants (n = 15) from uncomplicated pregnancies were cultured for up to 4 days in 21% or 1% O2, or with 200 μM cobalt chloride (CoCl2), or treated with the ESRRG agonists DY131 under different oxygen concentrations. RT-PCR, Western blotting, and immunochemistry were used to assess mRNA and protein levels of ESRRG and its localization in placental tissue from FGR or AGA pregnancies, and in cultured placental explants. ESRRG mRNA and protein expression were significantly reduced in FGR placentas, as was mRNA expression of the downstream targets of ESRRG, hydroxysteroid 11-beta dehydrogenase 2 (HSD11B2), and cytochrome P-450 (CYP19A1.1). Hypoxia-inducible factor 1-alpha protein localized to the nuclei of the cytotrophoblasts and stromal cells in the explants exposed to CoCl2 or 1% O2. Both hypoxia and CoCl2 treatment decreased ESRRG and its downstream genes’ mRNA expression, but not ESRRG protein expression. DY131 increased the expression of ESRRG signaling pathways and prevented abnormal cell turnover induced by hypoxia. These data show that placental ESRRG is hypoxia-sensitive and altered ESRRG-mediated signaling may contribute to hypoxia-induced placental dysfunction in FGR. Furthermore, DY131 could be used as a novel therapeutic approach for the treatment of placental dysfunction.

Here, we evaluated the feasibility of non-prodrug PEG-drug conjugates to decrease the accumulation of drugs within the placental tissues. The results showed that PEG was biocompatible with the human placenta with no alteration of the basal rate of proliferation or apoptosis in term placental explants. No significant changes in the released levels of lactate dehydrogenase and the human chorionic gonadotropin were observed after PEG treatment. The cellular uptake studies revealed that conjugating Cy5.5 and haloperidol to PEG significantly reduced (by up to ∼40-fold) their uptake by the placenta. These findings highlight the viability of novel non-prodrug polymer-drug conjugates to avoid the accumulation of drugs within the placenta.

Normal placental development and function is of key importance to fetal growth. Conversely aberrations of placental structure and function are evident in pregnancy complications including fetal growth restriction (FGR) and preeclampsia. Although trophoblast turnover and function is altered in these conditions, their underlying aetiologies and pathophysiology remains unclear, which hampers development of therapeutic interventions. Here we review evidence that supports a role for estrogen related receptor-gamma (ESRRG) in the development of placental dysfunction in FGR and preeclampsia. This relationship deserves particular consideration because ESRRG is highly expressed in normal placenta, is reduced in FGR and preeclampsia and its expression is altered by hypoxia, which is thought to result from deficient placentation seen in FGR and preeclampsia. Several studies have also found microRNA (miRNA) or other potential upstream regulators of ESRRG negatively influence trophoblast function which could contribute to placental dysfunction seen in FGR and preeclampsia. Interestingly, miRNAs regulate ESRRG expression in human trophoblast. Thus, if ESRRG is pivotally associated with the abnormal trophoblast turnover and function it may be targeted by microRNAs or other possible upstream regulators in the placenta. This review explores altered expression of ESRRG and upstream regulation of ESRRG-mediated pathways resulting in the trophoblast turnover, placental vascularisation, and placental metabolism underlying placental dysfunctions. This demonstrates that the ESRRG pathway merits further investigation as a potential therapeutic target in FGR and preeclampsia.

BACKGROUND Nanotechnology involves the engineering of structures on a molecular level. Nanomedicine and nano-delivery systems have been designed to deliver therapeutic agents to a target site or organ in a controlled manner, maximizing efficacy while minimizing off-target effects of the therapeutic agent administered. In both reproductive medicine and obstetrics, developing innovative therapeutics is often tempered by fears of damage to the gamete, embryo or developing foetus or of negatively impacting a woman’s reproductive potential. Thus, nanomedicine delivery systems may provide alternative targeted intervention strategies, treating the source of the disease and minimizing long-term consequences for the mother and/or her foetus. OBJECTIVE AND RATIONALE This review summarizes the current state of nanomedicine technology in reproductive medicine and obstetrics, including safety, potential applications, future directions and the hurdles for translation. SEARCH METHODS A comprehensive electronic literature search of PubMed and Web of Science databases was performed to identify studies published in English up until February 2020. Relevant keywords were used to obtain information regarding use of nanoparticle technology in fertility and gene therapy, early pregnancy complications (ectopic pregnancy and gestational trophoblastic disease) and obstetric complications (preeclampsia, foetal growth restriction, preterm birth and gestational diabetes) and for selective treatment of the mother or foetus. Safety of specific nanoparticles to the gamete, embryo and foetus was also investigated. OUTCOMES Pre-clinical research in the development of nanoparticle therapeutic delivery is being undertaken in many fields of reproductive medicine. Non-hormonal-targeted nanoparticle therapy for fibroids and endometriosis may provide fertility-sparing medical management. Delivery of interventions via nanotechnology provides opportunities for gene manipulation and delivery in mammalian gametes. Targeting cytotoxic treatments to early pregnancy tissue provides an alternative approach to manage ectopic pregnancies and gestational trophoblastic disease. In pregnancy, nanotherapeutic delivery offers options to stably deliver silencing RNA and microRNA inhibitors to the placenta to regulate gene expression, opening doors to novel genetic treatments for preeclampsia and foetal growth restriction. Restricting delivery of teratogenic drugs to the maternal compartment (such as warfarin) may reduce risks to the foetus. Alternatively, targeted delivery of drugs to the foetus (such as those to treat foetal arrythmias) may minimize side effects for the mother. WIDER IMPLICATIONS We expect that further development of targeted therapies using nanoparticles in a reproductive setting has promise to eventually allow safe and directed treatments for conditions impacting the health and reproductive capacity of women and for the management of pregnancy and serious pregnancy complications.

