Human placenta releases extracellular vesicles carrying corticotrophin releasing hormone mRNA into the maternal blood.

The human placenta releases diverse extracellular vesicles (EVs), including microvesicles (100-1000 nm) and exosomes (30-150 nm), into the maternal blood for feto-maternal communication. Exosomes and microvesicles contribute to normal pregnancy physiology and major pregnancy pathologies. Differences in miRNA expressions and protein content in placental exosomes have been reported in complicated pregnancies. During human pregnancy, Corticotropin-Releasing Hormone (CRH) is produced and released by the placenta into the maternal blood. CRH is involved in regulating gestational length and the initiation of labour. CRH mRNA levels in the maternal plasma rise with gestation. High levels of CRH mRNA are reported to be associated with preeclamptic and preterm pregnancies. However, the underlying mechanism of placental CRH mRNA secretion remains to be elucidated. We hypothesise that the placenta releases CRH mRNA packaged within extracellular vesicles (EVs) into the maternal blood. In this study, placental EVs (microvesicles and exosomes) were isolated from human term healthy placentas via villus washes and from explant culture media by differential centrifugation and purified by density gradient ultracentrifugation using a continuous sucrose gradient (0.25-2.5 M). Western blotting using placenta- and exosome-specific markers and electron microscopy confirmed exosomes and microvesicles in the placental wash and explant media samples. Real-time quantitative RT-PCR data detected CRH mRNA in placenta-derived EVs from placental washes and explants. We also sorted placenta-secreted EVs in maternal plasma samples (≥37 weeks) by high-resolution flow cytometry using a fluorescent-labelled PLAP antibody. CRH mRNA was demonstrated in placental EVs obtained from maternal blood plasma. We therefore show that human placental EVs carry CRH mRNA into the maternal blood. Our study implies that measuring CRH mRNA in placental EVs in the maternal plasma could beused for monitoring pregnancy.

Extracellular vesicles- crucial players in human pregnancy.

Extracellular vesicles (EVs) are lipid-bilayer enclosed membrane vesicles released by cells in physiological and pathological states. EVs are generated and released through a variety of pathways and mediate cellular communication by carrying and transferring signals to recipient cells. EVs are specifically loaded with proteins, nucleic acids (RNAs and DNA), enzymes and lipids, and carry a range of surface proteins and adhesion molecules. EVs contribute to intercellular signalling, development, metabolism, tissue homeostasis, antigen presentation, gene expression and immune regulation. EVs have been categorised into three different subgroups based on their size: exosomes (30-150 nm), microvesicles (100-1000 nm) and apoptotic bodies (1-5 µm). The status of the cells of origin of EVs influences their biology, heterogeneity and functions. EVs, especially exosomes, have been studied for their potential roles in feto-maternal communication and impacts on normal pregnancy and pregnancy disorders. This review presents an overview of EVs, emphasising exosomes and microvesicles in a general context, and then focusing on the roles of EVs in human pregnancy and their potential as diagnostics for adverse pregnancy outcomes.

Involvement of oxidative stress in placental dysfunction, the pathophysiology of fetal death and pregnancy disorders.

In brief: Placental oxidative stress contributes to both normal and abnormal placentation during pregnancy. This review discusses the potential consequence of oxidative stress-induced placental dysfunction on pregnancies complicated by fetal death and pregnancies with a high risk of fetal death. Abstract: The placenta is a source of reactive oxygen free radicals due to the oxidative metabolism required to meet the demands of the growing fetus. The placenta has an array of efficient antioxidant defense systems to deal with rising oxidative stress created by free radicals during pregnancy. Properly controlled physiological (low-level) free radical production is a necessary part of cellular signaling pathways and downstream activities during normal placental development; however, poorly controlled oxidative stress can cause aberrant placentation, immune disturbances and placental dysfunction. Abnormal placental function and immune disturbances are linked to many pregnancy-related disorders, including early and recurrent pregnancy loss, fetal death, spontaneous preterm birth, preeclampsia and fetal growth restriction. This review discusses the role of placental oxidative stress in both normal and pathological settings. Finally, based on previously published work, this review presents multiple lines of evidence for the strong association between oxidative stress and adverse pregnancy outcomes, including fetal death and pregnancies with a high risk of fetal death.

