Extracellular vesicle-associated miRNAs are an adaptive response to gestational diabetes mellitus.

BACKGROUND: Gestational diabetes mellitus (GDM) is a serious public health issue affecting 9-15% of all pregnancies worldwide. Recently, it has been suggested that extracellular vesicles (EVs) play a role throughout gestation, including mediating a placental response to hyperglycaemia. Here, we investigated the EV-associated miRNA profile across gestation in GDM, assessed their utility in developing accurate, multivariate classification models, and determined the signaling pathways in skeletal muscle proteome associated with the changes in the EV miRNA profile. METHODS: Discovery: A retrospective, case-control study design was used to identify EV-associated miRNAs that vary across pregnancy and clinical status (i.e. GDM or Normal Glucose Tolerance, NGT). EVs were isolated from maternal plasma obtained at early, mid and late gestation (n = 29) and small RNA sequencing was performed. Validation: A longitudinal study design was used to quantify expression of selected miRNAs. EV miRNAs were quantified by real-time PCR (cases = 8, control = 14, samples at three times during pregnancy) and their individual and combined classification efficiencies were evaluated. Quantitative, data-independent acquisition mass spectrometry was use to establish the protein profile in skeletal muscle biopsies from normal and GDM. RESULTS: A total of 2822 miRNAs were analyzed using a small RNA library, and a total of 563 miRNAs that significantly changed (p < 0.05) across gestation and 101 miRNAs were significantly changed between NGT and GDM. Analysis of the miRNA changes in NGT and GDM separately identified a total of 256 (NGT-group), and 302 (GDM-group) miRNAs that change across gestation. A multivariate classification model was developed, based on the quantitative expression of EV-associated miRNAs, and the accuracy to correctly assign samples was > 90%. We identified a set of proteins in skeletal muscle biopsies from women with GDM associated with JAK-STAT signaling which could be targeted by the miRNA-92a-3p within circulating EVs. Interestingly, overexpression of miRNA-92a-3p in primary skeletal muscle cells increase insulin-stimulated glucose uptake. CONCLUSIONS: During early pregnancy, differently-expressed, EV-associated miRNAs may be of clinical utility in identifying presymptomatic women who will subsequently develop GDM later in gestation. We suggest that miRNA-92a-3p within EVs might be a protected mechanism to increase skeletal muscle insulin sensitivity in GDM.

Hypoxia-induced small extracellular vesicle proteins regulate proinflammatory cytokines and systemic blood pressure in pregnant rats.

Small extracellular vesicles (sEVs) released from the extravillous trophoblast (EVT) are known to regulate uterine spiral artery remodeling during early pregnancy. The bioactivity and release of these sEVs differ under differing oxygen tensions and in aberrant pregnancy conditions. Whether the placental cell-derived sEVs released from the hypoxic placenta contribute to the pathophysiology of preeclampsia is not known. We hypothesize that, in response to low oxygen tension, the EVT packages a specific set of proteins in sEVs and that these released sEVs interact with endothelial cells to induce inflammation and increase maternal systemic blood pressure. Using a quantitative MS/MS approach, we identified 507 differentially abundant proteins within sEVs isolated from HTR-8/SVneo cells (a commonly used EVT model) cultured at 1% (hypoxia) compared with 8% (normoxia) oxygen. Among these differentially abundant proteins, 206 were up-regulated and 301 were down-regulated (P < 0.05), and they were mainly implicated in inflammation-related pathways. In vitro incubation of hypoxic sEVs with endothelial cells, significantly increased (P < 0.05) the release of GM-CSF, IL-6, IL-8, and VEGF, when compared with control (i.e. cells without sEVs) and normoxic sEVs. In vivo injection of hypoxic sEVs into pregnant rats significantly increased (P < 0.05) mean arterial pressure with increases in systolic and diastolic blood pressures. We propose that oxygen tension regulates the release and bioactivity of sEVs from EVT and that these sEVs regulate inflammation and maternal systemic blood pressure. This novel oxygen-responsive, sEVs signaling pathway, therefore, may contribute to the physiopathology of preeclampsia.

