RESUMEN
BACKGROUND: The onset of preterm labor is associated with inflammation. Previous studies suggested that this is distinct from the inflammation observed during term labor. Our previous work on 44 genes differentially expressed in myometria in term labor demonstrated a different pattern of gene expression from that observed in preterm laboring and nonlaboring myometria. We found increased expression of inflammatory genes in preterm labor associated with chorioamnionitis, but in the absence of chorioamnionitis observed no difference in gene expression in preterm myometria regardless of laboring status, suggesting that preterm labor is associated with different myometrial genes or signals originating from outside the myometrium. Given that a small subset of genes were assessed, this study aimed to use RNA sequencing and bioinformatics to assess the myometrial transcriptome during preterm labor in the presence and absence of chorioamnionitis. OBJECTIVE: This study aimed to comprehensively determine protein-coding transcriptomic differences between preterm nonlaboring and preterm laboring myometria with and without chorioamnionitis. STUDY DESIGN: Myometria were collected at cesarean delivery from preterm patients not in labor (n=16) and preterm patients in labor with chorioamnionitis (n=8) or without chorioamnionitis (n=6). Extracted RNA from myometrial tissue was prepared and sequenced using Illumina NovaSeq. Gene expression was quantified by mapping the sequence reads to the human reference genome (hg38). Differential gene expression analysis, gene set enrichment analysis, and weighted gene coexpression network analysis were used to comprehensively interrogate transcriptomic differences and their associated biology. RESULTS: Differential gene expression analysis comparing preterm patients in labor with chorioamnionitis with preterm patients not in labor identified 931 differentially expressed genes, whereas comparing preterm patients in labor without chorioamnionitis with preterm patients not in labor identified no statistically significant gene expression changes. In contrast, gene set enrichment analysis and weighted gene coexpression network analysis demonstrated that preterm labor with and without chorioamnionitis was associated with enrichment of pathways involved in activation of the innate immune system and inflammation, and activation of G protein-coupled receptors. Key genes identified included chemotactic CYP4F3, CXCL8, DOCK2, and IRF1 in preterm labor with chorioamnionitis and CYP4F3, FCAR, CHUK, and IL13RA2 in preterm labor without chorioamnionitis. There was marked overlap in the pathways enriched in both preterm labor subtypes. CONCLUSION: Differential gene expression analysis demonstrated that myometria from preterm patients in labor without chorioamnionitis and preterm patients not in labor were transcriptionally similar, whereas the presence of chorioamnionitis was associated with marked gene changes. In contrast, comprehensive bioinformatic analysis indicated that preterm labor with or without chorioamnionitis was associated with innate immune activation. All causes of preterm labor were associated with activation of the innate immune system, but this was more marked in the presence of chorioamnionitis. These data suggest that anti-inflammatory therapy may be relevant in managing preterm labor of all etiologies.
Asunto(s)
Corioamnionitis , Trabajo de Parto , Trabajo de Parto Prematuro , Embarazo , Femenino , Recién Nacido , Humanos , Miometrio/metabolismo , Corioamnionitis/genética , Corioamnionitis/metabolismo , Transcriptoma , Trabajo de Parto Prematuro/genética , Trabajo de Parto Prematuro/metabolismo , Trabajo de Parto/genética , Trabajo de Parto/metabolismo , Inflamación/genética , Inflamación/metabolismo , Perfilación de la Expresión GénicaRESUMEN
BACKGROUND: The onset of the term human parturition involves myometrial gene expression changes to transform the uterus from a quiescent to a contractile phenotype. It is uncertain whether the same changes occur in the uterus during preterm labor. OBJECTIVE: This study aimed to compare the myometrial gene expression between term and preterm labor and to determine whether the presence of acute clinical chorioamnionitis or twin gestation affects these signatures. STUDY DESIGN: Myometrial specimens were collected during cesarean delivery from the following 7 different groups of patients: term not in labor (n=31), term labor (n=13), preterm not in labor (n=21), preterm labor with acute clinical chorioamnionitis (n=6), preterm labor with no acute clinical chorioamnionitis (n=9), twin preterm not in labor (n=8), and twin preterm labor with no acute clinical chorioamnionitis (n=5). RNA was extracted, reverse transcribed and quantitative polymerase chain reactions were performed on 44 candidate genes (with evidence for differential expression in human term labor) using the Fluidigm platform. Computational analysis was performed using 2-class unpaired Wilcoxon tests and principal component analysis. RESULTS: Computational analysis revealed that gene expression in the preterm myometrium, irrespective of whether in labor or not in labor, clustered tightly and is clearly different from the term labor and term not-in-labor groups. This was true for both singleton and twin pregnancies. Principal component analysis showed that 57% of the variation was explained by 3 principal components. These 44 genes interact in themes of prostaglandin activity and inflammatory signaling known to be important during term labor, but are not a full representation of the myometrium transcriptional activity. CONCLUSION: The myometrial contractions associated with preterm labor are associated with a pattern of gene expression that is distinct from term labor. Therefore, preterm labor may be initiated by a different myometrial process or processes outside the myometrium.
