Regulation of 20α-Hydroxysteroid Dehydrogenase Expression in Term Pregnant Human Myometrium Ex Vivo.

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.

Clopidogrel Administration Impairs Post-Stroke Learning and Memory Recovery in Mice.

Clopidogrel, which is one of the most prescribed antiplatelet medications in the world, is given to stroke survivors for the prevention of secondary cardiovascular events. Clopidogrel exerts its antiplatelet activity via antagonism of the P2Y12 receptor (P2RY12). Although not widely known or considered during the initial clinical trials for clopidogrel, P2RY12 is also expressed on microglia, which are the brain's immune cells, where the receptor facilitates chemotactic migration toward sites of cellular damage. If microglial P2RY12 is blocked, microglia lose the ability to migrate to damaged sites and carry out essential repair processes. We aimed to investigate whether administering clopidogrel to mice post-stroke was associated with (i) impaired motor skills and cognitive recovery; (ii) physiological changes, such as survival rate and body weight; (iii) changes in the neurovascular unit, including blood vessels, microglia, and neurons; and (iv) changes in immune cells. Photothrombotic stroke (or sham surgery) was induced in adult male mice. From 24 h post-stroke, mice were treated daily for 14 days with either clopidogrel or a control. Cognitive performance (memory and learning) was assessed using a mouse touchscreen platform (paired associated learning task), while motor impairment was assessed using the cylinder task for paw asymmetry. On day 15, the mice were euthanized and their brains were collected for immunohistochemistry analysis. Clopidogrel administration significantly impaired learning and memory recovery, reduced mouse survival rates, and reduced body weight post-stroke. Furthermore, clopidogrel significantly increased vascular leakage, significantly increased the number and appearance of microglia, and significantly reduced the number of T cells within the peri-infarct region post-stroke. These data suggest that clopidogrel hampers cognitive performance post-stroke. This effect is potentially mediated by an increase in vascular permeability post-stroke, providing a pathway for clopidogrel to access the central nervous system, and thus, interfere in repair and recovery processes.

20α-Hydroxysteroid Dehydrogenase Expression in the Human Myometrium at Term and Preterm Birth: Relationships to Fetal Sex and Maternal Body Mass Index.

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.

Assessing the Potency of the Novel Tocolytics 2-APB, Glycyl-H-1152, and HC-067047 in Pregnant Human Myometrium.

The intracellular signaling pathways that regulate myometrial contractions can be targeted by drugs for tocolysis. The agents, 2-APB, glycyl-H-1152, and HC-067047, have been identified as inhibitors of uterine contractility and may have tocolytic potential. However, the contraction-blocking potency of these novel tocolytics was yet to be comprehensively assessed and compared to agents that have seen greater scrutiny, such as the phosphodiesterase inhibitors, aminophylline and rolipram, or the clinically used tocolytics, nifedipine and indomethacin. We determined the IC50 concentrations (inhibit 50% of baseline contractility) for 2-APB, glycyl-H-1152, HC-067047, aminophylline, rolipram, nifedipine, and indomethacin against spontaneous ex vivo contractions in pregnant human myometrium, and then compared their tocolytic potency. Myometrial strips obtained from term, not-in-labor women, were treated with cumulative concentrations of the contraction-blocking agents. Comprehensive dose-response curves were generated. The IC50 concentrations were 53 µM for 2-APB, 18.2 µM for glycyl-H-1152, 48 µM for HC-067047, 318.5 µM for aminophylline, 4.3 µM for rolipram, 10 nM for nifedipine, and 59.5 µM for indomethacin. A single treatment with each drug at the determined IC50 concentration was confirmed to reduce contraction performance (AUC) by approximately 50%. Of the three novel tocolytics examined, glycyl-H-1152 was the most potent inhibitor. However, of all the drugs examined, the overall order of contraction-blocking potency in decreasing order was nifedipine > rolipram > glycyl-H-1152 > HC-067047 > 2-APB > indomethacin > aminophylline. These data provide greater insight into the contraction-blocking properties of some novel tocolytics, with glycyl-H-1152, in particular, emerging as a potential novel tocolytic for preventing preterm birth.