Quantitative Proteomics by SWATH-MS Suggest an Association Between Circulating Exosomes and Maternal Metabolic Changes in Gestational Diabetes Mellitus.

Several factors including placental hormones (PH) released from the human placenta have been associated with the development of insulin resistance and gestational diabetes mellitus (GDM). However, circulating levels of PH does not correlate well with maternal insulin sensitivity across gestation, suggesting that other, previously unrecognized, mechanisms may be involved. The levels of circulating exosomes are higher in GDM compared to normal. GDM derived exosomes produce greater release of pro-inflammatory cytokines from endothelial cells compared to exosomes from normal, suggesting that their contents may differ compared to normal pregnancies. Using a quantitative, information-independent acquisition (Sequential Windowed Acquisition of All Theoretical Mass Spectra [SWATH]) approach, differentially abundant circulating exosome proteins are identified in women with normal glucose tolerance (NGT) and GDM at the time of GDM diagnosis. A total of 78 statistically significant proteins in the relative expression of exosomal proteins in GDM are compared with NGT. Bioinformatic analysis shows that the exosomal proteins in GDM target pathways are mainly associated with energy production, inflammation, and metabolism. Finally, an independent cohort of patients is used to validate some of the proteins identified by SWATH. The data obtained may be of utility in elucidating the underlying physiological mechanisms associated with insulin resistance in GDM.

Adipose Tissue Exosomal Proteomic Profile Reveals a Role on Placenta Glucose Metabolism in Gestational Diabetes Mellitus.

CONTEXT: Molecules produced by adipose tissue (AT) function as an endocrine link between maternal AT and fetal growth by regulating placental function in normal women and women with gestational diabetes mellitus (GDM). OBJECTIVE: We hypothesized that AT-derived exosomes (exo-AT) from women with GDM would carry a specific set of proteins that influences glucose metabolism in the placenta. DESIGN: Exosomes were isolated from omental AT-conditioned media from normal glucose tolerant (NGT) pregnant women (n = 65) and pregnant women with GDM (n = 82). Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry was used to construct a small ion library from AT and exosomal proteins, followed by ingenuity pathway analysis to determine the canonical pathways and biofunctions. The effect of exosomes on human placental cells was determined using a Human Glucose Metabolism RT2 Profiler PCR array. RESULTS: The number of exosomes (vesicles/µg of tissue/24 hours) was substantially (1.7-fold) greater in GDM than in NGT, and the number of exosomes correlated positively with the birthweight Z score. Ingenuity pathway analysis of the exosomal proteins revealed differential expression of the proteins targeting the sirtuin signaling pathway, oxidative phosphorylation, and mechanistic target of rapamycin signaling pathway in GDM compared with NGT. GDM exo-AT increased the expression of genes associated with glycolysis and gluconeogenesis in placental cells compared with the effect of NGT exo-AT. CONCLUSIONS: Our findings are consistent with the possibility that AT exosomes play an important role in mediating the changes in placental function in GDM and might be responsible for some of the adverse consequences in this pregnancy complication, such as fetal overgrowth.

Human placental exosomes in gestational diabetes mellitus carry a specific set of miRNAs associated with skeletal muscle insulin sensitivity.

There is increasing evidence that miRNAs, which are enriched in nanovesicles called exosomes, are important regulators of gene expression. When compared with normal pregnancies, pregnancies with gestational diabetes mellitus (GDM) are associated with skeletal muscle insulin resistance as well as increased levels of circulating placental exosomes. Here we investigated whether placental exosomes in GDM carry a specific set of miRNAs associated with skeletal muscle insulin sensitivity. Exosomes were isolated from chorionic villous (CV) explants from both women with Normal Glucose Tolerant (NGT) and GDM pregnancies. Using miRNA sequencing, we identified a specific set of miRNAs selectively enriched with exosomes and compared with their cells of origin indicating a specific packaging of miRNAs into exosomes. Gene target and ontology analysis of miRNA differentially expressed in exosomes secreted in GDM compared with NGT are associated with pathways regulating cell migration and carbohydrate metabolism. We determined the expression of a selected set of miRNAs in placenta, plasma, and skeletal muscle biopsies from NGT and GDM. Interestingly, the expression of these miRNAs varied in a consistent pattern in the placenta, in circulating exosomes, and in skeletal muscle in GDM. Placental exosomes from GDM pregnancies decreased insulin-stimulated migration and glucose uptake in primary skeletal muscle cells obtained from patients with normal insulin sensitivity. Interestingly, placental exosomes from NGT increase migration and glucose uptake in response to insulin in skeletal muscle from diabetic subjects. These findings suggest that placental exosomes might have a role in the changes on insulin sensitivity in normal and GDM pregnancies.

