Preterm labor is characterized by a high abundance of amniotic fluid prostaglandins in patients with intra-amniotic infection or sterile intra-amniotic inflammation.

OBJECTIVE: To distinguish between prostaglandin and prostamide concentrations in the amniotic fluid of women who had an episode of preterm labor with intact membranes through the utilisation of liquid chromatography-tandem mass spectrometry. STUDY DESIGN: Liquid chromatography-tandem mass spectrometry analysis of amniotic fluid of women with preterm labor and (1) subsequent delivery at term (2) preterm delivery without intra-amniotic inflammation; (3) preterm delivery with sterile intra-amniotic inflammation (interleukin (IL)-6>2.6 ng/mL without detectable microorganisms); and (4) preterm delivery with intra-amniotic infection [IL-6>2.6 ng/mL with detectable microorganisms]. RESULTS: (1) amniotic fluid concentrations of PGE2, PGF2α, and PGFM were higher in patients with intra-amniotic infection than in those without intra-amniotic inflammation; (2) PGE2 and PGF2α concentrations were also greater in patients with intra-amniotic infection than in those with sterile intra-amniotic inflammation; (3) patients with sterile intra-amniotic inflammation had higher amniotic fluid concentrations of PGE2 and PGFM than those without intra-amniotic inflammation who delivered at term; (4) PGFM concentrations were also greater in women with sterile intra-amniotic inflammation than in those without intra-amniotic inflammation who delivered preterm; (5) amniotic fluid concentrations of prostamides (PGE2-EA and PGF2α-EA) were not different among patients with preterm labor; (6) amniotic fluid concentrations of prostaglandins, but no prostamides, were higher in cases with intra-amniotic inflammation; and (7) the PGE2:PGE2-EA and PGF2α:PGF2α-EA ratios were higher in patients with intra-amniotic infection compared to those without inflammation. CONCLUSIONS: Mass spectrometric analysis of amniotic fluid indicated that amniotic fluid concentrations of prostaglandins, but no prostamides, were higher in women with preterm labor and intra-amniotic infection than in other patients with an episode of preterm labor. Yet, women with intra-amniotic infection had greater amniotic fluid concentrations of PGE2 and PGF2α than those with sterile intra-amniotic inflammation, suggesting that these two clinical conditions may be differentiated by using mass spectrometric analysis of amniotic fluid.

The Role of Exosomal Epigenetic Modifiers in Cell Communication and Fertility of Dairy Cows.

Abnormal uterine function affects conception rate and embryo development, thereby leading to poor fertility and reproduction failure. Exosomes are a nanosized subclass of extracellular vesicles (EV) that have important functions as intercellular communicators. They contain and carry transferable bioactive substances including micro RNA (miRNA) for target cells. Elements of the cargo can provide epigenetic modifications of the recipient cells and may have crucial roles in mechanisms of reproduction. The dairy industry accounts for a substantial portion of the economy of many agricultural countries. Exosomes can enhance the expression of inflammatory mediators in the endometrium, which contribute to various inflammatory diseases in transition dairy cows. This results in reduced fertility which leads to reduced milk production and increased cow maintenance costs. Thus, gaining a clear knowledge of exosomal epigenetic modifiers is critical to improving the breeding success and profitability of dairy farms. This review provides a brief overview of how exosomal miRNA contributes to inflammatory diseases and hence to poor fertility, particularly in dairy cows.

Prostaglandin and prostamide concentrations in amniotic fluid of women with spontaneous labor at term with and without clinical chorioamnionitis.