Placental dysfunction is the underlying cause of pregnancy complications such as fetal growth restriction (FGR) and pre-eclampsia. No therapies are available to treat a poorly functioning placenta, primarily due to the risks of adverse side effects in both the mother and the fetus resulting from systemic drug delivery. The use of targeted liposomes to selectively deliver payloads to the placenta has the potential to overcome these issues. In this study, we assessed the safety and efficacy of epidermal growth factor (EGF)-loaded, peptide-decorated liposomes to improve different aspects of placental function, using tissue from healthy control pregnancies at term, and pregnancies complicated by FGR. Phage screening identified a peptide sequence, CGPSARAPC (GPS), which selectively homed to mouse placentas in vivo, and bound to the outer syncytiotrophoblast layer of human placental explants ex vivo. GPS-decorated liposomes were prepared containing PBS or EGF (50–100 ng/mL), and placental explants were cultured with liposomes for up to 48 h. Undecorated and GPS-decorated liposomes containing PBS did not affect the basal rate of amino acid transport, human chorionic gonadotropin (hCG) release or cell turnover in placental explants from healthy controls. GPS-decorated liposomes containing EGF significantly increased amino acid transporter activity in healthy control explants, but not in placental explants from women with FGR. hCG secretion and cell turnover were unaffected by EGF delivery, however, differential activation of downstream protein kinases was observed when EGF was delivered via GPS-decorated vs. undecorated liposomes. These data indicate that targeted liposomes represent a safe and useful tool for the development of new therapies for placental dysfunction, recapitulating the effects of free EGF.

Bisphenol A (BPA) exposure during pregnancy is associated with low fetal weight, particularly in male fetuses. The expression of estrogen-related receptor gamma (ESRRG), a receptor for BPA in the human placenta, is reduced in fetal growth restriction. This study sought to explore whether ESRRG signaling mediates BPA-induced placental dysfunction and determine whether changes in the ESRRG signaling pathway are sex-specific. Placental villous explants from 18 normal term pregnancies were cultured with a range of BPA concentrations (1 nM–1 μM). Baseline BPA concentrations in the placental tissue used for explant culture ranged from 0.04 to 5.1 nM (average 2.3 ±1.9 nM; n = 6). Expression of ESRRG signaling pathway constituents and cell turnover were quantified. BPA (1 μM) increased ESRRG mRNA expression after 24 h in both sexes. ESRRG mRNA and protein expression was increased in female placentas treated with 1 μM BPA for 24 h but was decreased in male placentas treated with 1 nM or 1 μM for 48 h. Levels of 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1) and placenta specific-1 (PLAC1), genes downstream of ESRRG, were also affected. HSD17B1 mRNA expression was increased in female placentas by 1 μM BPA; however, 1 nM BPA reduced HSD17B1 and PLAC1 expression in male placentas at 48 h. BPA treatment did not affect rates of proliferation, apoptosis, or syncytiotrophoblast differentiation in cultured villous explants. This study has demonstrated that BPA affects the ESRRG signaling pathway in a sex-specific manner in human placentas and a possible biological mechanism to explain the differential effects of BPA exposure on male and female fetuses observed in epidemiological studies.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2018 there were nine themed workshops, five of which are summarised in this report. These workshops discussed new perspectives and knowledge in the following areas of research: 1) preeclampsia; 2) abnormally invasive placenta; 3) placental infection; 4) gestational trophoblastic disease; 4) drug delivery to treat placental dysfunction.

Our understanding of the activity of cationic antimicrobial peptides (AMPs) has focused on well-characterized natural sequences, or limited sets of synthetic peptides designed de novo. We have undertaken a comprehensive investigation of the underlying primary structural features that give rise to the development of activity in AMPs. We consider a complete set of all possible peptides, up to 7 residues long, composed of positively charged arginine (R) and / or hydrophobic tryptophan (W), two features most commonly associated with activity. We found the shortest active peptides were 4 or 5 residues in length, and the overall landscapes of activity against gram-positive and gram-negative bacteria and a yeast were positively correlated. For all three organisms we found a single activity peak corresponding to sequences with around 40% R; the presence of adjacent W duplets and triplets also conferred greater activity. The mechanistic basis of these activities comprises a combination of lipid binding, particularly to negatively charged membranes, and additionally peptide aggregation, a mode of action previously uninvestigated for such peptides. The maximum specific antimicrobial activity appeared to occur in peptides of around 10 residues, suggesting ‘diminishing returns’ for developing larger peptides, when activity is considered per residue of peptide., Clark et al. comprehensively explore the primary structural features underlying the activity of a complete set of antimicrobial peptides (AMPs). They find that the shortest active peptides were 4 or 5 residues in length, with activity being associated with 40% arginine, and multiple adjacent tryptophan residues. This study provides insights into the design of effective AMPs.