Proteomic analysis of exosomes reveals an association between cell invasiveness and exosomal bioactivity on endothelial and mesenchymal cell migration in vitro.

Ovarian cancer has resulted in over 140 000 deaths reported annually worldwide. This is often attributed to cellular changes in the microenvironment, including increased migration of mesenchymal stem cells (MSCs) and endothelial cells (ECs) to facilitate metastasis. Recently, the ability of exosomes to communicate signals between cells (and promote cancer progression) has been established. In the present study, we explored the effect of exosomes on cells present in the tumour microenvironment. Exosomes were isolated from ovarian cancer cells with different invasive capacity (high = SKOV-3 and low = OVCAR-3) by differential and buoyant density centrifugation and characterised using nanoparticle tracking analysis (NTA), Western blot, and EM. Exosome secretion was positively correlated with invasiveness of releasing cells. Proteomic analyses identified common and unique proteins between exosomes from SKOV-3 and OVCAR-3 with gene ontology analyses revealing that these exosomes are involved in the regulation of cell migration. Since the tumour microenvironment contains multiple cell types, including MSCs and ECs, we examined the effect of these exosomes on MSC and EC migration. Exosomes promoted MSC and EC migration in a time- and concentration-dependent manner. The effect of exosomes isolated from SKOV-3 on cell migration was significantly higher compared with exosomes from OVCAR-3. Thus, we suggest that exosomes from ovarian cancer cells contain a specific set of proteins that are representative of its cell of origin and the invasive capacity.

Placental exosomes profile in maternal and fetal circulation in intrauterine growth restriction - Liquid biopsies to monitoring fetal growth.

INTRODUCTION: Placenta-derived exosomes may represent an additional pathway by which the placenta communicates with the maternal system to induce maternal vascular adaptations to pregnancy and it may be affected during Fetal growth restriction (FGR). The objective of this study was to quantify the concentration of total and placenta-derived exosomes in maternal and fetal circulation in small fetuses classified as FGR or small for gestational age (SGA). METHODS: Prospective cohort study in singleton term gestations including 10 normally grown fetuses and 20 small fetuses, sub-classified into SGA and FGR accordingly to birth weight (BW) percentile and fetoplacental Doppler. Exosomes were isolated from maternal and fetal plasma and characterized by morphology, enrichment of exosomal proteins, and size distribution by electron microscopy, western blot, and nanoparticle tracking analysis, respectively. Total and specific placenta-derived exosomes were determined using quantum dots coupled with CD63+ve and placental-type alkaline phosphatase (PLAP)+ve antibodies, respectively. RESULTS: Maternal concentrations of CD63+ve and PLAP+ve exosomes were similar between the groups (all p > 0.05). However, there was a significant positive correlation between the ratio of placental-derived to total exosomes (PLAP+ve ratio) and BW percentile, [rho = 0.77 (95% CI: 0.57 to 0.89); p = 0.0001]. The contribution of placental exosomes to the total exosome concentration in maternal and fetal circulation showed a significant decrease among cases, with lower PLAP+ve ratios in FGR compared to controls and SGA cases. DISCUSSION: Quantification of placental exosomes in maternal plasma reflects fetal growth and it may be a useful indicator of placental function.

Methods to Enrich Exosomes from Conditioned Media and Biological Fluids.