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Trabajo de Parto/metabolismo , Miometrio/metabolismo , Trabajo de Parto Prematuro/metabolismo , Embarazo Gemelar , Contracción Uterina/metabolismo , Adulto , Simulación por Computador , Femenino , Expresión Génica , Edad Gestacional , Humanos , EmbarazoRESUMEN
Inflammatory genes are expressed increasingly in the foetal membranes at late gestation triggering birth. Here we have examined whether epigenetic histone modifications contribute to the upregulation of proinflammatory genes in the amnion in late pregnancy and at labour. Amnion samples were collected from early pregnancy, at term in the absence of labour and after spontaneous birth. The expression of the labour-associated proinflammatory genes PTGS2, BMP2 and NAMPT was determined by reverse transcription-coupled quantitative real-time PCR (qRT-PCR). Chromatin immunoprecipitation (ChIP) and sequential double ChIP were performed to determine the levels and co-occurrence of activating histone-3, lysine-4 trimethylation (H3K4me3) and repressive histone-3, lysine-27 trimethylation (H3K27me3) at the gene promoters. H3K4 methyltransferase, H3K27me3 demethylase and H3K27 methyltransferase expression was determined by qRT-PCR and immunofluorescence confocal microscopy. PTGS2, BMP2 and NAMPT expression was upregulated robustly between early pregnancy and term (P < 0.05). The promoters were marked bivalently by both the H3K4me3 and H3K27me3 modifications. Bivalence was reduced at term by the decrease of the H3K27me3-modified fraction of promoter copies marked by H3K4me3 indicating epigenetic activation. Messenger RNAs encoding the H3K4-specific methyl transferases MLL1,-2,-3,-4, SETD1A,-B and the H3K27me3-specific demethylases KDM6A,-B were expressed increasingly while the H3K27 methyl transferase EZH2 was expressed decreasingly at term. Histone modifying enzyme proteins were detected in amnion epithelial and mesenchymal cells. These results with prototypical proinflammatory genes suggest that nucleosomes at labour-promoting genes are marked bivalently in the amnion, which is shifted towards monovalent H3K4me3 modification at term when the genes are upregulated. Bivalent epigenetic regulation by histone modifying enzymes may control the timing of labour.
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Amnios/metabolismo , Epigénesis Genética , Histona Demetilasas/genética , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Procesamiento Proteico-Postraduccional , Amnios/citología , Amnios/crecimiento & desarrollo , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Citocinas/genética , Citocinas/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Edad Gestacional , Histona Demetilasas/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Células Madre Mesenquimatosas , Nicotinamida Fosforribosiltransferasa/genética , Nicotinamida Fosforribosiltransferasa/metabolismo , Parto/genética , Embarazo , Tercer Trimestre del Embarazo/genética , Regiones Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Progesterone plays a crucial role in maintaining pregnancy by promoting myometrial quiescence. The withdrawal of progesterone action signals the end of pregnancy and, in most mammalian species, this is achieved by a rapid fall in progesterone concentrations. However, in humans circulating progesterone concentrations remain high up to and during labour. Efforts to understand this phenomenon led to the 'functional progesterone withdrawal' hypothesis, whereby the pro-gestation actions of progesterone are withdrawn, despite circulating concentrations remaining elevated. The exact mechanism of functional progesterone withdrawal is still unclear and in recent years has been the focus of intense research. Emerging evidence now indicates that epigenetic regulation of progesterone receptor isoform expression may be the crucial mechanism by which functional progesterone withdrawal is achieved, effectively precipitating human labour despite high concentrations of circulating progesterone. This review examines current evidence that epigenetic mechanisms play a role in determining whether the pro-gestation or pro-contractile isoform of the progesterone receptor is expressed in the pregnant human uterus. We explore the mechanism by which these epigenetic modifications are achieved and, importantly, how these underlying epigenetic mechanisms are influenced by known regulators of uterine physiology, such as prostaglandins and oestrogens, in order to phenotypically transform the pregnant uterus and initiate labour.