Microfluidic enables precise control over the continuous mixing of fluid phases at the micrometre scale, aiming to optimize the processing parameters and to facilitate scale-up feasibility. The optimization of parameters to obtain monodispersed drug-loaded liposomes however is challenging. In this work, two phosphatidylcholines (PC) differing in acyl chain length were selected, and used to control the release of the chemotherapeutic agent doxorubicin hydrochloride, an effective drug used to treat breast cancer. Microfluidics was used to rapidly screen manufacturing parameters and PC formulations to obtain monodispersed unilamellar liposomal formulations with a reproducible size (i.e. 200 nm). Cholesterol was included in all liposomal formulations; some formulations also contained DMPC(1,2-dimyristoyl-sn-glycero-3-phosphocholine) and/or DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine). Systematic variations in microfluidics total flow rate (TFR) settings were performed, while keeping a constant flow rate ratio (FRR). A total of six PC-based liposomes were fabricated using the optimal manufacturing parameters (TFR 500 μL/min, FRR 0.1) for the production of reproducible, stable liposome formulations with a narrow size distribution. Liposomes actively encapsulating doxorubicin exhibited high encapsulation efficiencies (80%) for most of the six formulations, and sustained drug release profiles in vitro over 48 h. Drug release profiles varied as a function of the DMPC/DSPC mol content in the lipid bilayer, with DMPC-based liposomes exhibiting a sustained release of doxorubicin when compared to DSPC liposomes. The PC-based liposomes, with a slower release of doxorubicin, were tested in vitro, as to investigate their cytotoxic activity against three human breast cancer cell lines: the non-metastatic ER+/PR + MCF7 cells, the triple-negative aggressive MDA-MB 231 cells, and the metastatic HER2-overexpressing/PR + BT474 cells. Similar cytotoxicity levels to that of free doxorubicin were reported for DMPC

Revaluation of the association of the STOX1 (STORKHEAD_BOX1 PROTEIN 1) transcription factor mutation (Y153H, C allele) with the early utero-vascular origins of placental pathology is warranted. To investigate if placental STOX1 Y153H genotype affects utero-vascular remodeling—compromised in both preterm birth and preeclampsia—we utilized extravillous trophoblast (EVT) explant and placental decidual coculture models, transfection of STOX1 wild-type and mutant plasmids into EVT-like trophoblast cell lines, and a cohort of 75 placentas from obstetric pathologies. Primary EVT and HTR8/SVneo cells carrying STOX1 Y153H secreted lower levels of IL (interleukin) 6, and IL-8, and higher CXCL16 (chemokine [C-X-C motif] ligand 16) and TRAIL (tumor necrosis factor–related apoptosis-inducing ligand) than wild-type EVT and Swan71 cells. Media from wild-type EVT or Swan71 cells transfected with wild-type STOX1 stimulated: endothelial chemokine expression, angiogenesis, and decidual natural killer cell and monocyte migration. In contrast, Y153H EVT conditioned medium, Swan71 transfected with the Y153H plasmid, or HTR8/SVneo media had no effect. Genotyping of placental decidual cocultures demonstrated association of the placental STOX1 CC allele with failed vascular remodeling. Decidual GG NODAL R165H increased in failed cocultures carrying the placental CC alleles of STOX1. Multivariate analysis of the placental cohort showed that the STOX1 C allele correlated with premature birth, with or without severe early-onset preeclampsia, and small for gestational age babies. In conclusion, placental STOX1 Y153H is a precipitating factor in preterm birth and placental preeclampsia due to defects in early utero-placental development.

Developing more efficient manufacturing methods for nano therapeutic systems is becoming important, not only to better control their physico-chemical characteristics and therapeutic efficacy but also to ensure scale-up is cost-effective. The principle of cross-flow chemistry allows precise control over manufacturing parameters for the fabrication of uniform liposomal formulations, as well as providing reproducible manufacturing scale-up compared to conventional methods. We have herein investigated the use of microfluidics to produce PEGylated DSPC liposomes loaded with doxorubicin and compared their performance against identical formulations prepared by the thin-film method. The isoprenylated coumarin umbelliprenin was selected as a co-therapeutic. Umbelliprenin-loaded and doxorubicin:umbelliprenin co-loaded liposomes were fabricated using the optimised microfluidic set-up. The role of umbelliprenin as lipid bilayer fluidity modulation was characterized, and we investigated its role on liposomes size, size distribution, shape and stability compared to doxorubicin-loaded liposomes. Finally, the toxicity of all liposomal formulations was tested on a panel of human breast cancer cells (MCF-7, MDA-MB 231, BT-474) to identify the most potent formulation by liposomal fabrication method and loaded compound(s). We herein show that the microfluidic system is an alternative method to produce doxorubicin:umbelliprenin co-loaded liposomes, allowing fine control over liposome size (100–250 nm), shape, uniformity and doxorubicin drug loading (>80%). Umbelliprenin was shown to confer fluidity to model lipid biomembranes, which helps to explain the more homogeneous size and shape of co-loaded liposomes compared to liposomes without umbelliprenin. The toxicity of doxorubicin:umbelliprenin co-loaded liposomes was lower than that of free doxorubicin, due to the delayed release of doxorubicin from liposomes. An alternative, rapid and easy manufacturing method for the production of liposomes has been established using microfluidics to effectively produce uniform doxorubicin:umbelliprenin co-loaded liposomal formulations with proven cytotoxicity in human breast cancer cell lines in vitro.