OBJECTIVE: Prostaglandins (PGs) are considered universal mediators for the process of physiological parturition. This is based on observations that amniotic fluid concentrations of PGs are elevated prior to and during the onset of labor (mostly utilizing immunoassays). Distinguishing PGs from similarly structured molecules (i.e. prostamides; PG-EA) is difficult given the cross-reactivity of available antibodies and the chemical similarity between these compounds. Herein, this limitation was overcome by utilizing mass spectrometry to determine PG and PG-EA concentrations in amniotic fluid of women with spontaneous labor at term and in those with clinical chorioamnionitis (CHAM), the most common infection-related diagnosis made in labor and delivery units worldwide. STUDY DESIGN: Liquid chromatography-tandem mass spectrometry (LC MS/MS) was used to determine the PG and PG-EA content in amniotic fluid samples of women with spontaneous labor at term with (n = 14) or without (n = 28) CHAM. Controls included women who delivered at term without labor (n = 10). RESULTS: PGE2, PGF2α, and 13,14-dihydro-15-keto-PGF2α (PGFM) were higher in amniotic fluid of women with spontaneous labor at term than in those without labor. PGE2, PGF2α, and PGFM were also higher in amniotic fluid of women with CHAM than in those without labor. However, PGE2-EA and PGF2α-EA were lower in amniotic fluid of women with CHAM than in those without CHAM. The ratios of PGE2 to PGE2-EA and PGF2α to PGF2α-EA were higher in amniotic fluid of women with spontaneous labor at term with or without CHAM than in those without labor; yet, the ratio of PGF2α to PGF2α-EA was greater in women with CHAM than in those without this clinical condition. CONCLUSIONS: Spontaneous labor at term with or without CHAM is characterized by elevated amniotic fluid concentrations of prostaglandins (PGE2, PGF2α, and PGFM) but not prostamides. Quantification of these products by LC MS/MSlc==may potentially be of utility in identifying their physiological functions relevant to parturition. SUMMARY: Prostaglandins (PGs) are critical for the onset and progression of labor. Structural similarities of PGs and prostamides (PG-EA) prevents their specific identification by immunoassay. We utilized LC MS/MS to determine PG and PG-EA content in amniotic fluid (AF) of women with spontaneous labor at term with or without CHAM and women who delivered at term without labor. Higher aamniotic ffluid PG levels were observed in women with spontaneous labor with and without CHAM compared to women delivering without labor. PG-EA levels in amniotic fluid of women with spontaneous labor and CHAM were lower than in women with spontaneous labor without CHAM but not those without labor. Ratios of PGs to PG-EAs were higher in AF of women with labor and CHAM compared to those without labor. Delineation of these products by LC MS/MS may potentially be of utility in identifying their physiological functions relevant to parturition.

Altered productions of prostaglandins and prostamides by human amnion in response to infectious and inflammatory stimuli identified by mutliplex mass spectrometry.

INTRODUCTION: Prostaglandins are critical for the onset and progression of labor in mammals, and are formed by the metabolism of arachidonic acid. The products of arachidonic acid, 2-arachidonoylglycerol (2-AG), and anandamide (AEA) have a similar lipid back bone but differing polar head groups, meaning that identification of these products by immunoassay can be difficult. MATERIALS AND METHODS: In the current study, we present the use of mass spectrometry as multiplex method of identifying the specific end products of arachidonic and anandamide metabolism by human derived amnion explants treated with either an infectious agent (LPS) or inflammatory mediator (IL-1ß or TNF-α). RESULTS: Human amnion tissue explants treated with LPS, IL-1ß, or TNF-α increased production of prostaglandin E2 (PGE2; p < 0.05) but decreased PGFM. Overall, PGE2 production was greater compared to the other prostaglandins and prostamides irrespective of treatment. CONCLUSIONS: The findings of the current study are in keeping with the literature which describes amnion tissues as predominantly producing PGE2. The use of mass spectrometry for the differential identification of prostaglandins, prostamides, and other eicosanoids may help better elucidate mechanisms of preterm labor, and lead to new targets for the prediction of risk for preterm labor and/or birth.

The Role(s) of Eicosanoids and Exosomes in Human Parturition.