Preterm Birth and Corticotrophin-Releasing Hormone as a Placental Clock.

Preterm birth worldwide remains a significant cause of neonatal morbidity and mortality, yet the exact mechanisms of preterm parturition remain unclear. Preterm birth is not a single condition, but rather a syndrome with a multifactorial etiology. This multifactorial nature explains why individual predictive measures for preterm birth have had limited sensitivity and specificity. One proposed pathway for preterm birth is via placentally synthesized corticotrophin-releasing hormone (CRH). CRH is a peptide hormone that increases exponentially in pregnancy and has been implicated in preterm birth because of its endocrine, autocrine, and paracrine roles. CRH has actions that increase placental production of estriol and of the transcription factor nuclear factor-κB, that likely play a key role in activating the myometrium. CRH has been proposed as part of a placental clock, with early activation of placental production resulting in preterm birth. This article will review the current understanding of preterm birth, CRH as an initiator of human parturition, and the evidence regarding the use of CRH in the prediction of preterm birth.

Potential pharmacologic interventions targeting TLR signaling in placental malaria.

Complications from placental malaria cause poor pregnancy outcomes, including low birthweight, preterm delivery, and stillbirths. Many of these complications are driven by maternal innate proinflammatory responses to the sequestration of Plasmodium falciparum in the placenta. However, recent studies show that, in reaction to maternal innate immune responses that are detrimental to the fetus, the fetus mounts innate immune counter-responses that ameliorate pregnancy outcomes. Such fetal-maternal conflict in placental malaria has potential for pharmacologic modulation for better pregnancy outcomes. Here, we discuss placental malaria pathogenesis, its complications, and the role of innate immunity and fetal-maternal innate immune conflict in placental malaria. Finally, we discuss pharmacologic immunomodulatory strategies and agents with the potential to improve placental malaria outcomes.

Insights into fetal death-a patient resource.

Evidence supports a role for placental aging in the etiology of the majority of fetal deaths. This knowledge may reduce maternal feelings of guilt following fetal death that frequently exacerbates the distress caused by grief. The accompanying video may be a useful resource for women who have experienced a fetal death.

Inhibition of vertebrate aldehyde oxidase as a therapeutic treatment for cancer, obesity, aging and amyotrophic lateral sclerosis.

The aldehyde oxidases (AOXs) are a small sub-family of cytosolic molybdo-flavoenzymes, which are structurally conserved proteins and broadly distributed from plants to animals. AOXs play multiple roles in both physiological and pathological processes and AOX inhibition is of increasing significance in the development of novel drugs and therapeutic strategies. This review provides an overview of the evolution and the action mechanism of AOX and the role of each domain. The review provides an update of the polymorphisms in the human AOX. This review also summarises the physiology of AOX in different organs and its role in drug metabolism. The inhibition of AOX is a promising therapeutic treatment for cancer, obesity, aging and amyotrophic lateral sclerosis.

Is there a role for placental senescence in the genesis of obstetric complications and fetal growth restriction?

The placenta ages as pregnancy advances, yet its continued function is required for a successful pregnancy outcome. Placental aging is a physiological phenomenon; however, there are some placentas that show signs of aging earlier than others. Premature placental senescence and aging are implicated in a number of adverse pregnancy outcomes, including fetal growth restriction, preeclampsia, spontaneous preterm birth, and intrauterine fetal death. Here we discuss cellular senescence, a state of terminal proliferation arrest, and how senescence is regulated. We also explore the role of physiological placental senescence and how aberrant placental senescence alters placental function, contributing to the pathophysiology of fetal growth restriction, preeclampsia, spontaneous preterm labor/birth, and unexplained fetal death.