Exosomes are nano-vesicles which can transport a range of molecules including but not limited to proteins and miRNA. This ability of exosomes renders them useful in cellular communication often resulting in biological changes. They have several functions in facilitating normal biological processes such as immune responses and an involvement in pregnancy. However, they have also been linked to pathological conditions including cancer and pregnancy complications such as preeclampsia. An understanding for the role of exosomes in preeclampsia is based on the ability to purify and characterize exosomes. There have been several techniques proposed for the enrichment of exosomes such as ultracentrifugation, density gradient separation, and ultrafiltration although there is no widely accepted optimized technique. Here we describe a workflow for isolating exosomes from cell-conditioned media and biological fluids using a combination of centrifugation, buoyant density, and ultrafiltration approaches.

Placental Exosomes as Early Biomarker of Preeclampsia: Potential Role of Exosomal MicroRNAs Across Gestation.

Context: There is a need to develop strategies for early prediction of patients who will develop preeclampsia (PE) to establish preventive strategies to reduce the prevalence and severity of the disease and their associated complications. Objective: The objective of this study was to investigate whether exosomes and their microRNA cargo present in maternal circulation can be used as early biomarker for PE. Design, Setting, Patients, and Interventions: A retrospective stratified study design was used to quantify total exosomes and placenta-derived exosomes present in maternal plasma of normal (n = 32 per time point) and PE (n = 15 per time point) pregnancies. Exosomes present in maternal circulation were determined by nanoparticle tracking analysis. An Illumina TruSeq® Small RNA Library Prep Kit was used to construct a small RNA library from exosomal RNA obtained from plasma samples. Results: In presymptomatic women, who subsequently developed PE, the concentration of total exosomes and placenta-derived exosomes in maternal plasma was significantly greater than those observed in controls, throughout pregnancy. The area under the receiver operating characteristic curves for total exosome and placenta-derived exosome concentrations were 0.745 ± 0.094 and 0.829 ± 0.077, respectively. In total, over 300 microRNAs were identified in exosomes across gestation, where hsa-miR-486-1-5p and hsa-miR-486-2-5p were identified as the candidate microRNAs. Conclusions: Although the role of exosomes during PE remains to be fully elucidated, we suggest that the concentration and content of exosomes may be of diagnostic utility for women at risk for developing PE.

Oxygen tension regulates the miRNA profile and bioactivity of exosomes released from extravillous trophoblast cells - Liquid biopsies for monitoring complications of pregnancy.

Our understanding of how cells communicate has undergone a paradigm shift since the recent recognition of the role of exosomes in intercellular signaling. In this study, we investigated whether oxygen tension alters the exosome release and miRNA profile from extravillous trophoblast (EVT) cells, modifying their bioactivity on endothelial cells (EC). Furthermore, we have established the exosomal miRNA profile at early gestation in women who develop pre-eclampsia (PE) and spontaneous preterm birth (SPTB). HTR-8/SVneo cells were used as an EVT model. The effect of oxygen tension (i.e. 8% and 1% oxygen) on exosome release was quantified using nanocrystals (Qdot®) coupled to CD63 by fluorescence NTA. A real-time, live-cell imaging system (Incucyte™) was used to establish the effect of exosomes on EC. Plasma samples were obtained at early gestation (<18 weeks) and classified according to pregnancy outcomes. An Illumina TrueSeq Small RNA kit was used to construct a small RNA library from exosomal RNA obtained from EVT and plasma samples. The number of exosomes was significantly higher in EVT cultured under 1% compared to 8% oxygen. In total, 741 miRNA were identified in exosomes from EVT. Bioinformatic analysis revealed that these miRNA were associated with cell migration and cytokine production. Interestingly, exosomes isolated from EVT cultured at 8% oxygen increased EC migration, whilst exosomes cultured at 1% oxygen decreased EC migration. These changes were inversely proportional to TNF-α released from EC. Finally, we have identified a set of unique miRNAs in exosomes from EVT cultured at 1% oxygen and exosomes isolated from the circulation of mothers at early gestation, who later developed PE and SPTB. We suggest that aberrant exosomal signalling by placental cells is a common aetiological factor in pregnancy complications characterised by incomplete SpA remodeling and is therefore a clinically relevant biomarker of pregnancy complications.

