Roles of N-linked glycosylation and glycan-binding proteins in placentation: trophoblast infiltration, immunomodulation, angiogenesis, and pathophysiology.

Protein N-linked glycosylation is a structurally diverse post-translational modification that stores biological information in a larger order of magnitude than other post-translational modifications such as phosphorylation, ubiquitination and acetylation. This gives N-glycosylated proteins a diverse range of properties and allows glyco-codes (glycan-related information) to be deciphered by glycan-binding proteins (GBPs). The intervillous space of the placenta is richly populated with membrane-bound and secreted glycoproteins. Evidence exists to suggest that altering the structural nature of their N-glycans can impact several trophoblast functions, which include those related to interactions with decidual cells. This review summarizes trophoblast-related activities influenced by N-glycan-GBP recognition, exploring how different subtypes of trophoblasts actively adapt to characteristics of the decidualized endometrium through cell-specific expression of N-glycosylated proteins, and how these cells receive decidua-derived signals via N-glycan-GBP interactions. We highlight work on how changes in N-glycosylation relates to the success of trophoblast infiltration, interactions of immunomodulators, and uterine angiogenesis. We also discuss studies that suggest aberrant N-glycosylation of trophoblasts may contribute to the pathogenesis of pregnancy complications (e.g. pre-eclampsia, early spontaneous miscarriages and hydatidiform mole). We propose that a more in-depth understanding of how N-glycosylation shapes trophoblast phenotype during early pregnancy has the potential to improve our approach to predicting, diagnosing and alleviating poor maternal/fetal outcomes associated with placental dysfunction.

Transcription factors regulated by cAMP in smooth muscle of the myometrium at human parturition.

Cyclic adenosine monophosphate (cAMP) contributes to maintenance of a quiescent (relaxed) state in the myometrium (i.e. uterine smooth muscle) during pregnancy, which most commonly has been attributed to activation of protein kinase A (PKA). PKA-mediated phosphorylation of cytosolic contractile apparatus components in myometrial smooth muscle cells (mSMCs) are known to promote relaxation. Additionally, PKA also regulates nuclear transcription factor (TF) activity to control expression of genes important to the labour process; these are mostly involved in actin-myosin interactions, cell-to-cell connectivity and inflammation, all of which influence mSMC transition from a quiescent to a contractile (pro-labour) phenotype. This review focuses on the evidence that cAMP modulates the activity of TFs linked to pro-labour gene expression, predominantly cAMP response element (CRE) binding TFs, nuclear factor κB (NF-κB), activator protein 1 (AP-1) family and progesterone receptors (PRs). This review also considers the more recently described exchange protein directly activated by cAMP (EPAC) that may oppose the pro-quiescent effects of PKA, as well as explores findings from other cell types that have the potential to be of novel relevance to cAMP action on TF function in the myometrium.

Aminophylline and progesterone prevent inflammation-induced preterm parturition in the mouse†.

Although progesterone (P4) supplementation is the most widely used therapy for the prevention of preterm labor (PTL), reports of its clinical efficacy have been conflicting. We have previously shown that the anti-inflammatory effects of P4 can be enhanced by increasing intracellular cyclic adenosine monophosphate (cAMP) levels in primary human myometrial cells. Here, we have examined whether adding aminophylline (Am), a non-specific phosphodiesterase inhibitor that increases intracellular cAMP levels, to P4 might improve its efficacy using in vivo and in vitro models of PTL. In a mouse model of lipopolysaccharide (LPS)-induced PTL, we found that the combination of P4 and Am delayed the onset of LPS-induced PTL, while the same dose of P4 and Am alone had no effect. Pup survival was not improved by either agent alone or in combination. Myometrial prolabor and inflammatory cytokine gene expression was reduced, but the reduction was similar in P4 and P4/Am treated mice. There was no effect of the combination of P4 and Am on an ex vivo assessment of myometrial contractility. In human myometrial cells and myometrial tissue explants, we found that the combination had marked anti-inflammatory effects, reducing cytokine and COX-2 mRNA and protein levels to a greater extent than either agent alone. These data suggest that the combination of P4 and Am has a more potent anti-inflammatory effect than either agent alone and may be an effective combination in women at high-risk of PTL.

Differential impact of acute and prolonged cAMP agonist exposure on protein kinase A activation and human myometrium contractile activity.