The roles that eicosanoids play during pregnancy and parturition are crucial to a successful outcome. A better understanding of the regulation of eicosanoid production and the roles played by the various end products during pregnancy and parturition has led to our view that accurate measurements of a panel of those end products has exciting potential as diagnostics and prognostics of preterm labor and delivery. Exosomes and their contents represent an exciting new area for research of movement of key biological factors circulating between tissues and organs akin to a parallel endocrine system but involving key intracellular mediators. Eicosanoids and enzymes regulating their biosynthesis and metabolism as well as regulatory microRNAs have been identified within exosomes. In this review, the regulation of eicosanoid production, abundance and actions during pregnancy will be explored. Additionally, the functional significance of placental exosomes will be discussed.

Exosomes from dairy cows of divergent fertility; Action on endometrial cells.

Abnormalities in endometrial function contribute to poor fertility and reproductive failure. Exosomes are small lipid vesicles that contain transferable bioactive substances; they participate in intercellular signaling and may have critical roles in reproductive mechanisms, including endometrial remodeling in preparation for pregnancy. In this study, we evaluated the effects of exosomes from heifers with high and low genetic merit for fertility on inflammatory mediator expression by bovine endometrial epithelial and stromal cell lines. Co-incubation of exosomes from low, compared with high, fertility heifers upregulated the gene expression of pro-inflammatory IL1A and IL8 (CXCL8) but downregulated IL4 gene expression in epithelial cells. In contrast, stromal cells co-incubated with exosomes from low, compared with high, fertility heifers downregulated the gene expression of CXCL9, CXCL10, and CX3CL1. Our findings demonstrated that circulating exosomes from high fertility heifers did not alter endometrial inflammatory mediator gene expression. In contrast, circulating exosomes from low fertility heifers enhanced endometrial expression of inflammatory mediators, which may contribute to aberrant inflammation, leading to a reduced fertility in low fertility heifers. However, an in-depth investigation is required to elucidate the role of exosomes in regulating endometrial remodeling events required for enhanced reproductive performance and fertility in dairy cows.

Circulating exosomes may identify biomarkers for cows at risk for metabolic dysfunction.

Disease susceptibility of dairy cows is greatest during the transition from pregnancy to lactation. Circulating exosomes may provide biomarkers to detect at-risk cows to enhance health and productivity. From 490 cows, animals at high- (n = 20) or low-risk (n = 20) of transition-related diseases were identified using plasma non-esterified fatty acid and ß-hydroxybutyrate concentrations and liver triacylglyceride concentrations during the two weeks post-calving. We isolated circulating exosomes from plasma of dairy cows at low-risk (LR-EXO) and high-risk (HR-EXO), and analyzed their proteome profiles to determine markers for metabolic dysfunction. We evaluated the effects of these exosomes on eicosanoid pathway expression by bovine endometrial stromal (bCSC) and epithelial (bEEL) cells. HR-EXO had significantly lower yield of circulating exosomes compared with LR-EXO, and unique proteins were identified in HR-EXO and LR-EXO. Exposure to LR-EXO or HR-EXO differentially regulated eicosanoid gene expression and production in bCSC and bEEL cells. In bCSC, LR-EXO exposure increased PGE2 and PGD2 production, whereas HR-EXO exposure increased PTGS2 gene expression. In bEEL, HR-EXO exposure caused a decrease in PGE2, PGF2α, PGD2, PGFM and TXB2 production. The unique presence of serpin A3-7, coiled-coil domain containing 88A and inhibin/activin ß A chain in HR-EXO, indicates potential biomarkers for cows at-risk for metabolic diseases. Our results are in line with the health status of the cow indicating a potential diagnostic role for exosomes in enhancing cows' health and fertility.

Eicosanoid pathway expression in bovine endometrial epithelial and stromal cells in response to lipopolysaccharide, interleukin 1 beta, and tumor necrosis factor alpha.