Preterm deliveries remain the leading cause of neonatal morbidity and mortality. Current therapies target only myometrial contractions and are largely ineffective. As labor involves multiple coordinated events across maternal and fetal tissues, identifying fundamental regulatory pathways of normal term labor is vital to understanding successful parturition and consequently labor pathologies. We aimed to identify transcriptomic signatures of human normal term labor of two tissues: in the fetal-facing choriodecidua and the maternal myometrium. Microarray transcriptomic data from choriodecidua and myometrium following term labor were analyzed for functional hierarchical networks, using Cytoscape 2.8.3. Hierarchically high candidates were analyzed for their regulatory casual relationships using Ingenuity Pathway Analysis. Selected master regulators were then chemically inhibited and effects on downstream targets were assessed using real-time quantitative PCR (RT-qPCR). Unbiased network analysis identified upstream molecular components in choriodecidua including vimentin, TLR4, and TNFSF13B. In the myometrium, candidates included metallothionein 2 (MT2A), TLR2, and RELB. These master regulators had significant differential gene expression during labor, hierarchically high centrality in community cluster networks, interactions amongst the labor gene set, and strong causal relationships with multiple downstream effects. In vitro experiments highlighted MT2A as an effective regulator of labor-associated genes. We have identified unique potential regulators of the term labor transcriptome in uterine tissues using a robust sequence of unbiased mathematical and literature-based in silico analyses. These findings encourage further investigation into the efficacy of predicted master regulators in blocking multiple pathways of labor processes across maternal and fetal tissues, and their potential as therapeutic approaches.

The methylated analogue of the polyphenol resveratrol (RV), 2,3′,4,5′-tetramethoxystilbene (TMS) displays potent antioxidant properties and is an effective cytochrome P450 (CYP) 1B1 inhibitor. The bioavailability of TMS is low. Therefore, the use of liposomes for the encapsulation of TMS is a promising delivery modality for enhanced uptake into tissues. We examined the effect of delivery of TMS in liposomes on the restoration of vasodilator responses of isolated aortic vessels after acute tension elevation ex vivo. Aortic vessels from young male Wistar rats were isolated, and endothelial-dependent (acetylcholine, ACh) and -independent (sodium nitroprusside, SNP) responses assessed. Acute tension elevation (1 h) significantly reduced ACh dilator responses, which were restored following incubation with superoxide dismutase or apocynin (an NADPH oxidase inhibitor). Incubation with TMS-loaded liposomes (mean diameter 157 ± 6 nm; PDI 0.097) significantly improved the attenuated dilator responses following tension elevation, which was sustained over a longer period (4 h) when compared to TMS solution. Endothelial denudation or co-incubation with L-NNA (Nω-nitro-l-arginine; nitric oxide synthase inhibitor) resulted in loss of dilator function. Our findings suggest that TMS-loaded liposomes can restore attenuated endothelial-dependent dilator responses induced by an oxidative environment by reducing NADPH-oxidase-derived ROS and potentiating the release of the vasodilator nitric oxide. TMS-loaded liposomes may be a promising therapeutic strategy to restore vasodilator function in vascular disease.

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 β-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

A complex combination of trafficking and signalling occurs at the surface of the placenta. The system delivers maternal nutrients to the fetus and facilitates gaseous exchange, whilst mediating signal transduction to support and stimulate the growth of the placenta itself. IGF-I is acknowledged as a maternally-derived ligand important in the regulation of placental growth. Here we show that quantum dots bearing IGF can stimulate IGF receptor (IGF1R) phosphorylation in the syncytio- (maternal-facing) and cyto- (fetal-facing) trophoblast bilayer that forms the outer boundary of the placenta, in a distribution similar to the one resulting from exposure to a soluble ligand. The conjugates are internalised by a clathrin-dependent pathway and delivered to a syncytioplasmic compartment that differs from conventional late endosomes and lysosomes. Two discrete downstream responses are evident in different cellular compartments: phosphorylation of P70S6K in the non-proliferative syncytiotrophoblast and of AKT in the cytotrophoblast. Co-conjugation of IGF-quantum dots with an RGD-containing ligand permits penetration beyond the syncytium, into the cytoplasm of the underlying cytotrophoblast. These data reveal the existence of a trans-syncytial pathway that allows maternal mitotic signals to penetrate to the inner progenitor cells, which must proliferate to support placental and consequently fetal growth.

Research on the placenta as the interface between the mother and the fetus has been undertaken for some 150 years, and in 2 subsequent reviews, we attempted to summarize the situation. In the first part, we described the discovery of unique physiological modifications of the uteroplacental spiral arteries, enabling them to cope with a major increase in blood flow necessary to ensure proper growth of the fetus. These consist of an invasion of the arterial walls by trophoblast and a progressive disappearance of its normal structure. Researchers then turned to the pathophysiology of the placental bed and in particular to its maternal vascular tree. This yielded vital information for a better understanding of the so-called great obstetrical syndromes (preeclampsia, fetal growth restriction, premature labor and delivery, placenta accreta). Systematic morphological investigations of the uteroplacental vasculature showed that preeclampsia is associated with decreased or failed transformation of spiral arteries and the persistence of endothelial and smooth muscle cells in segments of their myometrial portion. Here we report on recent functional investigations of the placental bed, including in situ biophysical studies of uteroplacental blood flow and vascular resistance, and manipulation of uteroplacental perfusion. These new methodologies have provided a novel way of identifying pregnancies in which remodeling is impaired. In animals it is now possible to manipulate uteroplacental blood flow, leading to an enhancement of fetal growth; this opens the way to trials in abnormal human pregnancies. In this second part, we explored a new, extremely important area of research that deals with the role of specific subsets of leukocytes and macrophages in the placental bed. The human first-trimester decidua is rich in leukocytes called uterine natural killer cells. Both macrophages and uterine natural killer cells increase in number from the secretory endometrium to early pregnancy and play a critical role in mediating the process of spiral artery transformation by inducing initial structural changes. It seems therefore that vascular remodeling of spiral arteries is initiated independently of trophoblast invasion. Dysregulation of the immune system may lead to reproductive failure or pregnancy complications, and in this respect, recent studies have advanced our understanding of the mechanisms regulating immunological tolerance during pregnancy, with several mechanisms being proposed for the development of tolerance to the semiallogeneic fetus. In particular, these include several strategies by which the trophoblast avoids maternal recognition. Finally, an important new dimension is being explored: the likelihood that pregnancy syndromes and impaired uteroplacental vascular remodeling may be linked to future maternal and even the child's cardiovascular disease risk. The functional evidence underlying these observations will be discussed.