Gestational Diabetes Mellitus Is Associated With Changes in the Concentration and Bioactivity of Placenta-Derived Exosomes in Maternal Circulation Across Gestation.

Although there is significant interest in elucidating the role of placenta-derived exosomes (PdEs) during pregnancy, the exosomal profile in pregnancies complicated by gestational diabetes mellitus (GDM) remains to be established. The aim of this study was to compare the gestational-age profile of PdEs in maternal plasma of GDM with normal pregnancies and to determine the effect of exosomes on cytokine release from human umbilical vein endothelial cells. A prospective cohort of patients was sampled at three time points during pregnancy for each patient (i.e., 11-14, 22-24, and 32-36 weeks' gestation). A retrospective stratified study design was used to quantify exosomes present in maternal plasma of normal (n = 13) and GDM (n = 7) pregnancies. Gestational age and pregnancy status were identified as significant factors contributing to variation in plasma exosome concentration (ANOVA, P < 0.05). Post hoc analyses established that PdE concentration increased during gestation in both normal and GDM pregnancies; however, the increase was significantly greater in GDM (∼2.2-fold, ∼1.5-fold, and ∼1.8-fold greater at each gestational age compared with normal pregnancies). Exosomes isolated from GDM pregnancies significantly increased the release of proinflammatory cytokines from endothelial cells. Although the role of exosomes during GDM remains to be fully elucidated, exosome profiles may be of diagnostic utility for screening asymptomatic populations.

Placental lipase expression in pregnancies complicated by preeclampsia: a case-control study.

BACKGROUND: Preeclampsia (PE) is associated with maternal and neonatal morbidity and mortality. In PE, the physiological hyperlipidaemia of pregnancy is exaggerated. The purpose of this study was to examine the expression of adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), lipoprotein lipase (LPL) and endothelial lipase (EL) in pregnancies complicated by PE. METHODS: Placentae were collected from 16 women with PE and 20 women with uncomplicated pregnancies matched for maternal prepregnancy BMI and gestational age of delivery. Gene and protein expression of the placental lipases were measured by Q-PCR and Western blot. DNA methylation of the promoter of LPL was assessed by bisulfite sequencing. Lipase localisation and activity were analysed. RESULTS: Gene expression of all lipases was significantly reduced, as was HSL protein level in women with PE. All lipases were localised to trophoblasts and endothelial cells in PE and control placentae. There was no difference in methylation of the LPL promoter between PE and control placentae. Lipase activity was not altered in placentae from women with PE. CONCLUSION: These results suggest that the decreased placental lipase gene but not protein expression or lipase activity, which is associated with late-onset PE is not a major contributor to the abnormal lipids seen in PE.

The Effect of Glucose on the Release and Bioactivity of Exosomes From First Trimester Trophoblast Cells.

CONTEXT: Hyperglycemia and hypoxia are risk factors of metabolic complication during pregnancy. The interactions between oxygen and glucose-sensing pathways that regulate exosome bioactivity from placental cells, however, have not been established. OBJECTIVE: The aim of this study was to test the hypothesis that exosomal signaling by placental cells (defined as the number of exosomes released per unit time and their bioactivity) is responsive to extracellular glucose concentration. METHODS: First-trimester primary trophoblast cells were incubated with D-glucose (5 mM or 25 mM) under 1%, 3%, or 8% O2 for 48 hours. Exosomes were isolated from cell-conditioned media by differential and buoyant density centrifugation. The total number of exosome vesicles was determined by quantifying immunoreactive exosomal CD63. The effect of exosomes on cytokine (granulocyte macrophage colony-stimulating factor, IL-2, IL-4, IL-6. IL-8, IL-10, interferon-γ, and TNF-α) release from endothelial cells was established by a protein solution array analysis. RESULTS: Glucose (25 mM) significantly increased the release of exosomes from trophoblast cells at all oxygen tensions tested (by approximately 2-fold when compared with controls, P < .001). Exosomes (100 µg/mL exosomal protein) released from trophoblast cells significantly increased (P < .05) the release of all cytokines from human umbilical vein endothelial cells when compared with the control (ie, cells without exosomes), with the exception of IL-2 and IL-10 (P > .05). CONCLUSIONS: The effects of high glucose on exosomes bioactivity may be recapitulated in vivo and is of clinical relevance in association with maternal insulin resistance (resulting in hyperglycemia) and preeclampsia (associated with placental insufficiency and hypoxia).