Preterm labor with and without chorioamnionitis is associated with activation of myometrial inflammatory networks: a comprehensive transcriptomic analysis.

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.

Histone Deacetylase Inhibitors: Providing New Insights and Therapeutic Avenues for Unlocking Human Birth.

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.

Preterm labor is a distinct process from term labor following computational analysis of human myometrium.

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.

Landscape of Preterm Birth Therapeutics and a Path Forward.

Preterm birth (PTB) remains the leading cause of infant morbidity and mortality. Despite 50 years of research, therapeutic options are limited and many lack clear efficacy. Tocolytic agents are drugs that briefly delay PTB, typically to allow antenatal corticosteroid administration for accelerating fetal lung maturity or to transfer patients to high-level care facilities. Globally, there is an unmet need for better tocolytic agents, particularly in low- and middle-income countries. Although most tocolytics, such as betamimetics and indomethacin, suppress downstream mediators of the parturition pathway, newer therapeutics are being designed to selectively target inflammatory checkpoints with the goal of providing broader and more effective tocolysis. However, the relatively small market for new PTB therapeutics and formidable regulatory hurdles have led to minimal pharmaceutical interest and a stagnant drug pipeline. In this review, we present the current landscape of PTB therapeutics, assessing the history of drug development, mechanisms of action, adverse effects, and the updated literature on drug efficacy. We also review the regulatory hurdles and other obstacles impairing novel tocolytic development. Ultimately, we present possible steps to expedite drug development and meet the growing need for effective preterm birth therapeutics.

Fetal Membrane Epigenetics.

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.

Innervation of papillary thyroid cancer and its association with extra-thyroidal invasion.

Nerves are emerging regulators of cancer progression and in several malignancies innervation of the tumour microenvironment is associated with tumour aggressiveness. However, the innervation of thyroid cancer is unclear. Here, we investigated the presence of nerves in thyroid cancers and the potential associations with clinicopathological parameters. Nerves were detected by immunohistochemistry using the pan-neuronal marker PGP9.5 in whole-slide sections of papillary thyroid cancer (PTC) (n = 75), compared to follicular thyroid cancer (FTC) (n = 13), and benign thyroid tissues (n = 26). Nerves were detected in most normal thyroid tissues and thyroid cancers, but nerve density was increased in PTC (12 nerves/cm2 [IQR 7-21]) compared to benign thyroid (6 nerves/cm2 [IQR: 3-10]) (p = 0.001). In contrast, no increase in nerve density was observed in FTC. In multivariate analysis, nerve density correlated positively with extrathyroidal invasion (p < 0.001), and inversely with tumour size (p < 0.001). The majority of nerves were adrenergic, although cholinergic and peptidergic innervation was detected. Perineural invasion was present in 35% of PTC, and was independently associated with extrathyroidal invasion (p = 0.008). This is the first report of infiltration of nerves into the tumour microenvironment of thyroid cancer and its association with tumour aggressiveness. The role of nerves in thyroid cancer pathogenesis should be further investigated.

The Regulation of Uterine Function During Parturition: an Update and Recent Advances.

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.

A comparison of uterine contractile responsiveness to arginine vasopressin in oviparous and viviparous lizards.

Nonapeptides and their receptors regulate a diverse range of physiological processes. We assessed the contractile responsiveness of uteri from the squamate viviparous-oviparous species pair, Pseudemoia entrecasteauxii and Lampropholis guichenoti, as well as the bimodally reproductive species, Saiphos equalis, to arginine vasopressin (AVP). We assessed the resulting uterine contractility as a function of pregnancy status, species and parity mode. We also measured mRNA abundance for the nonapeptide receptor, oxytocin receptor (oxtr), in uteri from P. entrecasteauxii and L. guichenoti and compared expression across pregnancy status and parity mode. We found that pregnant uteri exhibited a significantly greater contractile response to AVP than non-pregnant uteri in all three lizard species studied. Cross-species comparisons revealed that uteri from viviparous P. entrecasteauxii were significantly more responsive to AVP than uteri from oviparous L. guichenoti during both pregnant and non-pregnant states. Conversely, for non-pregnant S. equalis, uteri from viviparous individuals were significantly less responsive to AVP than uteri from oviparous individuals, while during pregnancy, there was no difference in AVP contractile responsiveness. There was no difference in expression of oxtr between L. guichenoti and P. entrecasteauxii, or between pregnant and non-pregnant individuals within each species. We found no significant correlation between oxtr expression and AVP contractile responsiveness. These findings indicate that there are differences in nonapeptide signalling across parity mode and suggest that in these lizards, labour may be triggered either by an increase in plasma nonapeptide concentration, or by an increase in expression of a different nonapeptide receptor from the vasopressin-like receptor family.