KEY POINTS: Over 15 million babies are born prematurely each year with approximately 1 million of these babies dying as a direct result of preterm delivery. ß2 -Adrenoreceptor agonists that act via cAMP can reduce uterine contractions to delay preterm labour, but their ability to repress uterine contractions lasts ≤ 48 h and their use does not improve neonatal outcomes. Previous research has suggested that cAMP inhibits myometrial contractions via protein kinase A (PKA) activation, but this has yet to be demonstrated with PKA-specific agonists. We investigated the role of PKA in mediating cAMP-induced human myometrial relaxation, and the impact of prolonged cAMP elevation on myometrial contractility. Our findings suggest that PKA is not the sole mediator of cAMP-induced myometrial relaxation and that prolonged prophylactic elevation of cAMP alone is unlikely to prevent preterm labour (PTL). ABSTRACT: Acute cAMP elevation inhibits myometrial contractility, but the mechanisms responsible are not fully elucidated and the long-term effects are uncertain. Both need to be defined in pregnant human myometrium before the therapeutic potential of cAMP-elevating agents in the prevention of preterm labour can be realised. In the present study, we tested the hypotheses that PKA activity is necessary for cAMP-induced myometrial relaxation, and that prolonged cAMP elevation can prevent myometrial contractions. Myometrial tissues obtained from term, pre-labour elective Caesarean sections were exposed to receptor-independent cAMP agonists to determine the relationship between myometrial contractility (spontaneous and oxytocin-induced), PKA activity, HSP20 phosphorylation and expression of contraction-associated and cAMP signalling proteins. Acute (1 h) application of cAMP agonists promoted myometrial relaxation, but this was weakly related to PKA activation. A PKA-specific activator, 6-Bnz-cAMP, increased PKA activity (6.8 ± 2.0 mean fold versus vehicle; P = 0.0313) without inducing myometrial relaxation. Spontaneous myometrial contractility declined after 24 h but was less marked when tissues were constantly exposed to cAMP agonists, especially for 8-bromo-cAMP (4.3 ± 1.2 mean fold versus vehicle; P = 0.0043); this was associated with changes to calponin, cofilin and HSP20 phosphorylated/total protein levels. Oxytocin-induced contractions were unaffected by pre-incubation with cAMP agonists despite treatments being able to enhance PKA activity and HSP20 phosphorylation. These data suggest that cAMP-induced myometrial relaxation is not solely dependent on PKA activity and the ability of cAMP agonists to repress myometrial contractility is lost with prolonged exposure. We conclude that cAMP agonist treatment alone may not prevent preterm labour.

Isolation of single cells from human uterus in the third trimester of pregnancy: myometrium, decidua, amnion and chorion.

During pregnancy, interactions between uterine immune cells and cells of the surrounding reproductive tissues are thought to be vital for regulating labour. The mechanism that specifically initiates spontaneous labour has not been determined, but distinct changes in uterine immune cell populations and their activation status have been observed during labour at term gestation. To understand the regulation of human labour by the immune system, the ability to isolate both immune cells and non-immune cells from the uterus is required. Here, we describe protocols developed in our laboratory to isolate single cells from uterine tissues, which preserve both immune and non-immune cell populations for further analysis. We provide detailed methods for isolating immune and non-immune cells from human myometrium, chorion, amnion and decidua, together with representative flow cytometry analysis of isolated cell populations present. The protocols can be completed in tandem and take approximately 4-5 h, resulting in single-cell suspensions that contain viable leucocytes, and non-immune cells in sufficient numbers for single-cell analysis approaches such as flow cytometry and single cell RNA sequencing (scRNAseq).

Targeting an Initiator Allergen Provides Durable and Expansive Protection against House Dust Mite Allergy.

Whereas treatment of allergic diseases such as asthma relies largely on the targeting of dysregulated effector pathways, the conceptually attractive alternative of preventing them by a pharmaceutical, at-source intervention has been stymied until now by uncertainties about suitable targets and the challenges facing drug design. House dust mites (HDMs) are globally significant triggers of allergy. Group 1 HDM allergens, exemplified by Der p 1, are cysteine proteases. Their degradome has a strong disease linkage that underlies their status as risk and initiator allergens acting directly and through bystander effects on other allergens. Our objective was to test whether target-selective inhibitors of group 1 HDM allergens might provide a viable route to novel therapies. Using structure-directed design to optimize a series of pyruvamides, we undertook the first examination of whether pharmaceutically developable inhibitors of group 1 allergens might offer protection against HDM exposure. Developability criteria included durable inhibition of clinically relevant signals after a single aerosolized dose of the drug. The compounds suppressed acute airway responses of rats and mice when challenged with an HDM extract representing the HDM allergome. Inhibitory effects operated through a miscellany of downstream pathways involving, among others, IL-33, thymic stromal lymphopoietin, chemokines, and dendritic cells. IL-13 and eosinophil recruitment, indices of Th2 pathway activation, were strongly attenuated. The surprisingly expansive benefits arising from a unique at-source intervention suggest a novel approach to multiple allergic diseases in which HDMs play prominent roles and encourage exploration of these pharmaceutically developable molecules in a clinical setting.

Evaluating aminophylline and progesterone combination treatment to modulate contractility and labor-related proteins in pregnant human myometrial tissues.