During endometrial inflammation, bovine endometrium responds by increasing the production of pro-inflammatory mediators, such as interleukin 1 beta (IL-1ß), tumor necrosis factor alpha (TNFα), and eicosanoids. The purpose of this study was to establish and characterize an in vitro model of endometrial inflammation using bovine endometrial epithelial (bEEL) and stromal (bCSC) cell lines. We evaluated the effects of the infectious agent (bacterial lipopolysaccharide; LPS) and pro-inflammatory mediators (IL-1ß and TNFα) on eicosanoid biosynthesis pathway gene expression and production by bEEL and bCSC cells. Based on concentration-response experiments, the optimal concentrations for responses were 1 µg/mL LPS, 10 ng/mL IL-1ß and 50 ng/mL TNFα. Real-time PCR results show that there was an upregulation of relative mRNA expression of PTGS2 when bEEL and bCSC were treated with LPS, IL-1ß and TNFα. An increase in PTGES3 expression was observed when bEEL cells were treated with LPS and IL-1ß and PTGES2 when treated with IL-1ß. In bCSC cells, FAAH relative mRNA was decreased upon treatments. Rate of production of PGE2, PGF2α, PGE2-EA and PGF2α-EA were also determined using liquid chromatography tandem mass spectrometry. Our results show that eicosanoid production was increased in both cell lines in response to LPS, IL-1ß, and TNFα. We suggest that the characteristics of bEEL and bCSC cell lines mimic the physiological responses found in mammals with endometrial infection, making them excellent in vitro models for intrauterine environment studies.

Regulation of inflammatory mediator expression in bovine endometrial cells: effects of lipopolysaccharide, interleukin 1 beta, and tumor necrosis factor alpha.

An abnormal uterine environment can influence maternal-fetal communication, conception rate and disrupt normal embryo development, thereby affecting fertility and the reproductive performance of dairy cows. Animal variability means that development of endometrial cell lines with appropriate characteristic are required. We evaluated the effect of an infectious agent (i.e., bacterial lipopolysaccharide; LPS) and proinflammatory mediators (i.e., Interleukin 1 beta; IL-1ß, and tumor necrosis factor alpha; TNFα) on inflammatory mediator gene expression and production by bovine endometrial epithelial (bEEL) and stromal (bCSC) cell lines. Expression of CXCL8/IL8, IL1A, IL1B, and IL6 cytokine genes was significantly upregulated in both epithelial and stromal cells when treated with LPS and IL-1ß. LPS treatment of epithelial cells (compared with treatment by IL-1ß and TNFα) exhibited greater CXCL8/IL8, IL1A, IL1B, and IL6 cytokine gene expression. Whereas, in stromal cells, IL-1ß treatment (compared with LPS and TNFα) exhibited greater CXCL8/IL8, IL1A, IL1B, and IL6 cytokine gene expression. Interestingly, bEEL and bCSC cells treated with IL-1ß increased IL1B gene expression, suggesting that IL-1ß may act unusually in an autocrine-positive feedback loop. Cytokine production was stimulated by these agents in both cell types. We suggest that the characteristics of these two cell lines make them excellent tools for the study of intrauterine environment.

Proteome profiling of exosomes derived from plasma of heifers with divergent genetic merit for fertility.

The current study evaluated exosomes isolated from plasma of heifers bred to have high or low fertility through developing extreme diversity in fertility breeding values, however, key animal traits (e.g., body weight, milk production, and percentage of North American genetics) remained similar between the 2 groups. The exosomes were isolated by a combined ultracentrifugation and size exclusion chromatography approach and characterized by their size distribution (nanoparticle tracking analysis), morphology (transmission electron microscopy), and presence of exosomal markers (immunoblotting). In addition, a targeted mass spectrometry approach was used to confirm the presence of 2 exosomal markers, tumor susceptibility gene 101 and flotillin 1. The number of exosomes from plasma of high fertility heifers was greater compared with low fertility heifers. Interestingly, the exosomal proteomic profile, evaluated using mass spectrometry, identified 89 and 116 proteins in the high and low fertility heifers respectively, of which 4 and 31 were unique, respectively. These include proteins associated with specific biological processes and molecular functions of fertility. Most notably, the tetratricopeptide repeat protein 41-related, glycodelin, and kelch-like protein 8 were identified in plasma exosomes unique to the low fertility heifers. These proteins are suggested to play a role in reproduction; however, the role of these proteins in dairy cow reproduction remains to be elucidated. Their identification underscores the potential for proteins within exosomes to provide information on the fertility status and physiological condition of the cow. This may potentially lead to the development of prognostic tools and interventions to improving dairy cow fertility.