Sharpey-Schafer Lecture 2019: From retroviruses to human birth.

NEW FINDINGS: What is the topic of this review? The timing of birth is an important determinant of future health and well-being. This review examines the role of endogenous retroviruses as upstream regulators of key biological functions of the placenta, including cell-cell fusion, modulation of the maternal immune system, and the production of key pregnancy hormones. What advances does it highlight? Endogenous retroviruses are an obligate requirement for successful human reproduction. The products of retroviral elements, incorporated into the germline millions of years ago, have been co-opted to serve vital biological roles within the placenta that ultimately dictate the length of human pregnancy and therefore well-being trajectories. ABSTRACT: Gestational length at the time of birth is an important determinant of future health and well-being, yet the physiological regulation of the onset of labour in humans remains obscure. The evolution of egg formation and internal fertilisation in amniotes required a mechanism to suppress the contractile activity of the oviduct that is provided by progesterone. Delivery of the egg is then associated with the withdrawal of progesterone and a return of contractile activity to the reproductive tract. In mammals, the process of pregnancy is complicated further by the need to protect the fetus from potential attack by the maternal immune system. There is increasing evidence that retroviruses incorporated into the mammalian germline in the evolutionary past play a key role in suppressing the maternal immune reaction to the developing conceptus, organising the development of the placenta and perhaps, in humans, modulating the action of progesterone, determining gestational length and the onset of labour. It seems that the presence of an endogenous retrovirus is an obligate requirement for human reproduction.

The precursor for nerve growth factor (proNGF) is not a serum or biopsy-rinse biomarker for thyroid cancer diagnosis.

BACKGROUND: Nerves and neurotrophic growth factors are emerging promoters of cancer growth. The precursor for Nerve Growth Factor (proNGF) is overexpressed in thyroid cancer, but its potential role as a clinical biomarker has not been reported. Here we have examined the value of proNGF as a serum and biopsy-rinse biomarker for thyroid cancer diagnosis. METHODS: Patients presenting for thyroid surgery or biopsy were enrolled in separate cohorts examining serum (n = 204, including 46 cases of thyroid cancer) and biopsy-rinse specimens (n = 188, including 26 cases of thyroid cancer). ProNGF levels in clinical samples were analysed by ELISA. Univariate and multivariate statistical analyses were used to compare proNGF levels with malignancy status and clinicopathological parameters. RESULTS: ProNGF was not detected in the majority of serum samples (176/204, 86%) and the detection of proNGF was not associated with thyroid cancer diagnosis. In the few cases where proNGF was detected in the serum, thyroidectomy did not affect proNGF concentration, demonstrating that the thyroid was not the source of serum proNGF. Intriguingly, an association between hyperthyroidism and serum proNGF was observed (OR 3.3, 95% CI 1.6-8.7 p = 0.02). In biopsy-rinse, proNGF was detected in 73/188 (39%) cases, with no association between proNGF and thyroid cancer. However, a significant positive association between follicular lesions and biopsy-rinse proNGF was found (OR 3.3, 95% CI 1.2-8.7, p = 0.02). CONCLUSIONS: ProNGF levels in serum and biopsy-rinse are not increased in thyroid cancer and therefore proNGF is not a clinical biomarker for this condition.

The Precursor for Nerve Growth Factor (proNGF) in Thyroid Cancer Lymph Node Metastases: Correlation with Primary Tumour and Pathological Variables.