Workshops are an important part of the IFPA annual meeting, as they allow for discussion of specialized topics. At the IFPA meeting 2015 there were twelve themed workshops, three of which are summarized in this report. These workshops were related to various aspects of placental biology but collectively covered areas of pregnancy pathologies and placental metabolism: 1) nanomedicine applications and exosome biology; 2) xenobiotics and endocrine disruptors and pregnancy; 3) lipid mediators and placental function. Fil: Albrecht, Claudia. University of Berne; Suiza. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Caniggia, I.. Lunenfeld-Tanenbaum Research Institute; Canadá Fil: Clifton, V.. Matter Hospital; Australia Fil: Göhner, C.. University of Groningen; Países Bajos Fil: Harris, L.. University of Manchester; Reino Unido Fil: Hemmings, D.. University of Alberta; Canadá Fil: Jawerbaum, Alicia Sandra. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Johnstone, E.. University of Manchester; Reino Unido Fil: Jones, H.. Cincinnati Children's Hospital Medical Center; Estados Unidos Fil: Keelan, J.. University of Western Australia; Australia Fil: Lewis, R.. University of Southampton; Reino Unido Fil: Mitchell, M.. University of Queensland; Australia Fil: Murthi, P.. Monash University; Australia Fil: Powell, T.. University of Colorado Anschutz Medical Campus; Estados Unidos Fil: Saffery, R.. The University of Melbourne; Australia Fil: Smith, R.. University of Newcastle; Reino Unido Fil: Vaillancourt, C.. Université du Québec a Montreal; Canadá Fil: Wadsack, C.. University of Graz; Austria Fil: Salomon, C.. University of Queensland; Australia

Homeobox genes regulate embryonic and placental development, and are widely expressed in the human placenta, but their regulatory control by DNA methylation is unclear. DNA methylation analysis was performed on human placentae from first, second and third trimesters to determine methylation patterns of homeobox gene promoters across gestation. Most homeobox genes were hypo-methylated throughout gestation, suggesting that DNA methylation is not the primary mechanism involved in regulating HOX genes expression in the placenta. Nevertheless, several genes showed variable methylation patterns across gestation, with a general trend towards an increase in methylation over gestation. Three genes (TLX1, HOXA10 and DLX5) showed inverse gains of methylation with decreasing mRNA expression throughout pregnancy, supporting a role for DNA methylation in their regulation. Proteins encoded by these genes were primarily localised to the syncytiotrophoblast layer, and showed decreased expression later in gestation. siRNA mediated downregulation of DLX5, TLX1 and HOXA10 in primary term villous cytotrophoblast resulted in decreased proliferation and increased expression of differentiation markers, including ERVW-1. Our data suggest that loss of DLX5, TLX1 and HOXA10 expression in late gestation is required for proper placental differentiation and function.

p32 is a conserved eukaryotic protein which is primarily expressed in the mitochondria and regulates cell proliferation, migration and metabolism in various tissues. In this study, we sought to examine the expression and function of p32 in the human placenta. p32 was highly expressed in the syncytiotrophoblast, the underlying cytotrophoblast (CTB), the vascular endothelium and by a proportion of cells in the villous stroma in first trimester and term placenta. p32 mRNA and protein expression was significantly higher in the first trimester of pregnancy than at term, and expression in the trophoblast was significantly reduced in placentas from women with fetal growth restriction (FGR). Small interfering RNA (siRNA)-mediated knockdown of p32 in term placental explants significantly reduced the number of Ki67-positive CTB, but did not alter CTB apoptosis or necrosis. p32 knockdown increased lactate production, reduced glucose extraction from culture medium and was associated with reduced MitoTracker dye accumulation in trophoblast mitochondria. p32 knockdown was also associated with a significant reduction in expression of the mitochondrial respiratory complexes I and IV. These data suggest that p32 expression is important for CTB proliferation, via a mechanism involving regulation of normal mitochondrial function. As p32 expression is reduced in FGR placentas, this may contribute to some of the observed placental pathology, such as reduced CTB proliferation and mitochondrial dysfunction.