Proteomic content of circulating exosomes in dairy cows with or without uterine infection.

In the past few decades, there has been a global decrease in dairy cow reproductive performance. An activated inflammatory system, due to uterine infection, has been associated with decreased cow fertility and as such, there is a need to detect uterine disease earlier. Early detection could be achieved by identifying biomarkers for uterine disease. Exosomes are small nanovesicles known to package and deliver protein, mRNA, and miRNAs to near and distant sites. Therefore, the content of circulating exosomes may have the potential to carry biomarkers for earlier diagnosis of disease. We hypothesized that circulating exosomes from cows with and without uterine infection may contain information representative of endometrial health or disease. We compared the proteomic content of circulating exosomes derived from plasma of dairy cows with (n = 10) or without (n = 10) induced uterine infection, using high-performance liquid chromatography tandem mass spectrometry (HPLC MS/MS). Our results demonstrate that there were a total of 103 bovine and 9 Trueperella pyogenes proteins found in plasma exosomes derived from infected cows (infected exosomes), and 90 bovine and 5 T. pyogenes proteins found in exosomes derived from plasma of non-infected cows (non-infected exosomes). 71 bovine proteins were found to be unique to the infected exosomes while only 4 bovine proteins were found to be unique to the non-infected exosomes. 8 unique T. pyogenes proteins were identified in infected exosomes and 4 were found to be unique to the non-infected exosomes. Pathway analysis showed that infected exosomes had more proteins involved in structural molecule activity and immune system processes than non-infected exosomal protein. Additionally, proteins from infected exosomes were involved in unique pathways: angiogenesis and integrin signaling pathway. Our data provide preliminary evidence of a potential role for exosomes in the early diagnosis of uterine infection in dairy cows.

Mid-to-Late Gestational Changes in Inflammatory Gene Expression in the Rat Placenta.

BACKGROUND: The placenta plays an important role during pregnancy providing maternal blood supply from the uterus to the developing fetus. The structure and function of the placenta changes with gestation, as the fetus develops and its demands change. This study aims to elucidate changes in cytokine and chemokine gene expression throughout mid-to-late gestation in rat placenta. METHODS: Sprague Dawley rats were time-mated, and placentae were obtained from 6 pregnant dams at 4 different gestational periods: E14.25, E15.25, E17.25, and E20. Changes in placental gene expression were measured by microarray analysis. Differentially expressed inflammatory genes were functionally categorized by pathway analysis. To validate the microarray results, a subset of genes was analyzed by quantitative real-time polymerase chain reaction (qPCR) in a validation cohort of 22 rats. RESULTS: Changes in messenger RNA (mRNA) expression of various cytokines, chemokines, and genes of the tumor growth factor ß and tumor necrosis factor family were analyzed in rat placentae at E14.25, E15.25, E17.25, and E20. Forty-six genes were differentially expressed, and of these 21 genes had increased expression in late gestation (E20). The gestational age pattern of gene expression was confirmed by qPCR in the validation cohort. CONCLUSION: The observed acute, prelabor changes in the expression of these genes during gestation warrant further investigation to elucidate their role in pregnancy and parturition.

Exosome enrichment by ultracentrifugation and size exclusion chromatography.

Exosomes are a subset of extracellular vesicles (EVs) that have important roles in intercellular communication. They contain and carry bioactive molecules within their membranes which are delivered to target cells. Reproducible isolation and enrichment of these exosomes will aid in evaluation of cellular communication. We present an approach that involved the pre-processing of plasma, combined with ultracentrifugation (UC) and size exclusion chromatography (SEC) to isolate EVs and subsequently enrich exosomes. Four variations of this approach (denoted methods I to IV) were compared. Coupling an ultracentrifugation method with size exclusion chromatography (Method II) provided the best yield by nanoparticle tracking analyses (NTA), the presence of the exosomal markers CD63, Flotillin-1 and TSG-101 (immunoblotting) and showed exosome morphology using transmission electron microscopy (TEM). This method provides an efficient way to enrich the exosomes from blood (plasma), which could be potentially employed for clinical diagnostic assessment and therapeutic intervention.