Evidence that fetal death is associated with placental aging.

BACKGROUND: The risk of unexplained fetal death or stillbirth increases late in pregnancy, suggesting that placental aging is an etiological factor. Aging is associated with oxidative damage to DNA, RNA, and lipids. We hypothesized that placentas at >41 completed weeks of gestation (late-term) would show changes consistent with aging that would also be present in placentas associated with stillbirths. OBJECTIVE: We sought to determine whether placentas from late-term pregnancies and unexplained stillbirth show oxidative damage and other biochemical signs of aging. We also aimed to develop an in vitro term placental explant culture model to test the aging pathways. STUDY DESIGN: We collected placentas from women at 37-39 weeks' gestation (early-term and term), late-term, and with unexplained stillbirth. We used immunohistochemistry to compare the 3 groups for: DNA/RNA oxidation (8-hydroxy-deoxyguanosine), lysosomal distribution (lysosome-associated membrane protein 2), lipid oxidation (4-hydroxynonenal), and autophagosome size (microtubule-associated proteins 1A/1B light chain 3B, LC3B). The expression of aldehyde oxidase 1 was measured by real-time polymerase chain reaction. Using a placental explant culture model, we tested the hypothesis that aldehyde oxidase 1 mediates oxidative damage to lipids in the placenta. RESULTS: Placentas from late-term pregnancies show increased aldehyde oxidase 1 expression, oxidation of DNA/RNA and lipid, perinuclear location of lysosomes, and larger autophagosomes compared to placentas from women delivered at 37-39 weeks. Stillbirth-associated placentas showed similar changes in oxidation of DNA/RNA and lipid, lysosomal location, and autophagosome size to placentas from late-term. Placental explants from term deliveries cultured in serum-free medium also showed evidence of oxidation of lipid, perinuclear lysosomes, and larger autophagosomes, changes that were blocked by the G-protein-coupled estrogen receptor 1 agonist G1, while the oxidation of lipid was blocked by the aldehyde oxidase 1 inhibitor raloxifene. CONCLUSION: Our data are consistent with a role for aldehyde oxidase 1 and G-protein-coupled estrogen receptor 1 in mediating aging of the placenta that may contribute to stillbirth. The placenta is a tractable model of aging in human tissue.

Oxidative stress, placental ageing-related pathologies and adverse pregnancy outcomes.

Oxidative stress (OS), an imbalance between free radical generation and antioxidant defence, is recognized as a key factor in the pathogenesis of adverse pregnancy outcomes. Although OS is a common future of normal pregnancy, persistent, overwhelming OS leads to consumption and decline of antioxidants, affecting placental antioxidant capacity and reducing systems. The accumulation of OS causes damage to lipids, proteins and DNA in the placental tissue that induces a form of accelerated ageing. Premature ageing of the placenta is associated with placental insufficiency that prevents the organ meeting the needs of the foetus, and as a consequence, the viability of the foetus is compromised. This review summarizes the literature regarding the role of OS and premature placental ageing in the pathophysiology of pregnancy complications.

Diminished hERG K+ channel activity facilitates strong human labour contractions but is dysregulated in obese women.

Human ether-a-go-go-related gene (hERG) potassium channels determine cardiac action potential and contraction duration. Human uterine contractions are underpinned by an action potential that also possesses an initial spike followed by prolonged depolarization. Here we show that hERG channel proteins (α-conducting and ß-inhibitory subunits) and hERG currents exist in isolated patch-clamped human myometrial cells. We show that hERG channel activity suppresses contraction amplitude and duration before labour, thereby facilitating quiescence. During established labour, expression of ß-inhibitory protein is markedly enhanced, resulting in reduced hERG activity that is associated with an increased duration of uterine action potentials and contractions. Thus, changes in hERG channel activity contribute to electrophysiological mechanisms that produce contractions during labour. We also demonstrate that this system fails in women with elevated BMI, who have enhanced hERG activity as a result of low ß-inhibitory protein expression, which likely contributes to the weak contractions and poor labour outcomes observed in many obese women necessitating caesarean delivery.