Suboptimal placental growth and development are the underlying cause of many pregnancy complications. No treatments are available, primarily due to the risk of causing fetal teratogenicity. microRNAs (miRNAs) are short, non-coding RNA sequences that regulate multiple downstream genes; miR-145 and miR675 have previously been identified as negative regulators of placental growth. In this proof of principle study, we explored the feasibility of delivering miRNA inhibitors to the placentas of pregnant mice and developed novel placental homing peptide-microRNA inhibitor conjugates for targeted enhancement of intrinsic placental growth signalling. Scrambled-, miR-145- or miR-675 inhibitor sequences were synthesised from peptide nucleic acids and conjugated to the placental homing peptide CCGKRK. Intravenous administration of the miR-145- and miR-675 conjugates to pregnant C57BL/6J mice significantly increased fetal and placental weights compared to controls; the miR-675 conjugate significantly reduced placental miR-675 expression. When applied to human first trimester placental explants, the miR-145 conjugate significantly reduced placental miR-145 expression, and both conjugates induced significant enhancement of cytotrophoblast proliferation; no effect was observed in term placental explants. This study demonstrates that homing peptide-miRNA inhibitor conjugates can be exploited to promote placental growth; these novel therapeutics may represent an innovative strategy for targeted treatment of compromised placental development.

Decidual spiral arteriole (SpA) remodeling is essential to ensure optimal uteroplacental blood flow during human pregnancy, yet very little is known about the regulatory mechanisms. Uterine decidual NK (dNK) cells and macrophages infiltrate the SpAs and are proposed to initiate remodeling before colonization by extravillous trophoblasts (EVTs); however, the trigger for their infiltration is unknown. Using human first trimester placenta, decidua, primary dNK cells, and macrophages, we tested the hypothesis that EVTs activate SpA endothelial cells to secrete chemokines that have the potential to recruit maternal immune cells into SpAs. Gene array, real-time PCR, and ELISA analyses showed that treatment of endothelial cells with EVT conditioned medium significantly increased production of two chemokines, CCL14 and CXCL6. CCL14 induced chemotaxis of both dNK cells and decidual macrophages, whereas CXCL6 also induced dNK cell migration. Analysis of the decidua basalis from early pregnancy demonstrated expression of CCL14 and CXCL6 by endothelial cells in remodeling SpAs, and their cognate receptors are present in both dNK cells and macrophages. Neutralization studies identified IL-6 and CXCL8 as factors secreted by EVTs that induce endothelial cell CCL14 and CXCL6 expression. This study has identified intricate crosstalk between EVTs, SpA cells, and decidual immune cells that governs their recruitment to SpAs in the early stages of remodeling and has identified potential key candidate factors involved. This provides a new understanding of the interactions between maternal and fetal cells during early placentation and highlights novel avenues for research to understand defective SpA remodeling and consequent pregnancy pathology.

Tumor-homing peptides have been exploited to create nanocarriers for targeted delivery of therapeutic agents to the placenta., The availability of therapeutics to treat pregnancy complications is severely lacking mainly because of the risk of causing harm to the fetus. As enhancement of placental growth and function can alleviate maternal symptoms and improve fetal growth in animal models, we have developed a method for targeted delivery of payloads to the placenta. We show that the tumor-homing peptide sequences CGKRK and iRGD bind selectively to the placental surface of humans and mice and do not interfere with normal development. Peptide-coated nanoparticles intravenously injected into pregnant mice accumulated within the mouse placenta, whereas control nanoparticles exhibited reduced binding and/or fetal transfer. We used targeted liposomes to efficiently deliver cargoes of carboxyfluorescein and insulin-like growth factor 2 to the mouse placenta; the latter significantly increased mean placental weight when administered to healthy animals and significantly improved fetal weight distribution in a well-characterized model of fetal growth restriction. These data provide proof of principle for targeted delivery of drugs to the placenta and provide a novel platform for the development of placenta-specific therapeutics.

Insulin-like growth factor-II (IGF-II) affects many aspects of cellular function through its ability to activate several different receptors and, consequently, numerous intracellular signalling molecules. Thus, IGF-II is a key regulator of normal foetal development and growth. However, abnormalities in IGF-II function are associated with cardiovascular disease and cancer. Here, we review the cellular mechanisms by which IGF-II's physiological and pathophysiological actions are exerted by discussing the involvement of the type 1 and type 2 IGF receptors (IGF1R and IGF2R), the insulin receptor and the downstream MAP kinase, PI-3 kinase and G-protein-coupled signalling pathways in mediating IGF-II stimulated cellular proliferation, survival, differentiation and migration. © 2011 Informa UK, Ltd.

Insulin-like growth factor 2 (IGF2) enhances proliferation and survival of human first-trimester cytotrophoblasts (CTB) by signaling through the insulin-like growth factor 1 receptor (IGF1R). However, the role of the IGF2 receptor (IGF2R) in regulating trophoblast kinetics is unclear: It could act as a clearance receptor for trafficking excess ligand to lysosomes for degradation and/or directly mediate IGF2 signaling. We used an IGF2R knockdown strategy in BeWo cells and placental villous explants to investigate trophoblast proliferation and survival in response to stimulation by IGF. Both IGF1 and IGF2 significantly (P < 0.001) increased mitosis and reduced apoptosis in serum-starved BeWo cells. Small interfering RNA (siRNA)-mediated knockdown of IGF2R further enhanced IGF2-stimulated mitosis (P < 0.01), and IGF2-mediated rescue of apoptosis (P < 0.001) in these cells. Leu27IGF2, an IGF2 analogue that binds to IGF2R but not IGF1R, also protected IGF2R-expressing BeWo cells from apoptosis but did not increase mitosis. IGF treatment of term placental villous explants with reduced syncytial expression of IGF2R increased CTB proliferation (P < 0.001) and decreased apoptosis (P < 0.01) compared to untreated controls. Moreover, IGF2-mediated rescue of CTB apoptosis was significantly greater than that in tissue with normal IGF2R expression. Leu27IGF2 promoted mitogenesis and survival only in explants with intact IGF2R expression. Given that altered CTB turnover is observed in pregnancies complicated by fetal growth restriction, the development of strategies to manipulate the IGF2R signaling axis in the syncytiotrophoblast may provide a therapeutic avenue for treating this condition.