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Histonas/metabolismo , Trabajo de Parto/metabolismo , Parto/metabolismo , Receptores de Progesterona/metabolismo , Útero/metabolismo , Epigénesis Genética , Femenino , Histonas/genética , Humanos , Trabajo de Parto/genética , Parto/genética , Embarazo , Progesterona/metabolismo , Receptores de Progesterona/genéticaRESUMEN
Preterm birth (PTB) is the single largest cause of death in infants and young children. The rate of PTB is significantly higher in male infants, particularly those that are born very preterm. Here we present evidence to suggest that the decidual renin-angiotensin system may play a role in inhibiting inflammation and maintaining the integrity of the fetal membranes during pregnancy, and that sex-specific alterations in the intrauterine RAS could contribute to the increased risk of PTB in male babies. Women carrying female fetuses have high levels of expression of decidual prorenin at term. Decidua from 'female' pregnancies also have greater expression of the anti-inflammatory angiotensin (Ang)-(1-7) pathway, than decidua from 'male' pregnancies, and have lower levels of the pro-inflammatory Ang II pathway. We propose that in 'female' pregnancies, the very high levels of decidual prorenin drive the anti-inflammatory Ang-(1-7) pathway, thus reducing the likelihood of PTB. In addition, the high levels of prorenin produced by the decidua in 'female' pregnancies are able to diffuse into the amnion and bind to the PRR. We postulate that PRR/prorenin interactions, possibly through both angiotensin dependent and independent pathways, stimulate the production of ECM proteins, inhibit ECM degradation and prevent apoptosis, thus strengthening the amnion. Thus control of the inflammatory signature and the integrity of the fetal membranes prior to parturition may partly depend on the sexually determined activity of the decidual and amniotic renin-angiotensin system pathways.
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Nacimiento Prematuro/epidemiología , Nacimiento Prematuro/metabolismo , Sistema Renina-Angiotensina/fisiología , Caracteres Sexuales , Femenino , Humanos , Recién Nacido , Masculino , Embarazo , PrevalenciaRESUMEN
Metabolic inactivation of progesterone within uterine myocytes by 20α-hydroxysteroid dehydrogenase (20α-HSD) has been postulated as a mechanism contributing to functional progesterone withdrawal at term. In humans, 20α-HSD is encoded by the gene AKR1C1. Myometrial AKR1C1 mRNA abundance has been reported to increase significantly during labor at term. In spontaneous preterm labor, however, we previously found no increase in AKR1C1 mRNA level in the myometrium except for preterm labor associated with clinical chorioamnionitis. This suggests that increased 20α-HSD activity is a mechanism through which inflammation drives progesterone withdrawal in preterm labor. In this study, we have determined the effects of various treatments of therapeutic relevance on AKR1C1 expression in pregnant human myometrium in an ex vivo culture system. AKR1C1 expression increased spontaneously during 48 h culture (p < 0.0001), consistent with the myometrium transitioning to a labor-like phenotype ex vivo, as reported previously. Serum supplementation, prostaglandin F2α, phorbol myristate acetate, and mechanical stretch had no effect on the culture-induced increase, whereas progesterone (p = 0.0058) and cAMP (p = 0.0202) further upregulated AKR1C1 expression. In contrast, culture-induced upregulation of AKR1C1 expression was dose-dependently repressed by three histone/protein deacetylase inhibitors: trichostatin A at 5 (p = 0.0172) and 25 µM (p = 0.0115); suberoylanilide hydroxamic acid at 0.5 (p = 0.0070), 1 (p = 0.0045), 2.5 (p = 0.0181), 5 (p = 0.0066) and 25 µM (p = 0.0014); and suberoyl bis-hydroxamic acid at 5 (p = 0.0480) and 25 µM (p = 0.0238). We propose the inhibition of histone/protein deacetylation helps to maintain the anti-inflammatory, pro-quiescence signaling of progesterone in pregnant human myometrium by blocking its metabolic inactivation. Histone deacetylase inhibitors may represent a class of agents that preserve or restore the progesterone sensitivity of the pregnant uterus.