A method for the isolation and enrichment of purified bovine milk exosomes.

Exosomes are nanovesicles that play important roles in intercellular communication as they carry information to target cells. Isolation of high purity exosomes will aid in studying the exosomal cargo and quantity as well as how cell-specific messages are carried. We describe a new method incorporating size exclusion chromatography (SEC) to enrich milk-derived exosomes from extracellular vesicles (EVs). This involved the initial isolation of EVs from bovine milk via milk processing and ultracentrifugation; followed by a new method to enrich exosomes using SEC. This method was compared to buoyant density gradient centrifugation, a widely used method of enrichment. Exosomes were characterised by particle concentration and size (nanoparticle tracking analysis, NTA), morphology (transmission electron microscopy, TEM), presence of exosomal markers (immunoblotting) and protein concentration (bicinchoninic acid assay, BCA). Proteomic profiles of exosomal fractions were analyzed by mass spectrometry using Information Dependant Acquisition. Milk exosomal fractions were shown to contain exosomal markers flotillin-1 (FLOT-1) and tumor susceptibility gene-101 (TSG-101). The new method produced a higher yield of exosomes compared to buoyant density gradient centrifugation. Pooled exosomal fractions exhibited intact morphology by TEM. The use of SEC confirmed the fractionation of exosomes based on size while minimizing the interference with proteins. Tetraspanins CD9 and CD81 were observed via mass spectrometry in exosomal fractions. This new and efficient method confirmed the signatures for exosomes derived from unpasteurized bovine milk. Purification of exosomes is a foundational technique in the study of biomarkers for pathological conditions and effective drug delivery systems.

Effect of exosomes from plasma of dairy cows with or without an infected uterus on prostaglandin production by endometrial cell lines.

A contributing factor to declining fertility in dairy cows is an activated inflammatory system associated with uterine infection. Detecting uterine disease using biomarkers may allow earlier diagnosis and intervention with resultant improvements in fertility. Exosomes are known to participate in intercellular communication, paracrine, and endocrine signaling. Exosomes carry a cargo of proteins, lipids, and nucleic acids that represent specific cellular sources. Prostaglandins are lipids that are critical determinants of bovine fertility. In this study exosomes were isolated from the plasma of cows before (d 0) and during (d 10) the study in healthy animals or those with an induced uterine infection in a 2 × 2 factorial design. Exosomes were characterized for size and number (nanoparticle tracking analysis), exosomal marker expression (Western blot), and morphology (transmission electron microscopy). No significant differences were observed in exosome size or number. The abundance of exosome-enriched markers was confirmed in noninfected and infected animals. Transmission electron microscopy confirmed the morphology of the exosomes. These exosomes were co-incubated with bovine endometrial epithelial and stromal cells. Exosomes from d-10-infected animal plasma decreased PGF2α production in endometrial epithelial but not stromal cells. For future research, the identification of effectors in the cargo may provide a useful basis for early diagnosis of uterine infection using an exosomal characterization approach.

Characterization of exosomal release in bovine endometrial intercaruncular stromal cells.