Metastases in thyroid cancer are associated with aggressive disease and increased patient morbidity, but the factors driving metastatic progression are unclear. The precursor for nerve growth factor (proNGF) is increased in primary thyroid cancers, but its expression or significance in metastases is not known. In this study, we analysed the expression of proNGF in a retrospective cohort of thyroid cancer lymph node metastases (n = 56), linked with corresponding primary tumours, by automated immunohistochemistry and digital quantification. Potential associations of proNGF immunostaining with clinical and pathological parameters were investigated. ProNGF staining intensity (defined by the median h-score) was significantly higher in lymph node metastases (h-score 94, interquartile range (IQR) 50-147) than in corresponding primary tumours (57, IQR 42-84) (p = 0.002). There was a correlation between proNGF expression in primary tumours and corresponding metastases, where there was a 0.68 (95% CI 0 to 1.2) increase in metastatic tumour h-score for each unit increase in the primary tumour h-score. However, larger tumours (both primary and metastatic) had lower proNGF expression. In a multivariate model, proNGF expression in nodal metastases was negatively correlated with lateral neck disease and being male. In conclusion, ProNGF is expressed in locoregional metastases of thyroid cancer and is higher in lymph node metastases than in primary tumours, but is not associated with high-risk clinical features.

Epigenetic regulation of progesterone receptors and the onset of labour.

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.

Misleading Westerns: Common Quantification Mistakes in Western Blot Densitometry and Proposed Corrective Measures.

Densitometry data generated for Western blots are commonly used to compare protein abundance between samples. In the last decade, it has become apparent that assumptions underpinning these comparisons are often violated in studies reporting Western blot data in the literature. These violations can lead to erroneous interpretations of data and may contribute to poor reproducibility of research. We assessed the reliability of Western blot data obtained to study human myometrial tissue proteins. We ran dilution series of protein lysates to explore the linearity of densitometry data. Proteins analysed included αSMA, HSP27, ERK1/2, and GAPDH. While ideal densitometry data are directly proportional to protein abundance, our data confirm that densitometry data often deviate from this ideal, in which case they can fit nonproportional linear or hyperbolic mathematical models and can reach saturation. Nonlinear densitometry data were observed when Western blots were detected using infrared fluorescence or chemiluminescence, and under different SDS-PAGE conditions. We confirm that ghosting artefacts associated with overabundance of proteins of interest in Western blots can skew findings. We also confirm that when data to be normalised are not directly proportional to protein abundance, it is a mistake to use the normalisation technique of dividing densitometry data from the protein-of-interest with densitometry data from loading control protein(s), as this can cause the normalised data to be unusable for making comparisons. Using spiked proteins in a way that allowed us to control the total protein amount per lane, while only changing the amount of spiked proteins, we confirm that nonlinearity and saturation of densitometry data, and errors introduced from normalisation processes, can occur in routine assays that compare equal amounts of lysate. These findings apply to all Western blot studies, and we highlight quality control checks that should be performed to make Western blot data more quantitative.

Preventing preterm birth: New approaches to labour therapeutics using Nanoparticles.

Preterm birth remains a major obstetric problem with ramifications that extend beyond immediate health and safety concerns for the newborn to include massive societal and economic burden. Although three quarters of preterm birth-related deaths could be prevented with cost-effective interventions, there has been little progress towards achieving sustained tocolysis that translates into improved outcomes for the newborn. With private enterprise reluctant to venture into the sphere of tocolysis, due to potential litigation, advances in the field may fall to new approaches using existing tocolytic resources more effectively. An emerging approach is the utilisation of nanoparticles, which have been established as versatile drug carriers with the power to modify the pharmacokinetics of entrapped therapeutics. In this article, we examine the development of nanoparticle-based drug delivery in pregnancy, with a focus on new approaches to therapeutics for preterm birth and modifying the labour process more generally.

The expression of genes involved in myometrial contractility changes during ex situ culture of pregnant human uterine smooth muscle tissue.

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 48 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.

Modulation of Progesterone Receptor Isoform Expression in Pregnant Human Myometrium.

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.