Asunto(s)
Trabajo de Parto Prematuro , Progesterona , Femenino , Humanos , Recién Nacido , Embarazo , Histonas/metabolismo , Hidroxiesteroide Deshidrogenasas/genética , Hidroxiesteroide Deshidrogenasas/metabolismo , Miometrio/metabolismo , Trabajo de Parto Prematuro/metabolismo , Progesterona/metabolismo , ARN Mensajero/metabolismoRESUMEN
The decidua undergoes proinflammatory activation in late pregnancy, promoting labor. Bromodomain and Extra-Terminal (BET) family proteins interact with acetylated histones and may control gene expression in inflammation. Here, we assessed whether BETs are involved in inflammatory gene regulation in human decidual cells. We have treated primary cultures of decidual stromal cells (DSCs) from term pregnancies with endotoxin (LPS) and measured the expression of a panel of pro-and anti-inflammatory genes. BET involvement was assessed using the selective BET inhibitors (+)-JQ1 and I-BET-762 or the negative control compound (-)-JQ1. Histone 3 and -4 acetylation and BETs binding at the target gene promoters were determined to assess whether these processes are involved in the actions of LPS, BETs, and BET inhibitors. LPS increased the expression of the proinflammatory (PTGS2, IL6, CXCL8/IL8, TNF) and the anti-inflammatory (IL10, IDO1) genes of the panel. The constitutively expressed inflammatory genes (PTGS1, PTGES) were unaffected. The BET inhibitors, but not the control compound, reduced the basal and LPS-induced expression of PTGS1, PTGS2, IL6, CXCL8/IL8, IL10, and IDO1. TNF expression was not changed by BET inhibition. The dominant BETs were Bromodomain-containing protein -2 (BRD2) and -4L (BRD4L) in DSCs. LPS increased histone 4 acetylation at the CXCL8/IL8 and TNF promoters and histone 3 and -4 acetylation at the IDO1 promoter, while (+)-JQ1 abrogated histone acetylation at several promoters. Overall, histone acetylation and promoter binding of BETs showed no consistent relationship with gene expression across the gene panel and the treatments. BET proteins, predominantly BRD2 and BRD4L, control critical pro- and anti-inflammatory genes in DSCs. TNF induction exemplifies a BET-independent pathway. Changing histone acetylation at the promoters is not a general obligatory requirement for inflammatory gene expression in response to LPS. BETs likely act at chromatin loci separate from the examined promoters. BET inhibitors may block decidual activation at labor.
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Histonas , Interleucina-8 , Femenino , Humanos , Embarazo , Histonas/metabolismo , Interleucina-8/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos , Ciclooxigenasa 2/metabolismo , Interleucina-10/metabolismo , Factores de Transcripción/genética , Antiinflamatorios , Azepinas/farmacologíaRESUMEN
The mechanism by which human labor is initiated in the presence of elevated circulating progesterone levels remains unknown. Recent evidence indicates that the progesterone-metabolizing enzyme, 20α-hydroxysteroid dehydrogenase (20α-HSD), encoded by the gene AKR1C1, may contribute to functional progesterone withdrawal. We found that AKR1C1 expression significantly increased with labor onset in term myometrium, but not in preterm myometrium. Among preterm laboring deliveries, clinically diagnosed chorioamnionitis was associated with significantly elevated AKR1C1 expression. AKR1C1 expression positively correlated with BMI before labor and negatively correlated with BMI during labor. Analysis by fetal sex showed that AKR1C1 expression was significantly higher in women who delivered male babies compared to women who delivered female babies at term, but not preterm. Further, in pregnancies where the fetus was female, AKR1C1 expression positively correlated with the mother's age and BMI at the time of delivery. In conclusion, the increase in myometrial AKR1C1 expression with term labor is consistent with 20α-HSD playing a role in local progesterone metabolism to promote birth. Interestingly, this role appears to be specific to term pregnancies where the fetus is male. Upregulated AKR1C1 expression in the myometrium at preterm in-labor with clinical chorioamnionitis suggests that increased 20α-HSD activity is a mechanism through which inflammation drives progesterone withdrawal in preterm labor. The link between AKR1C1 expression and maternal BMI may provide insight into why maternal obesity is often associated with dysfunctional labor. Higher myometrial AKR1C1 expression in male pregnancies may indicate fetal sex-related differences in the mechanisms that precipitate labor onset at term.