During human pregnancy, the uterine spiral arteries are progressively remodelled to form dilated conduits lacking maternal vasomotor control. This phenomenon ensures that a constant supply of blood is delivered to the materno-fetal interface at an optimal velocity for nutrient exchange. Conversion of a tonic maternal arteriole composed of multiple layers of vascular smooth muscle, elastin and numerous other extracellular matrix components, into a highly dilated yet durable vessel, requires tight regulatory control and the coordinated actions of multiple cell types. Initial disruption of the vascular wall, characterised by foci of endothelial cell loss, and separation and misalignment of vascular smooth muscle cells (VSMC), is coincident with an influx of uterine natural killer (uNK) cells and macrophages. uNK cells are a source of angiogenic growth factors and matrix degrading proteases, thus they possess the capacity to initiate changes in VSMC phenotype and instigate extracellular matrix catabolism. However, complete vascular cell loss, mediated in part by apoptosis and dedifferentiation, is only achieved following colonisation of the arteries by extravillous trophoblast (EVT). EVT produce a variety of chemokines, cytokines and matrix degrading proteases, enabling them to influence the fate of other cells within the placental bed and complete the remodelling process. The complex interplay of cell-cell and cell-matrix interactions required for effective vascular transformation will be examined, with a particular focus on the role of (i) uNK cells and (ii) the enzyme matrix metalloproteinase-12 (MMP-12). Parallels with remodelling events occurring in other vascular beds will also be drawn.

Problem During pregnancy, the decidual spiral arterioles (SpAs) that supply maternal blood to the placenta undergo a series of changes to optimise the transfer of nutrients and oxygen to the developing foetus. Recent studies have shown that initiation of SpA transformation coincides with decidual leucocyte infiltration. Leucocytes are known to be a source of matrix metalloproteinases (MMPs); however, the complete profile of MMPs expressed by decidual NK cells (dNK) and macrophages has not been characterised. We hypothesised that leucocyte-derived MMPs contribute to SpA remodelling. Methods Decidual NK cells and macrophages were isolated from first trimester decidua and their MMP repertoire profiled by qRT-PCR (n = 10; 5-11 weeks). Dual immunofluorescence was used to localise MMP expression in situ (n = 3; 5-12 weeks). Gelatin zymography was carried out to assess whether leucocyte-derived MMPs can degrade ECM. In situ zymography and immunofluorescence identified MMP activity in tissue-resident dNK and macrophages. Results Decidual NK cells cells and macrophages expressed MMP2, -7, -9, -11, -16, -19 and tissue inhibitors of metalloproteinase-1, -2, and -3. Both cell types degraded gelatin using MMP2 and MMP9 and broke down collagen in an in vitro model of the SpA. Extravillous trophoblasts (EVTs) expressed a similar repertoire of MMPs. Conclusion We suggest that matrix remodelling in SpA is initiated by infiltrating leucocytes, while EVTs become involved at later stages.

During the first twenty weeks of human pregnancy, extravillous trophoblasts (EVT) colonise the decidua and remodel the uterine spiral arteries as far as the first third of the myometrium. This process leads to an irreversible vasodilatation, ensuring that maximal blood flow is delivered to the materno-fetal interface at an optimal velocity for nutrient exchange. There is accumulating evidence that subtle changes in vascular structure precede EVT colonisation; however, full physiological transformation is only achieved in the presence of trophoblast. This review discusses the mechanisms employed to facilitate arterial dilatation, including recent data regarding the contribution of vascular cell apoptosis, the importance of elastin catabolism and the source of candidate elastases. It also examines how the complex interplay between EVT, endothelial cells, smooth muscle cells and decidual leukocytes (macrophages and uterine natural killer cells) leads to enhanced receptivity to invasion, vascular cell loss and extracellular matrix remodelling.

A recent study of human placental villi [Mori et al., The cytotrophoblast layer of human chorionic villi becomes thinner but maintains its structural integrity during gestation, Biol Reprod 76 (2007) 164-172] concluded that cytotrophoblast (CT) cells occupy 80% of the basal lamina (BL) surface at term and that syncytiotrophoblast (ST) does not make direct contact with the BL. Based on SPINT-1 localisation using immunofluorescence on cryosections, these conclusions run counter to previous light and electron microscopic data suggesting that term CT cells cover no more than about 24% of the BL surface. To resolve these discrepancies, we have undertaken a stereological study of term placenta using transmission electron microscopy (TEM) and a novel immunofluorescence approach. Test line lattices were randomly superimposed on TEM images of villous trophoblast from 13 normal term placentae. Intersections with the test lines were counted to assess the fractional surface of BL occupied by CT cells. After trypsin-mediated removal of syncytium, cells in whole-mounted term and first trimester villi were stained with cytokeratin 7 to identify CT and then visualised by confocal microscopy. CT formed an almost continuous layer in the first trimester. In contrast, term CT cells and their processes were found to cover only 44% (SD 14%) of the BL surface with intervening regions occupied by ST. TEM and confocal images were consistent with the concept of a network of 'octopoid' CT cells with fine processes extending from a central cell body. Our estimates of CT coverage are lower than the recent immunofluorescence estimate but greater than earlier TEM estimates. The former may have been biased by overprojection (section thickness) effects whilst the latter may be underestimates due to failure to include the fine CT cell processes. We conclude that CT cells transform from a cuboidal phenotype early in gestation to flattened cells with multiple interconnecting processes. The CT layer thins but maintains a functional network within which cells intercommunicate without compromising substance transfer via the syncytium.