Effects of corticotrophin releasing hormone (CRH) on cell viability and differentiation in the human BeWo choriocarcinoma cell line: a potential syncytialisation inducer distinct from cyclic adenosine monophosphate (cAMP).

BACKGROUND: Placental production of corticotrophin releasing hormone (CRH) rises exponentially as pregnancy progresses, and has been linked with the onset of normal and preterm labour. CRH is produced in syncytiotrophoblast cells and production is increased by glucocorticoids and cAMP. It remains unclear whether cAMP acts by inducing differentiation of cytotrophoblasts and/or through induction of syncytialisation. As CRH can stimulate cAMP pathways we have tested whether a feed-forward system may exist in placental cells during syncytialisation. METHODS: The choriocarcinoma BeWo cell line was treated with cAMP, CRH or vehicle. Cell viability was determined by MTT assay, while apoptosis was analysed by DAPI staining and by FACS. Differentiation was measured by assaying message for hCG and ERVW-1 (syncytin1) by qRT-PCR, as well as the respective protein by ELISA. Fusion of BeWo cells was assessed by co-staining cell membrane and nuclei with CellMask and Hoechst 33342. CRHR1 and CRHR2 mRNA levels were measured by qRT-PCR. RESULTS: We show that cAMP has an inductive effect on syncytialisation, as evidenced by induction of hCG secretion, by ERVW-1 mRNA expression and by formation of multinuclear cells. CRH mRNA expression was found to increase prior to the changes in the other syncytialisation markers. cAMP had an inhibitory effect on BeWo cell viability, but exogenous CRH did not. However, CRH did mimic the differentiation inducing effect of cAMP, suggesting a link between CRH and cAMP signalling in syncytialisation. We also found that treatment of BeWo cells with exogenous CRH resulted in elevated cellular CRHR1 levels. CONCLUSIONS: This study suggests a positive feed-forward role exists for CRH in trophoblast cell differentiation, which may underlie the exponential rise in CRH observed as gestation advances.

Recent advances in understanding the endocrinology of human birth.

The timing of human birth has a crucial impact upon the survival of the fetus. New knowledge on the regulation of human birth includes the role of endogenous retroviruses in the formation of the syncytiotrophoblast cells and consequently the secretion of corticotrophin releasing hormone, a hormone linked to gestational length determination. miRNAs have been identified that mediate progesterone withdrawal at labor by suppressing progesterone-induced transcription factors. Progress has also been made in understanding how the contractile machinery of the uterine myocytes is activated at labor and the role of small heat-shock proteins in this process. From this work, new therapeutic targets have been identified that may be used to regulate the onset of labor and improve neonatal mortality.

Phasic phosphorylation of caldesmon and ERK 1/2 during contractions in human myometrium.

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.

A gonadotropin-releasing hormone-II antagonist induces autophagy of prostate cancer cells.

Gonadotropin-releasing hormone-I (GnRH-I) is known to directly regulate prostate cancer cell proliferation. However, the role of GnRH-II in prostate cancer is unclear. Here, we investigated the effect of the GnRH-II antagonist trptorelix-1 (Trp-1) on growth of PC3 prostate cancer cells. Trp-1 induced growth inhibition of PC3 cells in vitro and inhibited growth of PC3 cells xenografted into nude mice. FITC-N3, an FITC-conjugated Trp-1 analogue, was largely present in the mitochondria of prostate cancer cells, but not in other cells that are not derived from the prostate. Trp-1-induced PC3 growth inhibition was associated with decreased mitochondrial membrane potential and increased levels of mitochondrial and cytosolic reactive oxygen species (ROS). Growth inhibition was partially prevented by cotreating cells with N-acetyl cysteine, an antioxidant. Cytochrome c release and caspase-3 activation were not detected in Trp-1-treated cells. However, Trp-1 induced autophagosome formation, as seen by increased LysoTracker staining and recruitment of microtubule-associated protein 1 light chain 3 to these new lysosomal compartments. Trp-1-induced autophagy was accompanied by decreased AKT phosphorylation and increased c-Jun NH(2) terminal kinase phosphorylation, two events known to be linked to autophagy. Taken together, these data suggest that Trp-1 directly induces mitochondrial dysfunction and ROS increase, leading to autophagy of prostate cancer cells. GnRH-II antagonists may hold promise in the treatment of prostate cancer.