Asunto(s)
Corioamnionitis , Trabajo de Parto Prematuro , Nacimiento Prematuro , Recién Nacido , Humanos , Femenino , Masculino , Embarazo , Progesterona/metabolismo , Miometrio/metabolismo , Índice de Masa Corporal , Nacimiento Prematuro/metabolismo , Corioamnionitis/metabolismo , Trabajo de Parto Prematuro/metabolismo , Hidroxiesteroide Deshidrogenasas/genética , Hidroxiesteroide Deshidrogenasas/metabolismoRESUMEN
The pregnant uterus remains relaxed throughout fetal gestation before transforming to a contractile phenotype at term to facilitate birth. Despite ongoing progress, the precise mechanisms that regulate this phenotypic transformation are not yet understood. This knowledge gap limits our understanding of how dysregulation of uterine smooth muscle biology contributes to life-threatening obstetric complications, including preterm birth, and hampers our ability to develop effective therapeutic intervention strategies. Protein acetylation plays a vital role in regulating protein structure, function, and subcellular localization, as well as gene transcription availability through regulating chromatin condensation. Histone deacetylase inhibitors (HDACis) are a class of compounds that block the removal of acetyl functional groups from proteins and, as such, have profound effects on important cellular events, including phenotypic transformation. A large body of data now demonstrates that HDACis have profound effects on pregnant human myometrium. Studies to date show that HDACis operate through both genomic and non-genomic mechanisms to affect myometrial function and phenotype. Interestingly, the effects of HDACis on pregnant myometrium are largely "pro-relaxation," including the direct inhibition of contractile machinery as well as repression of pro-labor genes. The "dual action" effects of HDACis make them a powerful tool for unlocking the regulatory processes that underpin myometrial phenotypic transformation and raises prospects of their therapeutic applications. Here, we review the new insights into human myometrial biology that have garnered through the application of HDACis and explore their potential therapeutic application toward the development of novel preterm birth prevention strategies.
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Trabajo de Parto , Nacimiento Prematuro , Recién Nacido , Embarazo , Femenino , Humanos , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Nacimiento Prematuro/tratamiento farmacológico , Nacimiento Prematuro/prevención & control , Nacimiento Prematuro/metabolismo , Miometrio/metabolismo , Trabajo de Parto/fisiología , ÚteroRESUMEN
The characteristics of fetal membrane cells and their phenotypic adaptations to support pregnancy or promote parturition are defined by global patterns of gene expression controlled by chromatin structure. Heritable epigenetic chromatin modifications that include DNA methylation and covalent histone modifications establish chromatin regions permissive or exclusive of regulatory interactions defining the cell-specific scope and potential of gene activity. Non-coding RNAs acting at the transcriptional and post-transcriptional levels complement the system by robustly stabilizing gene expression patterns and contributing to ordered phenotype transitions. Here we review currently available information about epigenetic gene regulation in the amnion and the chorion laeve. In addition, we provide an overview of epigenetic phenomena in the decidua, which is the maternal tissue fused to the chorion membrane forming the anatomical and functional unit called choriodecidua. The relationship of gene expression with DNA (CpG) methylation, histone acetylation and methylation, micro RNAs, long non-coding RNAs and chromatin accessibility is discussed in the context of normal pregnancy, parturition and pregnancy complications. Data generated using clinical samples and cell culture models strongly suggests that epigenetic events are associated with the phenotypic transitions of fetal membrane cells during the establishment, maintenance and termination of pregnancy potentially driving and consolidating the changes as pregnancy progresses. Disease conditions and environmental factors may produce epigenetic footprints that indicate exposures and mediate adverse pregnancy outcomes. Although knowledge is expanding rapidly, fetal membrane epigenetics is still in an early stage of development necessitating further research to realize its remarkable basic and translational potential.
RESUMEN
Successful pregnancy necessitates that the uterus is maintained in a relaxed, quiescent state for the majority of pregnancy, before being transformed to a contractile and excitable phenotype to facilitate parturition. There is now a substantial body of evidence highlighting key upstream regulators involved in this transformation. Despite our rapidly advancing knowledge of myometrial biology, the exact mechanisms that regulate parturition are not yet understood. Further work is necessary to define the complex interactions that form the key regulatory pathways controlling uterine quiescence, contractility, and the transition between the two states. Furthermore, new evidence continues to emerge implicating novel mechanisms that regulate uterine activity during normal and preterm birth. This review examines current evidence pertaining to key upstream regulators that have been implicated in human parturition over the past decades and surveys recent findings that are yet to be integrated into the paradigm of uterine regulation.
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Parto/fisiología , Nacimiento Prematuro/fisiopatología , Útero/fisiología , Animales , Parto Obstétrico , Femenino , Humanos , Contracción Uterina/fisiologíaRESUMEN
Extensive evidence now shows that adversity during the perinatal period is a significant risk factor for the development of neurodevelopmental disorders long after the causative event. Despite stemming from a variety of causes, perinatal compromise appears to have similar effects on the developing brain, thereby resulting in behavioural disorders of a similar nature. These behavioural disorders occur in a sex-dependent manner, with males affected more by externalising behaviours such as attention deficit hyperactivity disorder (ADHD) and females by internalising behaviours such as anxiety. Regardless of the causative event or the sex of the offspring, these disorders may begin in childhood or adolescence but extend into adulthood. A mechanism by which adverse events in the perinatal period impact later in life behaviour has been shown to be the changing epigenetic landscape. Methylation of the GAD1/GAD67 gene, which encodes the key glutamate-to-GABA-synthesising enzyme glutamate decarboxylase 1, resulting in increased levels of glutamate, is one epigenetic mechanism that may account for a tendency towards excitation in disorders such as ADHD. Exposure of the fetus or the neonate to high levels of cortisol may be the mediator between perinatal compromise and poor behavioural outcomes because evidence suggests that increased glucocorticoid exposure triggers widespread changes in the epigenetic landscape. This review summarises the current evidence and recent literature about the impact of various perinatal insults on the epigenome and the common mechanisms that may explain the similarity of behavioural outcomes occurring following diverse perinatal compromise.