Progesterone (P4) and cyclic adenosine monophosphate (cAMP) are regarded as pro-quiescent factors that suppress uterine contractions during pregnancy. We previously used human primary cells in vitro and mice in vivo to demonstrate that simultaneously enhancing myometrial P4 and cAMP levels may reduce inflammation-associated preterm labor. Here, we assessed whether aminophylline (Ami; phosphodiesterase inhibitor) and P4 can reduce myometrial contractility and contraction-associated proteins (CAPs) better together than individually; both agents are clinically used drugs. Myometrial tissues from pregnant non-laboring women were treated ex vivo with Ami acutely (while spontaneous contracting) or throughout 24-h tissue culture (±P4); isometric tension measurements, PKA assays, and Western blotting were used to assess tissue contractility, cAMP action, and inflammation. Acute (1 h) treatment with 250 and 750 µM Ami reduced contractions by 50% and 84%, respectively, which was not associated with a directly proportional increase in whole tissue PKA activity. Sustained myometrial relaxation was observed during 24-h tissue culture with 750 µM Ami, which did not require P4 nor reduce CAPs. COX-2 protein can be reduced by 300 nM P4 but this did not equate to myometrial relaxation. Ami (250 µM) and P4 (100 and 300 nM) co-treatment did not prevent oxytocin-augmented contractions nor reduce CAPs during interleukin-1ß stimulation. Overall, Ami and P4 co-treatment did not suppress myometrial contractions more than either agent alone, which may be attributed to low specificity and efficacy of Ami; cAMP and P4 action at in utero neighboring reproductive tissues during pregnancy should also be considered.

Labour classified by cervical dilatation & fetal membrane rupture demonstrates differential impact on RNA-seq data for human myometrium tissues.

High throughput sequencing has previously identified differentially expressed genes (DEGs) and enriched signalling networks in human myometrium for term (≥37 weeks) gestation labour, when defined as a singular state of activity at comparison to the non-labouring state. However, transcriptome changes that occur during transition from early to established labour (defined as ≤3 and >3 cm cervical dilatation, respectively) and potentially altered by fetal membrane rupture (ROM), when adapting from onset to completion of childbirth, remained to be defined. In the present study, we assessed whether differences for these two clinically observable factors of labour are associated with different myometrial transcriptome profiles. Analysis of our tissue ('bulk') RNA-seq data (NCBI Gene Expression Omnibus: GSE80172) with classification of labour into four groups, each compared to the same non-labour group, identified more DEGs for early than established labour; ROM was the strongest up-regulator of DEGs. We propose that lower DEGs frequency for early labour and/or ROM negative myometrium was attributed to bulk RNA-seq limitations associated with tissue heterogeneity, as well as the possibility that processes other than gene transcription are of more importance at labour onset. Integrative analysis with future data from additional samples, which have at least equivalent refined clinical classification for labour status, and alternative omics approaches will help to explain what truly contributes to transcriptomic changes that are critical for labour onset. Lastly, we identified five DEGs common to all labour groupings; two of which (AREG and PER3) were validated by qPCR and not differentially expressed in placenta and choriodecidua.

The impact of progesterone and RU-486 on classic pro-labour proteins & contractility in human myometrial tissues during 24-hour exposure to tension & interleukin-1ß.

Increased expression of pro-labour genes that encode cyclooxygenase-2 (COX-2), oxytocin receptor (OTR) and connexin-43 (Cx43) at parturition is often attributed to P4 functional withdrawal, based on findings from animal models and human primary myometrial cells. However, the cause of reduced myometrial P4 responsiveness that promotes contractions at labour is not fully determined. Uterine stretch occurs with advancing gestation but most in vitro experimental models do not take this into consideration. We aimed to examine whether tissue-level myometrial stretch influences the ability of P4 to regulate pro-labour protein abundance by using myometrial biopsies from term gestation pregnant women to assess the impact of 24 h exposure to combinations of (i) stretch-mediated tension, (ii) P4 (100 nM) and (iii) an anti-progestin, RU-486 (1 µM). Firstly, we observed baseline COX-2 and Cx43 protein levels increased, whereas P4 content along with calponin-1 and progesterone receptor (PR) protein abundance decreased, in vehicle-treated tissues. P4 supplementation subtly reduced COX-2 levels in un-stretched tissues. Spontaneous and oxytocin-augmented contractility were unchanged by tissue culture exposure to P4 and/or RU-486. Interleukin-1ß (IL-1ß; 1 ng/ml) enhanced COX-2 protein and PGE2 content in un-stretched tissues. Overall, tissue stretch may, in part, regulate P4-sensitive pro-labour protein levels, but this is likely to be reliant on interaction with other in utero factors that were absent in our tissue cultures. More complex culture conditions should be evaluated in future to aid further development of a physiologically relevant model to improve our understanding of in utero myometrial P4 responsiveness.