During pregnancy, trophoblasts invade and transform the maternal spiral arteries that supply blood to the placenta. Recent work has revealed that this process occurs in several stages, and details of the molecular and cellular mechanisms are beginning to emerge, including changes that precede or accompany trophoblastic colonization of the vascular media. Disruption and eventual loss of smooth muscle cells and their associated extracellular matrix are central to physiological transformation. Advances in understanding will lead to the identification of the causative factors involved in failure of remodeling in pathological pregnancies and suggest novel diagnostic and therapeutic avenues.

Remodeling of the uterine spiral arteries during pregnancy transforms them from high to low resistance vessels that lack vasoconstrictive properties. This process is essential to meet the demand for increased blood flow imposed by the growing fetus. Loss of endothelial and smooth muscle cells (SMC) is evident in remodeled arteries but the mechanisms underlying this transformation remain unknown. This study investigated the hypothesis that fetal trophoblast invading from the placenta instigate remodeling by triggering cell death in vascular SMC. Specifically, a role for trophoblast-derived death inducing cytokine tumor necrosis factor-α–related apoptosis-inducing ligand (TRAIL) was investigated. Expression of the activating TRAIL receptors R1 and R2 was detected by flow cytometry on human aortic SMC and by immunohistochemistry on spiral artery SMC. Recombinant human TRAIL induced human aortic SMC apoptosis, which was inhibited by antibodies against TRAIL-R1 or -R2. Perfusion of denuded spiral artery segments with recombinant human TRAIL also induced SMC apoptosis. Trophoblasts isolated from first trimester placenta expressed membrane-associated TRAIL and induced apoptosis of human aortic SMC; apoptosis was significantly inhibited by a recombinant human TRAIL-R1:Fc construct. Trophoblast within the first trimester placental bed also expressed TRAIL. These data show that: 1) TRAIL causes SMC death; 2) trophoblast produce the apoptotic cytokine TRAIL; and 3) trophoblast induce SMC apoptosis via a TRAIL-dependent mechanism. We conclude that TRAIL produced by trophoblast causes apoptosis of SMC and thus may contribute to SMC loss during spiral artery remodeling in pregnancy.

Uterine spiral arteriole (SA) remodeling in early pregnancy involves a coordinated series of events including decidual immune cell recruitment, vascular cell disruption and loss, and colonization by placental-derived extravillous trophoblast (EVT). During this process, decidual SA are converted from narrow, muscular vessels into dilated channels lacking vasomotor control. We hypothesized that this extensive alteration in SA architecture must require significant reorganization and/or breakdown of the vascular extracellular matrix (ECM). First trimester decidua basalis (30 specimens) was immunostained to identify spiral arterioles undergoing trophoblast-independent and -dependent phases of remodeling. Serial sections were then immunostained for a panel of ECM markers, to examine changes in vascular ECM during the remodeling process. The initial stages of SA remodeling were characterized by loss of laminin, elastin, fibrillin, collagen types III, IV and VI from the basement membrane, vascular media and/or adventitia, and surrounding decidual stromal cells. Loss of ECM correlated with disruption and disorganization of vascular smooth muscle cells, and the majority of changes occurred prior to extensive colonization of the vessel wall by EVT. The final stages of SA remodeling, characterized by the arrival of EVT, were associated with the increased mural deposition of fibronectin and fibrinoid. This study provides the first detailed analysis of the spatial and temporal loss of ECM from the walls of remodeling decidual SA in early pregnancy.

Elastin breakdown in the walls of uterine spiral arteries during early pregnancy facilitates their transformation into dilated, high-flow, low-resistance channels. Elastin-derived peptides (EDP) can influence cell migration, invasion and protease activity, and so we hypothesized that EDP released during elastolysis promote extravillous trophoblast (EVT) invasion and further elastin breakdown. Treatment of the trophoblast cell line SGHPL4 with the elastin-derived matrikine VGVAPG (1 μg/ml) significantly increased total elastase activity, promoted migration in a wound healing assay and increased invasion through Matrigel-coated transwells compared with vehicle control (0.1% DMSO) or the scrambled sequence VVGPGA. Furthermore, treatment of first-trimester placental villous explants with this EDP significantly increased both the area of trophoblast outgrowth and distance of migration away from the villous tips. Primary first-trimester cytotrophoblast exposed to VGVAPG (1 μg/ml) for 30 min showed increased phosphorylation of endothelial nitric oxide synthase and activation of the mitogen activated protein kinase pathway, events also associated with tumour cell migration and invasion. These in vitro observations suggest liberation of bioactive EDP during induction of elastolysis in the uterine spiral arteries may orchestrate a positive feedback loop that promotes EVT invasion and further elastin breakdown, contributing to the process of vascular remodelling.