Involvement of amino acids flanking Glu7.32 of the gonadotropin-releasing hormone receptor in the selectivity of antagonists.

The Glu/Asp(7.32) residue in extracellular loop 3 of the mammalian type-I gonadotropin-releasing hormone receptor (GnRHR) interacts with Arg(8) of GnRH-I, conferring preferential ligand selectivity for GnRH-I over GnRH-II. Previously, we demonstrated that the residues (Ser and Pro) flanking Glu/Asp(7.32) also play a role in the differential agonist selectivity of mammalian and non-mammalian GnRHRs. In this study, we examined the differential antagonist selectivity of wild type and mutant GnRHRs in which the Ser and Pro residues were changed. Cetrorelix, a GnRH-I antagonist, and Trptorelix-2, a GnRH-II antagonist, exhibited high selectivity for mammalian type-I and non-mammalian GnRHRs, respectively. The inhibitory activities of the antagonists were dependent on agonist concentration and subtype. Rat GnRHR in which the Ser-Glu-Pro (SEP) motif was changed to Pro-Glu-Val (PEV) or Pro-Glu-Ser (PES) had increased sensitivity to Trptorelix-2 but decreased sensitivity to Cetrorelix. Mutant bullfrog GnRHR-1 with the SEP motif had the reverse antagonist selectivity, with reduced sensitivity to Trptorelix-2 but increased sensitivity to Cetrorelix. These findings indicate that the residues flanking Glu(7.32) are important for antagonist as well as agonist selectivity.

Differential effects of gonadotropin-releasing hormone (GnRH)-I and GnRH-II on prostate cancer cell signaling and death.

CONTEXT: GnRH is known to directly regulate prostate cancer cell proliferation, but the precise mechanism of action of the peptide is still under investigation. OBJECTIVE: This study demonstrates differential effects of GnRH-I and GnRH-II on androgen-independent human prostate cancer cells. RESULTS: Both GnRH-I and GnRH-II increased the intracellular Ca(2+) concentration ([Ca(2+)](i)) either through Ca(2+) influx from external Ca(2+) source or via mobilization of Ca(2+) from internal Ca(2+) stores. Interestingly, the [Ca(2+)](i) increase was mediated by activation of the ryanodine receptor but not the inositol trisphosphate receptor. Trptorelix-1, a novel GnRH-II antagonist but not cetrorelix, a classical GnRH-I antagonist, completely inhibited the GnRH-II-induced [Ca(2+)](i) increase. Concurrently at high concentrations, trptorelix-1 and cetrorelix inhibited GnRH-I-induced [Ca(2+)](i) increase, whereas at low concentrations they exerted an agonistic action, inducing Ca(2+) influx. High concentrations of trptorelix-1 but not cetrorelix-induced prostate cancer cell death, probably through an apoptotic process. Using photoaffinity labeling with (125)I-[azidobenzoyl-D-Lys(6)]GnRH-II, we observed that an 80-kDa protein specifically bound to GnRH-II. CONCLUSIONS: This study suggests the existence of a novel GnRH-II binding protein, in addition to a conventional GnRH-I receptor, in prostate cancer cells. These data may facilitate the development of innovatory therapeutic drugs for the treatment of prostate cancer.