Progesterone-Related Immune Modulation of Pregnancy and Labor.

Pregnancy involves a complex interplay between maternal neuroendocrine and immunological systems in order to establish and sustain a growing fetus. It is thought that the uterus at pregnancy transitions from quiescent to laboring state in response to interactions between maternal and fetal systems at least partly via altered neuroendocrine signaling. Progesterone (P4) is a vital hormone in maternal reproductive tissues and immune cells during pregnancy. As such, P4 is widely used in clinical interventions to improve the chance of embryo implantation, as well as reduce the risk of miscarriage and premature labor. Here we review research to date that focus on the pathways through which P4 mediates its actions on both the maternal reproductive and immune system. We will dissect the role of P4 as a modulator of inflammation, both systemic and intrinsic to the uterus, during human pregnancy and labor.

Myometrial Transcriptional Signatures of Human Parturition.

The process of parturition involves the transformation of the quiescent myometrium (uterine smooth muscle) to the highly contractile laboring state. This is thought to be driven by changes in gene expression in myometrial cells. Despite the existence of multiple myometrial gene expression studies, the transcriptional programs that initiate labor are not known. Here, we integrated three transcriptome datasets, one novel (NCBI Gene Expression Ominibus: GSE80172) and two existing, to characterize the gene expression changes in myometrium associated with the onset of labor at term. Computational analyses including classification, singular value decomposition, pathway enrichment, and network inference were applied to individual and combined datasets. Outcomes across studies were integrated with multiple protein and pathway databases to build a myometrial parturition signaling network. A high-confidence (significant across all studies) set of 126 labor genes were identified and machine learning models exhibited high reproducibility between studies. Labor signatures included both known (interleukins, cytokines) and unknown (apoptosis, MYC, cell proliferation/differentiation) pathways while cyclic AMP signaling and muscle relaxation were associated with non-labor. These signatures accurately classified and characterized the stages of labor. The data-derived parturition signaling networks provide new genes/signaling interactions to understand phenotype-specific processes and aid in future studies of parturition.

Corrigendum: Myometrial Transcriptional Signatures of Human Parturition.

[This corrects the article DOI: 10.3389/fgene.2019.00185.].

Tetrabromobisphenol A (TBBPA), induces cell death in TM4 Sertoli cells by modulating Ca2+ transport proteins and causing dysregulation of Ca2+ homeostasis.

Tetrabromobisphenol A (TBBPA) is a commonly used brominated flame retardant (BFR) utilized to reduce the flammability of a variety of products. Studies have indicated that a number of BFRs are becoming widely distributed within the environment and are bio-accumulating within organisms. There has been much speculation that a variety of phenolic pollutants (including compounds chemically related to TBBPA, such as bisphenol A) may cause endocrine disruption and Ca2+ dysregulation in cells involved in spermatogenesis. In this study we therefore investigate the effects of TBBPA on mouse TM4 Sertoli cells (essential for sperm development). Results show that TBBPA increases Ca2+ within these cells in the 5-60 microM concentration range (EC50, 21 microM). TBBPA also causes cell death (LC50, 18 microM) partly via apoptosis, involving Ca2+-dependent mitochondrial depolarisation. Studies on intracellular Ca2+ transporters shows that TBBPA can inhibit sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA) at low concentrations (IC50, 0.4 to 1.2 microM) and also activate the Ryanodine receptor Ca2+ channel within the 0.4-4 microM concentration range. Therefore these studies suggest that the cytotoxic effects of TBBPA on cells is partly due to dysregulation of Ca2+ signalling, by directly affecting Ca2+ transport proteins.

Endocrine disrupting alkylphenols: structural requirements for their adverse effects on Ca2+ pumps, Ca2+ homeostasis & Sertoli TM4 cell viability.

Alkylphenols such as nonylphenol are pollutants that are widely dispersed within our environment. They bio-accumulate within man, with levels in the muM concentration range reported in human tissues. These chemicals act as endocrine disruptors, having xenoestrogenic activity. More recently alkylphenols have also been shown to affect Ca2+ signalling pathways. Here we show that alkylphenols are potent inhibitors of sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) activity. For linear chain alkylphenols the potency of inhibition is related to chain length, with the IC50 values for inhibition ranging from 8 microM for 4-n-nonylphenol (C9) to 1.3 mM for 4-n-propylphenol (C3). Branched chain alkylphenols generally had lower potencies than their linear chain counterparts, however, good correlations for all alkylphenols were observed between their Ca2+ pump inhibition and hydrophobicity, molecular volume and flexibility, indicating that these parameters are all important factors. Alkylphenols cause abnormal elevations of intracellular [Ca2+] within TM4 Sertoli cells (cells involved in sperm maturation) depolarise their mitochondria and induce cell death in these cells, in an alkyl chain size-dependent manner.