BACKGROUND: Cell-to-cell communication between the blastocyst and endometrium is critical for implantation. In recent years, evidence has emerged from studies in humans and several other animal species that exosomes are secreted from the endometrium and trophoblast cells and may play an important role in cell-to-cell communication maternal-fetal interface during early pregnancy. Exosomes are stable extracellular lipid bilayer vesicles that encapsulate proteins, miRNAs, and mRNAs, with the ability to deliver their cargo to near and distant sites, altering cellular function(s). Furthermore, the exosomal cargo can be altered in response to environmental cues (e.g. hypoxia). The current study aims to develop an in vitro system to evaluate maternal-embryo interactions via exosomes (and exosomal cargo) produced by bovine endometrial stromal cells (ICAR) using hypoxia as a known stimulus associated with the release of exosomes and alterations to biological responses (e.g. cell proliferation). METHODS: ICAR cells cultured under 8 % O2 or 1 % O2 for 48 h and changes in cell function (i.e. migration, proliferation and apoptosis) were evaluated. Exosome release was determined following the isolation (via differential centrifugation) and characterization of exosomes from ICAR cell-conditioned media. Exosomal proteomic content was evaluated by mass spectrometry. RESULTS: Under hypoxic conditions (i.e. 1 % O2), ICAR cell migration and proliferation was decreased (~20 and ~32 %, respectively) and apoptotic protein caspase-3 activation was increased (∼1.6 fold). Hypoxia increased exosome number by ~3.6 fold compared with culture at 8 % O2. Mass spectrometry analysis identified 128 proteins unique to exosomes of ICAR cultured at 1 % O2 compared with only 46 proteins unique to those of ICAR cultured at 8 % O2. Differential production of proteins associated with specific biological processes and molecular functions were identified, most notably ADAM10, pantetheinase and kininogen 2. CONCLUSIONS: In summary, we have shown that a stimulus such as hypoxia can alter both the cellular function and exosome release of ICAR cells. Alterations to exosome release and exosomal content in response to stimuli may play a crucial role in maternal-fetal crosstalk and could also affect placental development.

Placental exosomes in normal and complicated pregnancy.

While there is considerable contemporary interest in elucidating the role of placenta-derived extracellular vesicles in normal and complicated pregnancies and their utility as biomarkers and therapeutic interventions, progress in the field is hindered by a lack of standardized extracellular vesicle taxonomy and isolation protocols. The term "extracellular vesicle" is nonspecific and refers to all membrane-bound vesicles from nanometer to micrometer diameters and of different biogenic origins. To meaningfully ascribe biological function and/or diagnostic and therapeutic utility to extracellular vesicles, and in particular exosomes, greater specificity and vesicle characterization is required. The current literature relating to exosome biology must be interpreted in this context. Exosomes are a subtype of extracellular vesicle that are specifically defined by an endosomal biogenesis and particle size (40-120 nm) and density (1.13-1.19 g/mL(-1)). Exosomes are specifically package with signaling molecules (including protein, messenger RNA, microRNA, and noncoding RNA) and are released by exocytosis into biofluid compartments. Exosomes regulate the activity of both proximal and distal target cells, including translational activity, angiogenesis, proliferation, metabolism, and apoptosis. As such, exosomal signaling represents an integral pathway mediating intercellular communication. During pregnancy, the placenta releases exosomes into the maternal circulation from as early as 6 weeks of gestation. Release is regulated by factors that include both oxygen tension and glucose concentration and correlates with placental mass and perfusion. The concentration of placenta-derived exosomes in maternal plasma increases progressively during gestation. Exosomes isolated from maternal plasma are bioactive in vitro and are incorporated into target cells by endocytosis. While the functional significance of placental exosomes in pregnancy remains to be fully elucidated, available data support a role in normal placental development and maternal immunotolerance. Similarly, the role of exosomes in the etiology and progression of complications of pregnancy remains in a formative stage. Changes in the release of placenta- and nonplacenta-derived exosomes, their concentration in maternal plasma, composition, and bioactivity have been reported in association with pregnancies complicated by gestational diabetes and preeclampsia. The data, however, are confounded by the use of different isolation methodologies and vesicle subpopulations. The application of specific and well-characterized isolation methodologies is requisite to resolving the precise role of exosomes in complications of pregnancies and their ultimate clinical utility.

Gestation Related Gene Expression of the Endocannabinoid Pathway in Rat Placenta.