Placental fibroblast growth factor 21 is not altered in late-onset preeclampsia.

BACKGROUND: Preeclampsia (PE) is associated with alterations of placental function. The incidence of PE is higher in insulin resistant states. Women with PE have high circulating levels of the metabolic regulator fibroblast growth factor 21 (FGF21). FGF21 is synthesized in the placenta. The aim of this study was to compare the expression of FGF21, its receptors, downstream targets and transcriptional regulators in placental tissue from pregnancies with and without late-onset PE. Circulating FGF21 in maternal and cord blood was also studied. METHODS: mRNA expression was determined by semi-quantitative real-time PCR and normalized for cellular composition in 17 women with and 20 without PE. Protein expression was quantified by Western Blot. FGF21 levels were measured by ELISA in maternal and cord serum of ten mother-baby dyads per condition. RESULTS: Placental FGF21 mRNA and protein expression were similar in PE compared with control. Placental mRNA expression of the FGF receptors (1-4) and the co-receptor beta-Klotho was not different between the groups. There was no difference in the expression of the glucose transporters GLUT1, 3 or 4. PPAR-alpha but not PPAR-gamma expression was decreased in PE. Maternal FGF21 serum levels were not significantly different in PE. FGF21 was detected in cord blood of 6 infants (4 PE, 2 controls) but was undetectable in 14 infants. CONCLUSIONS: Late-onset PE is not associated with major changes to the expression of FGF21, its receptors or metabolic targets.

Extravillous trophoblast cells-derived exosomes promote vascular smooth muscle cell migration.

BACKGROUND: Vascular smooth muscle cells (VSMCs) migration is a critical process during human uterine spiral artery (SpA) remodeling and a successful pregnancy. Extravillous trophoblast cells (EVT) interact with VSMC and enhance their migration, however, the mechanisms by which EVT remodel SpA remain to be fully elucidated. We hypothesize that exosomes released from EVT promote VSMC migration. METHODS: JEG-3 and HTR-8/SVneo cell lines were used as models for EVT. Cells were cultured at 37°C and humidified under an atmosphere of 5% CO2-balanced N2 to obtain 8% O2. Cell-conditioned media were collected, and exosomes (exo-JEG-3 and exo- HTR-8/SVneo) isolated by differential and buoyant density centrifugation. The effects of exo-EVT on VSMC migration were established using a real-time, live-cell imaging system (Incucyte™). Exosomal proteins where identified by mass spectrometry and submitted to bioinformatic pathway analysis (Ingenuity software). RESULTS: HTR-8/SVneo cells were significantly more (~30%) invasive than JEG-3 cells. HTR-8/SVneo cells released 2.6-fold more exosomes (6.39 × 10(8) ± 2.5 × 10(8) particles/10(6) cells) compared to JEG-3 (2.86 × 10(8) ± 0.78 × 10(8) particles/10(6) cells). VSMC migration was significantly increased in the presence of exo-JEG-3 and exo-HTR-8/SVneo compared to control (-exosomes) (21.83 ± 0.49 h and 15.57 ± 0.32, respectively, vs. control 25.09 ± 0.58 h, p < 0.05). Sonication completely abolished the effect of exosomes on VSMC migration. Finally, mass spectrometry analysis identified unique exosomal proteins for each EVT cell line-derived exosomes. CONCLUSION: The data obtained in this study are consistent with the hypothesis that the release, content, and bioactivity of exosomes derived from EVT-like cell lines is cell origin-dependent and differentially regulates VSMC migration. Thus, an EVT exosomal signaling pathway may contribute to SpA remodeling by promoting the migration of VSMC out of the vessel walls.