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Epigénesis Genética , Desarrollo Fetal/genética , Glutamato Descarboxilasa/genética , Estrés Psicológico/genética , Animales , Femenino , Humanos , Hidrocortisona/sangre , Recién Nacido , Embarazo , Efectos Tardíos de la Exposición Prenatal/genéticaAsunto(s)
Progesterona/metabolismo , Contracción Uterina/metabolismo , Animales , Conexina 43/genética , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , MicroARNs/metabolismo , Miometrio/metabolismo , Embarazo , Receptores de Oxitocina/genética , Receptores de Progesterona/metabolismo , Proteínas Represoras/metabolismo , Regulación hacia Arriba , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
CONTEXT: Differential promoter use and alternative splicing generate a variety of glucocorticoid receptor (GR) mRNA transcripts, potentially altering the cortisol responsiveness of gestational tissues during pregnancy and labor. OBJECTIVE: We examined GR mRNA transcript expression in term placentae before and after labor, in association with fetal sex and after glucocorticoid treatment. DESIGN: RNA from 34 placentae and from eight placental explants incubated with glucocorticoids were analyzed for the GR mRNA variants GR-alpha, GR-beta, GR-P, and GR-gamma and the untranslated exon one variants 1A1, 1A2, 1A3, 1B, and 1C by quantitative RT-PCR. MAIN OUTCOME MEASURE: mRNA expression was assessed. RESULTS: All GR mRNA variants examined were detected in the human placenta, with GR-alpha and GR-1C mRNA having the highest expression of GR splice variants and exon 1 variants, respectively. GR-P mRNA abundance decreased with spontaneous labor (P < 0.01). GR-1A3 mRNA abundance changed with fetal sex, with a higher level in placentae of male fetuses (P < 0.05). GR-1C was the preferential promoter for GR-alpha, GR-gamma, and GR-P mRNA. GR-beta mRNA was preferentially associated with GR-1A1. GR-P mRNA transcription switched to the GR-1A1 promoter after labor and to the GR-1A3 promoter in placentae from male fetuses. Glucocorticoid treatment significantly reduced transcription from promoters GR-1B and -1C and decreased GR-alpha and GR-P mRNA abundance. CONCLUSIONS: The human placenta expresses a variety of GR mRNA transcripts. GR-alpha mRNA transcribed from the 1C promoter generates the majority of placental GR. However, alterations in promoter use and alternative splicing may modulate responses to cortisol during stressful events.
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Placenta/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Glucocorticoides/biosíntesis , Receptores de Glucocorticoides/genética , Adulto , Cartilla de ADN , Interpretación Estadística de Datos , Exones/genética , Femenino , Variación Genética , Glucocorticoides/farmacología , Humanos , Recién Nacido , Trabajo de Parto/metabolismo , Masculino , Embarazo , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Caracteres Sexuales , Adulto JovenRESUMEN
The guinea pig (Cavia porcellus) displays many features of gestational physiology that makes it the most translationally relevant rodent species. Progesterone production undergoes a luteal to placental shift as in human pregnancy with levels rising during gestation and with labor and delivery occurring without a precipitous decline in maternal progesterone levels. In contrast to other laboratory rodents, labor in guinea pigs is triggered by a functional progesterone withdrawal, which involves the loss of uterine sensitivity to progesterone like in women. In both species the amnion membrane is a major source of labor-inducing prostaglandins, which promote functional progesterone withdrawal by modifying myometrial progesterone receptor expression. These similar features appear to result from convergent evolution rather than closer evolutionally relationship to primates compared to other rodents. Nevertheless, the similarities in the production, metabolism and actions of progesterone and prostaglandins allow information gained in pregnant guinea pigs to be extended to pregnant women with confidence. This includes exploring the effects of pregnancy complications including growth restriction and the mechanisms by which stressful conditions increase the incidence of preterm labor. The relatively long gestation of the guinea pig and the maturity of the pups at birth particularly in brain development means that a greater proportion of brain development happens in utero. This allows adverse intrauterine conditions to make a sustained impact on the developing brain like in compromised human pregnancies. In addition, the brain is exposed to a protective neurosteroid environment in utero, which has been suggested to promote development in the guinea pig and the human. Moreover, in utero stresses that have been shown to adversely affect long term neurobehavioral outcomes in clinical studies, can be modeled successfully in guinea pigs. Overall, these parallels to the human have led to increasing interest in the guinea pig for translational studies of treatments and therapies that potentially improve outcomes following adverse events in pregnancy and after preterm birth.
