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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.

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.

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.

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.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.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.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.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.

The establishment and maintenance of pregnancy rely on a number of hormones, including progesterone and human chorionic gonadotrophin. Throughout gestation, pregnancy is under a progesterone block, which maintains uterine quiescence to permit fetal growth and maturation. As a woman approaches term, there is a functional progesterone withdrawal and functional estrogen activation that upregulate contraction-associated proteins. These hormonal changes appear to coincide with inflammatory changes that further encourage the transformation of the uterus from a resting to a contractile phenotype. During parturition, the release of oxytocin and prostaglandins enhances contractions, and increasing gap junctions allows the synchronization of myocyte activity and rhythmic contractions of the whole uterus. A number of factors can initiate preterm labor, including infection, sterile inflammation, and blood and cellular senescence.

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.

We present a hypothesis for a mechanism involving self-organization of small functional units that leads to organ-level synchronization of uterine contractions in human labor. This view is in contrast to the long-held presumption that the synchronized behavior of the uterus is subject to well-defined internal organization (as is found in the heart) that exists prior to the onset of labor. The contractile units of the uterus are myocytes, which contract in response to both mechanical stretch and electrical stimulation. Throughout pregnancy progesterone maintains quiescence by suppression of “contraction-associated proteins” (CAPs). At the end of pregnancy a functional withdrawal of progesterone and an increasingly estrogenic environment leads to an increase in the production of CAPs. One CAP of particular importance is connexin 43, which creates gap junctions between the myocytes that cause them to become electrically coupled. The electrical connectivity between myocytes, combined with an increase in intrauterine pressure at the end of pregnancy shifts the uterus towards an increasingly unstable critical point, characterized by irregular, uncoordinated contractions. We propose that synchronous, coordinated contractions emerge from this critical point through a process of self-organization, and that the search for a uterine pacemaker has been unfruitful for the sole reason that it is non-existent.

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.

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.

Background The ability to provide safe and effective pharmacotherapy during obstetric complications, such as preterm labor or postpartum hemorrhage, is hampered by the systemic toxicity of therapeutic agents leading to adverse side effects in the mother and fetus. Development of novel strategies to target tocolytic and uterotonic agents specifically to uterine myocytes would improve therapeutic efficacy while minimizing the risk of side effects. Ligand-targeted liposomes have emerged as a reliable and versatile platform for targeted drug delivery to specific cell types, tissues or organs. Objective Our objective was to develop a targeted drug delivery system for the uterus utilizing an immunoliposome platform targeting the oxytocin receptor. Study Design We conjugated liposomes to an antibody that recognizes an extracellular domain of the oxytocin receptor. We then examined the ability of oxytocin receptor–targeted liposomes to deliver contraction-blocking (nifedipine, salbutamol and rolipram) or contraction-enhancing (dofetilide) agents to strips of spontaneously contracting myometrial tissue in vitro (human and mouse). We evaluated the ability of oxytocin receptor–targeted liposomes to localize to uterine tissue in vivo, and assessed if targeted liposomes loaded with indomethacin were capable of preventing lipopolysaccharide-induced preterm birth in mice. Results Oxytocin receptor–targeted liposomes loaded with nifedipine, salbutamol or rolipram consistently abolished human myometrial contractions in vitro, while oxytocin receptor–targeted liposomes loaded with dofetilide increased contraction duration. Nontargeted control liposomes loaded with these agents had no effect. Similar results were observed in mouse uterine strips. Following in vivo administration to pregnant mice, oxytocin receptor–targeted liposomes localized specifically to the uterine horns and mammary tissue. Targeting increased localization to the uterus 7-fold. Localization was not detected in the maternal brain or fetus. Targeted and nontargeted liposomes also localized to the liver. Oxytocin receptor–targeted liposomes loaded with indomethacin were effective in reducing rates of preterm birth in mice, whereas nontargeted liposomes loaded with indomethacin had no effect. Conclusion Our results demonstrate that oxytocin receptor–targeted liposomes can be used to either inhibit or enhance human uterine contractions in vitro. In vivo, the liposomes localized to the uterine tissue of pregnant mice and were effective in delivering agents for the prevention of inflammation-induced preterm labor. The potential clinical advantage of targeted liposomal drug delivery to the myometrium is reduced dose and reduced toxicity to both mother and fetus.

Prostaglandins are key components of the parturition cascade; however, the mechanisms that regulate prostaglandin concentrations in the uterus during pregnancy are largely unknown. The purpose of this study was to determine the intrauterine expression of the chief prostaglandin-inactivating enzyme, 15-hydroxyprostaglandin dehydrogenase (PGDH), during gestation and labor in the guinea pig, an animal model in which the endocrine control of pregnancy and parturition is analogous to that of women. PGDH messenger RNA (mRNA) abundance decreased significantly in the visceral yolk sac membrane (VYS, the anatomical equivalent of the human chorion laeve) and the amnion throughout the last third of pregnancy. PGDH protein was robustly expressed in the VYS epithelium and mesoderm, correlated strongly with PGDH mRNA levels and exhibited a nadir at term prior to labor onset. PGDH protein was not detected in the amnion. PGDH mRNA and protein levels in the placenta and myoendometrium were variable throughout late gestation. In the placenta, PGDH protein was concentrated in the parietal yolk sac membrane (PYS) lining the placental surface and in placental blood vessels. We observed strong expression of PGDH protein in the endometrial epithelium with comparably little expression in the myometrium. These data indicate that metabolic inactivation of prostaglandins in the pregnant guinea pig uterus takes place in the VYS, PYS, and endometrium. Decreased PGDH expression in the fetal membranes may contribute to the increase in intrauterine prostaglandin concentrations at term, stimulating the onset of labor.