Placenta-derived exosomes continuously increase in maternal circulation over the first trimester of pregnancy.

BACKGROUND: Human placenta releases specific nanovesicles (i.e. exosomes) into the maternal circulation during pregnancy, however, the presence of placenta-derived exosomes in maternal blood during early pregnancy remains to be established. The aim of this study was to characterise gestational age related changes in the concentration of placenta-derived exosomes during the first trimester of pregnancy (i.e. from 6 to 12 weeks) in plasma from women with normal pregnancies. METHODS: A time-series experimental design was used to establish pregnancy-associated changes in maternal plasma exosome concentrations during the first trimester. A series of plasma were collected from normal healthy women (10 patients) at 6, 7, 8, 9, 10, 11 and 12 weeks of gestation (n = 70). We measured the stability of these vesicles by quantifying and observing their protein and miRNA contents after the freeze/thawing processes. Exosomes were isolated by differential and buoyant density centrifugation using a sucrose continuous gradient and characterised by their size distribution and morphology using the nanoparticles tracking analysis (NTA; Nanosight™) and electron microscopy (EM), respectively. The total number of exosomes and placenta-derived exosomes were determined by quantifying the immunoreactive exosomal marker, CD63 and a placenta-specific marker (Placental Alkaline Phosphatase PLAP). RESULTS: These nanoparticles are extraordinarily stable. There is no significant decline in their yield with the freeze/thawing processes or change in their EM morphology. NTA identified the presence of 50-150 nm spherical vesicles in maternal plasma as early as 6 weeks of pregnancy. The number of exosomes in maternal circulation increased significantly (ANOVA, p = 0.002) with the progression of pregnancy (from 6 to 12 weeks). The concentration of placenta-derived exosomes in maternal plasma (i.e. PLAP+) increased progressively with gestational age, from 6 weeks 70.6 ± 5.7 pg/ml to 12 weeks 117.5 ± 13.4 pg/ml. Regression analysis showed that weeks is a factor that explains for >70% of the observed variation in plasma exosomal PLAP concentration while the total exosome number only explains 20%. CONCLUSIONS: During normal healthy pregnancy, the number of exosomes present in the maternal plasma increased significantly with gestational age across the first trimester of pregnancy. This study is a baseline that provides an ideal starting point for developing early detection method for women who subsequently develop pregnancy complications, clinically detected during the second trimester. Early detection of women at risk of pregnancy complications would provide an opportunity to develop and evaluate appropriate intervention strategies to limit acute adverse sequel.

Placental lipases in pregnancies complicated by gestational diabetes mellitus (GDM).

Infants of women with gestational diabetes mellitus (GDM) are more likely to be born large for gestational age with a higher percentage body fat. Elevated maternal lipids may contribute to this. Placental lipases such as lipoprotein lipase (LPL), endothelial lipase (EL) and hormone sensitive lipase (HSL) are involved in transferring lipids from mother to fetus. Previous studies of expression of these lipases in placentae in women with diabetes in pregnancy have reported divergent results. Intracellular lipases such as adipose triglyceride lipase (ATGL), and HSL are central to lipid droplet metabolism. The activities of these lipases are both influenced by Perilipin 1, and ATGL is also activated by a co-factor comparative gene identification-58 (CGI-58) and inhibited by G0/G1 switch gene 2 (GS02). None of these modifying factors or ATGL have been examined previously in placenta. The purpose of this study was therefore to examine the expression of ATGL, HSL, LPL, EL, as well as Perilipin 1, GS02 and CGI-58 in term pregnancies complicated by GDM. mRNA and protein expression of the lipases were measured in placentae from 17 women with GDM and 17 normoglycaemic pregnancies, matched for maternal BMI and gestational age of delivery. ATGL mRNA expression was increased and HSL mRNA expression reduced in placentae from GDM although there was no differences in protein expression of any of the lipases. All lipases were localised to trophoblasts and endothelial cells. The expression of Perilipin 1 and CGI-58 mRNA was increased and GS02 not altered in GDM. These results suggest that there is no difference in expression in these four lipases between GDM and normoglycaemic placentae, and therefore altered lipid transfer via these lipases does not contribute to large for gestational age in infants of women with GDM.