Mammalian placentation is a vital facet of the development of a healthy and viable offspring. Throughout gestation the placenta changes to accommodate, provide for, and meet the demands of a growing fetus. Gestational gene expression is a crucial part of placenta development. The endocannabinoid pathway is activated in the placenta and decidual tissues throughout pregnancy and aberrant endocannabinoid signaling during the period of placental development has been associated with pregnancy disorders. In this study, the gene expression of eight endocannabinoid system enzymes was investigated throughout gestation. Rat placentae were obtained at E14.25, E15.25, E17.25, and E20, RNA was extracted, and microarray was performed. Gene expression of enzymes Faah, Mgll, Plcd4, Pld1, Nat1, Daglα, and Ptgs2 was studied (cohort 1, microarray). Biological replication of the results was performed by qPCR (cohort 2). Four genes showed differential expression (Mgll, Plcd4, Ptgs2, and Pld1), from mid to late gestation. Genes positively associated with gestational age were Ptgs2, Mgll, and Pld1, while Plcd4 was downregulated. This is the first comprehensive study that has investigated endocannabinoid pathway gene expression during rat pregnancy. This study provides the framework for future studies that investigate the role of endocannabinoid system during pregnancy.

The rat placental renin-angiotensin system - a gestational gene expression study.

BACKGROUND: The placenta is an essential organ that provides nutrients and oxygen to the developing fetus and removes toxic waste products from the fetal circulation. Maintaining placental blood osmotic pressure and blood flow is crucial for viable offspring. The renin-angiotensin system (RAS) in the placenta is a key player in the regulation of maternal-fetal blood flow during pregnancy. Therefore, the aim of this study was to determine if RAS genes are differentially expressed in mid to late gestation in rat placenta. METHODS: Whole placental tissue samples from pregnant Sprague Dawley rats at embryonic (E) days 14.25, 15.25, 17.25 and 20 (n = 6 for each gestational age) were used for genome-wide gene expression by microarray. RAS genes with expression differences of >2 fold were further analyzed. Quantitative Real-Time PCR (qPCR) was performed on independent samples to confirm and validate microarray data. Immunohistochemisty and Western blotting were performed on a differentially expressed novel RAS pathway gene (ANPEP). RESULTS: Six out of 17 genes of the RAS pathway were differentially expressed at different gestational ages. Gene expression of four genes (Angiotensin converting enzyme (Ace), angiotensin converting enzyme 2 (Ace2), membrane metalloendopeptidase (Mme) and angiotensin II receptor 1A (Agtr1a)) were significantly upregulated at E20 whereas two others (Thimet oligopeptidase 1 (Thop1) and Alanyl aminopeptidase (Anpep)) were downregulated at E20 prior to the onset of labour. These changes were confirmed by qPCR. Western blots revealed no overall differences in ANPEP protein expression in the placentae. Immunohistochemical studies, however, indicated that the localization of ANPEP differed at E17.25 and E20 as ANPEP localization in the giant trophoblast cell of the junctional zone was no longer detectable at E20. CONCLUSIONS: The current study investigated the expression of members of the RAS pathway in rat placentae and observed significantly altered expression of 6 RAS genes at 4 gestational ages. These findings present the need for further comprehensive investigation of RAS genes in normal and complicated pregnancies.

Expression of Myostatin in Intrauterine Growth Restriction and Preeclampsia Complicated Pregnancies and Alterations to Cytokine Production by First-Trimester Placental Explants Following Myostatin Treatment.

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are major obstetric health problems. Higher levels of T-helper (Th) 1 (proinflammatory) cytokines have been observed in pregnancies complicated with PE and IUGR; this is in contrast to the predominant Th2 (anti-inflammatory) cytokine environment found in uncomplicated pregnancies. Myostatin is best known as a negative regulator of muscle development and reportedly has a role in fat deposition, glucose metabolism, and cytokine modulation (outside the placenta). Myostatin concentrations in plasma and protein expression in placental tissue are significantly higher in women with PE. Expression of myostatin in IUGR and PE-IUGR and the effect of this protein on the cytokine production from the placenta is unknown. In the current study, significant differences were identified in the expression of myostatin in pregnancies complicated with IUGR, PE, and PE with IUGR. Furthermore, cytokine production by first-trimester placental tissues was altered following myostatin treatment.