RESUMEN
Whereas the essential role of progesterone in the maintenance of pregnancy is accepted generally, the mechanisms that suppress progesterone's function near term to allow labor and delivery of the conceptus are still shrouded in uncertainty. In most subprimate placental mammals, the withdrawal of progesterone before the initiation of labor is manifest by a significant drop in circulating progesterone levels, which is due to either luteolysis or changes in placental steroidogenesis, which shunts precursors towards estrogen production. No such events can be demonstrated in human pregnancy. In this review, we shall present a brief historic background of the research that led to the concepts of "progesterone block" and its withdrawal, based on experiments with rabbits and laboratory rodents, and discuss some of the more recent ideas about "functional progesterone withdrawal," in an attempt to bridge the apparent differences between the regulation of parturition in human and subprimate mammals.
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Mifepristona/farmacología , Parto/efectos de los fármacos , Preñez , Progesterona/metabolismo , Animales , Biomarcadores/metabolismo , Femenino , Miometrio/efectos de los fármacos , Miometrio/metabolismo , Parto/fisiología , Embarazo , Progesterona/antagonistas & inhibidores , Conejos , Ratas , Receptores de Progesterona/metabolismo , Sensibilidad y Especificidad , OvinosRESUMEN
The primary prostaglandins PGE(2) and PGF(2 alpha) are metabolized in tissues by a series of enzymatic and non-enzymatic reactions. To measure metabolic rates and individual reaction rates it is necessary to extract the parent prostaglandins and metabolites before the separation and quantification of each compound is achieved. Here we have established and optimized a solid phase extraction (SPE) procedure to recover PGE(2), PGF(2 alpha) and their six enzymatic and non-enzymatic tissue metabolites from aqueous solutions including urine, plasma and tissue homogenate. We have used octadecyl-bonded silica gel as the stationary phase and methanol-water mixtures as binary mobile phases. The volumes and concentrations of the washing and elution solutions were optimized individually for each PG. Recoveries of all PG standards were quantitative except for PGEM, which was recovered at 80% efficiency. Biological matrix components interfered with the extraction in a PG- and matrix-specific fashion. Inclusion of 1% formic acid in the loading mixture raised recoveries from urine, plasma and tissue homogenate to >or=90%. This SPE method is the first that has been optimized by systematic elution studies for PGE(2), PGF(2 alpha) and the complement of their tissue metabolites. The procedure is simple, robust and can serve as an effective pre-purification step before downstream separation and quantification of each tissue metabolite of PGE(2) and PGF(2 alpha) from complex biological matrices.
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Dinoprost/aislamiento & purificación , Dinoprostona/aislamiento & purificación , Extracción en Fase Sólida/métodos , Cromatografía en Capa Delgada , Dinoprost/química , Dinoprost/metabolismo , Dinoprostona/química , Dinoprostona/metabolismo , Humanos , Estructura Molecular , Reproducibilidad de los ResultadosRESUMEN
Background. Regulation of myometrial progesterone receptor (PR) expression is an unresolved issue central to understanding the mechanism of functional progesterone withdrawal and initiation of labor in women. Objectives. To determine whether pregnant human myometrium undergoes culture-induced changes in PR isoform expression ex situ and, further, to determine if conditions approaching the in vivo environment stabilise PR isoform expression in culture. Methods. Term nonlaboring human myometrial tissues were cultured under specific conditions: serum supplementation, steroids, stretch, cAMP, PMA, PGF2α , NF-κB inhibitors, or TSA. Following 48 h culture, PR-T, PR-A, and PR-B mRNA levels were determined using qRT-PCR. PR-A/PR-B ratios were calculated. Results. PR-T and PR-A expression and the PR-A/PR-B ratio significantly increased in culture. Steroids prevented the culture-induced increase in PR-T and PR-A expression. Stretch blocked the effects of steroids on PR-T and PR-A expression. PMA further increased the PR-A/PR-B ratio, while TSA blocked culture-induced increases of PR-A expression and the PR-A/PR-B ratio. Conclusion. Human myometrial tissue in culture undergoes changes in PR gene expression consistent with transition toward a laboring phenotype. TSA maintained the nonlaboring PR isoform expression pattern. This suggests that preserving histone and/or nonhistone protein acetylation is critical for maintaining the progesterone dependent quiescent phenotype of human myometrium in culture.