A gestational profile of placental exosomes in maternal plasma and their effects on endothelial cell migration.

Studies completed to date provide persuasive evidence that placental cell-derived exosomes play a significant role in intercellular communication pathways that potentially contribute to placentation and development of materno-fetal vascular circulation. The aim of this study was to establish the gestational-age release profile and bioactivity of placental cell-derived exosome in maternal plasma. Plasma samples (n = 20 per pregnant group) were obtained from non-pregnant and pregnant women in the first (FT, 6-12 weeks), second (ST, 22-24 weeks) and third (TT, 32-38 weeks) trimester. The number of exosomes and placental exosome contribution were determined by quantifying immunoreactive exosomal CD63 and placenta-specific marker (PLAP), respectively. The effect of exosomes isolated from FT, ST and TT on endothelial cell migration were established using a real-time, live-cell imaging system (Incucyte). Exosome plasma concentration was more than 50-fold greater in pregnant women than in non-pregnant women (p<0.001). During normal healthy pregnancy, the number of exosomes present in maternal plasma increased significantly with gestational age by more that two-fold (p<0.001). Exosomes isolated from FT, ST and TT increased endothelial cell migration by 1.9±0.1, 1.6±0.2 and 1.3±0.1-fold, respectively compared to the control. Pregnancy is associated with a dramatic increase in the number of exosomes present in plasma and maternal plasma exosomes are bioactive. While the role of placental cell-derived exosome in regulating maternal and/or fetal vascular responses remains to be elucidated, changes in exosome profile may be of clinical utility in the diagnosis of placental dysfunction.

Increased placental expression of fibroblast growth factor 21 in gestational diabetes mellitus.

BACKGROUND: Fibroblast growth factor 21 (FGF21) can regulate glucose and lipid metabolism. The placenta actively synthesizes and secretes many hormones, but it is unknown whether this includes FGF21. This study aimed to analyze the placental expression of FGF21 in women with or without gestational diabetes mellitus (GDM). METHODS: FGF21 and peroxisome proliferator-activated receptor (PPAR)-α mRNA and protein expression were measured in the placentae of 20 women with and 18 without GDM. mRNA expression of PPARα, FGF receptors 1-4, the coreceptor ß-klotho, and glucose transporter (GLUT)-1, -3, and -4 was investigated. Maternal and fetal circulating FGF21 levels were assessed in 10 mother-baby dyads per condition. RESULTS: FGF21 was expressed in the placenta and its mRNA expression increased in women with GDM [10.75 (interquartile range 3.28-125.6 AU)] vs control [0.83 (0.22-4.78), P < .001], as is its protein expression [GDM 2.89 (1.44-5.10)] vs control [0.42 (0.05-1.98), P < .05]. PPARα mRNA but not protein expression was increased in GDM [2.94 (0.70-7.26)] vs control [0.99 (0.43-2.17), P < .05] and was positively correlated to FGF21 mRNA expression (ρ = 0.43, P < .01). Placental mRNA expression of FGF receptors and GLUT1 was unchanged, and ß-klotho, GLUT3, and GLUT4 showed increased expression in GDM. Maternal circulating FGF21 levels were similar [GDM 323 (75-921) vs control 269 (49-731) pg/mL, P = .81]. FGF21 was undetected in fetal cord blood. CONCLUSIONS: FGF21 is expressed in the placenta and its expression is increased in GDM. The absence of FGF21 in fetal cord blood suggests that neither placental FGF21 nor maternal circulating FGF21 is secreted into the fetal circulation. Placental FGF21 may be a regulator of placental metabolism.