The objective of this study is to describe the fetal phenotype in utero and its associations with maternal pre-pregnancy weight and gestational weight gain. This prospective longitudinal cohort included 179 Australian women with singleton pregnancies. Serial ultrasound measurements were performed at 19, 25, 30 and 36 (±1) weeks gestation and maternal anthropometry were collected concurrently. The ultrasound scans included the standard fetal biometry of head circumference, biparietal diameter, abdominal circumference, and femur length, and body composition at the abdomen and mid-thigh, including fat and lean mass cross-sectional areas. Maternal gestational weight gain was compared to current clinical guidelines. The participants had an average of 3.7 ± 0.8 scans and birth data were available for 165 neonates. Fifty four per cent of the cohort gained weight in excess of current recommendations, according to pre-pregnancy body mass index (BMI). Maternal gestational weight positively predicted fetal abdominal circumference (P 0.029) and lean abdominal mass area (P 0.046) in linear mixed model regression analysis, adjusted for known and potential confounders. At any pre-pregnancy BMI gaining weight above the current recommendations resulted in a larger fetus according to standard biometry, because of significantly larger lean muscle mass at the abdomen (P 0.024) and not due to an increase in fat mass (P 0.463). We have demonstrated the importance of maternal weight gain, independent of pre-pregnancy BMI, to support the growth of a large but lean fetus. Prenatal counselling should focus on achieving a healthy BMI prior to conception so that gestational weight gain restrictions can be minimised.

Estrogens are key mediators of increased uterine contractility at labor. We sought to determine whether membrane-associated estrogen receptors, such as the recently described seven-transmembrane receptor G protein-coupled receptor 30 (GPR30), mediated some of this effect. Using human myometrium obtained at term cesarean section before or after the onset of labor, we demonstrated the presence of GPR30 mRNA and protein using quantitative RT-PCR and Western blotting. GPR30 receptor was localized to the cell membrane and often colocalized with calveolin-1. Using the specific estrogen membrane receptor agonist G-1 and myometrial explants, we showed that membrane receptor activation led to phosphorylation of MAPK and the actin-modifying small heat shock protein 27. Using myometrial strips incubated with G-1 or vehicle we demonstrated that estrogen membrane receptor activation increased the myometrial contractile response to oxytocin. These data suggest that activation of the plasma membrane estrogen receptor GPR30 likely participates in the physiology of the human myometrium during pregnancy and identifies it as a potential target to modify uterine activity.

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.

Throughout their evolution, animals have developed mechanisms which protect them against parasites or infections by detecting and destroying foreign biological material within their own bodies. These mechanisms for excluding “non-self” biological materials whilst at the same time maintaining the integrity of the “self” have evolved for hundreds of millions of years into a highly complex body system, the immune system (Bainbridge, 2000). Any disbalance or alteration of the mechanisms maintaining the dynamic equilibrium between the “self” and “non-self” recognition could translate into a pathological state (or condition) like autoimmune disease at one end of the scale or immunodeficiency at the other.

Human myometrium develops phasic contractions during labor. Phosphorylation of caldesmon (h-CaD) and extracellular signal-regulated kinase 1/2 (ERK 1/2) has been implicated in development of these contractions, however the phospho-regulation of these proteins is yet to be examined during periods of both contraction and relaxation. We hypothesized that protein phosphorylation events are implicated in the phasic nature of myometrial contractions, and aimed to examine h-CaD and ERK 1/2 phosphorylation in myometrium snap frozen at specific stages, including; (1) prior to onset of contractions, (2) at peak contraction and (3) during relaxation. We aimed to compare h-CaD and ERK 1/2 phosphorylation in vitro against results from in vivo studies that compared not-in-labor (NIL) and laboring (L) myometrium. Comparison of NIL (n = 8) and L (n = 8) myometrium revealed a 2-fold increase in h-CaD phosphorylation (ser-789; P = 0.012) during onset of labor in vivo, and was associated with significantly up-regulated ERK2 expression (P = 0.022), however no change in ERK2 phosphorylation was observed (P = 0.475). During in vitro studies (n = 5), transition from non-contracting tissue to tissue at peak contraction was associated with increased phosphorylation of both h-CaD and ERK 1/2. Furthermore, tissue preserved at relaxation phase exhibited diminished levels of h-CaD and ERK 1/2 phosphorylation compared to tissue preserved at peak contraction, thereby producing a phasic phosphorylation profile for h-CaD and ERK 1/2. h-CaD and ERK 1/2 are phosphorylated during myometrial contractions, however their phospho-regulation is dynamic, in that h-CaD and ERK 1/2 are phosphorylated and dephosphorylated in phase with contraction and relaxation respectively. Comparisons of NIL and L tissue are at risk of failing to detect these changes, as L samples are not necessarily preserved in the midst of an active contraction.