Asunto(s)
Progesterona/genética , Isoformas de Proteínas/genética , Receptores de Progesterona/genética , Acilación , Adulto , Femenino , Regulación de la Expresión Génica/genética , Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/administración & dosificación , Trabajo de Parto , Miometrio , Embarazo , Progesterona/metabolismo , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , Receptores de Progesterona/metabolismo , Nacimiento a TérminoRESUMEN
BACKGROUND: Ex situ analyses of human myometrial tissue has been used to investigate the regulation of uterine quiescence and transition to a contractile phenotype. Following concerns about the validity of cultured primary cells, we examined whether myometrial tissue undergoes culture-induced changes ex situ that may affect the validity of in vitro models. OBJECTIVES: To determine whether human myometrial tissue undergoes culture-induced changes ex situ in Estrogen receptor 1 (ESR1), Prostaglandin-endoperoxide synthase 2 (PTGS2) and Oxytocin receptor (OXTR) expression. Additionally, to determine whether culture conditions approaching the in vivo environment influence the expression of these key genes. METHODS: Term non-laboring human myometrial tissues were cultured in the presence of specific treatments, including; serum supplementation, progesterone and estrogen, cAMP, PMA, stretch or NF-κB inhibitors. ESR1, PTGS2 and OXTR mRNA abundance after 48â h culture was determined using quantitative RT-PCR. RESULTS: Myometrial tissue in culture exhibited culture-induced up-regulation of ESR1 and PTGS2 and down-regulation of OXTR mRNA expression. Progesterone prevented culture-induced increase in ESR1 expression. Estrogen further up-regulated PTGS2 expression. Stretch had no direct effect, but blocked the effects of progesterone and estrogen on ESR1 and PTGS2 expression. cAMP had no effect whereas PMA further up-regulated PTGS2 expression and prevented decline of OXTR expression. CONCLUSION: Human myometrial tissue in culture undergoes culture-induced gene expression changes consistent with transition toward a laboring phenotype. Changes in ESR1, PTGS2 and OXTR expression could not be controlled simultaneously. Until optimal culture conditions are determined, results of in vitro experiments with myometrial tissues should be interpreted with caution.
Asunto(s)
Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Expresión Génica/genética , Músculo Liso/fisiología , Receptores de Oxitocina/genética , Receptores de Oxitocina/metabolismo , Contracción Uterina/genética , Útero , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Estrógenos/farmacología , Femenino , Humanos , Embarazo , Progesterona/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Placental CRH production increases with advancing pregnancy in women and its course predicts gestational length. We hypothesized that CRH gene expression in the placenta is epigenetically controlled setting gestational trajectories characteristic of normal and pathological pregnancies. Here we determined histone modification and DNA methylation levels and DNA methylation patterns at the CRH promoter in primary trophoblast cultures by chromatin immunoprecipitation combined with clonal bisulfite sequencing and identified the transcriptionally active epialleles that associate with particular histone modifications and transcription factors during syncytialisation and cAMP-stimulation. CRH gene expression increased during syncytial differentiation and cAMP stimulation, which was associated with increased activating and decreased repressive histone modification levels at the promoter. DNA methylation levels remained unchanged. The nine CpGs of the CRH proximal promoter were partially and allele-independently methylated displaying many (>100) epialleles. RNA-polymerase-II (Pol-II) bound only to three particular epialleles in cAMP-stimulated cells, while phospho-cAMP response element-binding protein (pCREB) bound to only one epiallele, which was different from those selected by Pol-II. Binding of TATA-binding protein increased during syncytial differentiation preferentially at epialleles compatible with Pol-II and pCREB binding. Histone-3 acetylation was detected only at epialleles targeted by Pol-II and pCREB, while gene activating histone-4 acetylation and histone-3-lysine-4 trimethylation occurred at CRH epialleles not associated with Pol-II or pCREB. The suppressive histone-3-lysine-27 trimethyl and-lysine-9 trimethyl modifications showed little or no epiallele preference. The epiallele selectivity of activating histone modifications and transcription factor binding demonstrates the epigenetic and functional diversity of the CRH gene in trophoblasts, which is controlled predominantly by the patterns, not the overall extent, of promoter methylation. We propose that conditions impacting on epiallele distribution influence the number of transcriptionally active CRH gene copies in the trophoblast cell population determining the gestational trajectory of placental CRH production in normal and pathological pregnancies.