Extracellular loop 3 (EL3) and EL3-proximal transmembrane helix 7 of the mammalian type I and type II gonadotropin-releasing hormone (GnRH) receptors determine differential ligand selectivity to GnRH-I and GnRH-II.

Mammalian type I and II gonadotropin-releasing hormone (GnRH) receptors (GnRHRs) show differential ligand preference for GnRH-I and GnRH-II, respectively. Using a variety of chimeric receptors based on green monkey GnRHR-2 (gmGnRHR-2), a representative type II GnRHR, and rat GnRHR, a representative type I GnRHR, this study elucidated specific domains responsible for this ligand selectivity. A chimeric gmGnRHR-2 with the extracellular loop 3 (EL3) and EL3-proximal transmembrane helix 7 (TMH7) of rat GnRHR showed a great increase in ligand sensitivity to GnRH-I but not to GnRH-II. Point-mutation studies indicate that four amino acids, Leu/Phe(7.38), Leu/Phe(7.43), Ala/Pro(7.46), and Pro/Cys(7.47) in TMH7 are critical for ligand selectivity as well as receptor conformation. Furthermore, a combinatory mutation (Pro(7.31)-Pro(7.32)-Ser(7.33) motif to Ser-Glu-Pro in EL3 and Leu(7.38), Leu(7.43), Ala(7.46), and Pro(7.47) to those of rat GnRHR) in gmGnRH-2 exhibited an approximately 500-fold increased sensitivity to GnRH-I, indicating that these residues are critical for discriminating GnRH-II from GnRH-I. [Trp(7)]GnRH-I and [Trp(8)]GnRH-I but not [His(5)]GnRH-I exhibit a higher potency in activating wild-type gmGnRHR-2 than native GnRH-I, indicating that amino acids at positions 7 and 8 of GnRHs are more important than position 5 for differential recognition by type I and type II GnRHRs. As a whole, these data suggest a molecular coevolution of ligands and their receptors and facilitate the understanding of the molecular interaction between GnRHs and their cognate receptors.

Position of Pro and Ser near Glu7.32 in the extracellular loop 3 of mammalian and nonmammalian gonadotropin-releasing hormone (GnRH) receptors is a critical determinant for differential ligand selectivity for mammalian GnRH and chicken GnRH-II.

A Glu/Asp7.32 residue in the extracellular loop 3 of the mammalian GnRH receptor (GnRHR) is known to interact with Arg8 of mammalian GnRH (mGnRH), which may confer preferential ligand selectivity for mGnRH than for chicken GnRH-II (cGnRH-II). However, some nonmammalian GnRHRs also have the Glu/Asp residue at the same position, yet respond better to cGnRH-II than mGnRH. Amino acids flanking Glu/Asp7.32 are differentially arranged such that mammalian and nonmammalian GnRHRs have an S-E/D-P motif and P-X-S/Y motif, respectively. We presumed the position of Ser7.31 or Pro7.33 of rat GnRHR as a potential determinant for ligand selectivity. Either placing Pro before Glu7.32 or placing Ser after Glu7.32 significantly decreased the sensitivity and/or efficacy for mGnRH, but slightly increased that for cGnRH-II in several mutant receptors. Among them, those with a PEV, PES, or SES motif exhibited a marked decrease in sensitivity for mGnRH such that cGnRH-II had a higher potency than mGnRH, showing a reversed preferential ligand selectivity. Chimeric mGnRHs in which positions 5, 7, and/or 8 were replaced by those of cGnRH-II revealed a greater ability to activate these mutant receptors than mGnRH, whereas they were less potent to activate wild-type rat GnRHR than mGnRH. Interestingly, a mutant bullfrog type I receptor with the SEP motif exhibited an increased sensitivity for mGnRH but a decreased sensitivity for cGnRH-II. These results indicate that the position of Pro and Ser near Glu7.32 in the extracellular loop 3 is critical for the differential ligand selectivity between mammalian and nonmammalian GnRHRs.