Extracellular vesicles from maternal uterine cells exposed to risk factors cause fetal inflammatory response

Background Fetal cell-derived exosomes (extracellular vesicles, 40–160 nm) are communication channels that can signal parturition by inducing inflammatory changes in maternal decidua and myometrium. Little is known about maternal cell-derived exosomes and their functional roles on the fetal side. This study isolated and characterized exosomes from decidual and myometrial cells grown under normal and inflammatory/oxidative stress conditions and determined their impact on fetal membrane cells. Methods Decidual and myometrial cells were grown under standard culture conditions (control) or exposed for 48 h to cigarette smoke extract or tumor necrosis factor-α, as proxies for oxidative stress and inflammation, respectively. Exosomes were isolated from media (differential ultra-centrifugation followed by size exclusion chromatography), quantified (nano particle tracking analysis), and characterized in terms of their size and morphology (cryo-electron microscopy), markers (dot blot), and cargo contents (proteomics followed by bioinformatics analysis). Maternal exosomes (109/mL) were used to treat amnion epithelial cells and chorion trophoblast cells for 24 h. The exosome uptake by fetal cells (confocal microscopy) and the cytokine response (enzyme-linked immunosorbent assays for IL-6, IL-10, and TNF-α) was determined. Results Exosomes from both decidual and myometrial cells were round and expressed tetraspanins and endosomal sorting complexes required for transport (ESCRT) protein markers. The size and quantity was not different between control and treated cell exosomes. Proteomic analysis identified several common proteins in exosomes, as well as unique proteins based on cell type and treatment. Compared to control exosomes, pro-inflammatory cytokine release was higher in both amnion epithelial cell and chorion trophoblast cell media when the cells had been exposed to exosomes from decidual or myometrial cells treated with either cigarette smoke extract or tumor necrosis factor-α. In chorion trophoblast cells, anti-inflammatory IL-10 was increased by exosomes from both decidual and myometrial cells. Conclusion Various pathophysiological conditions cause maternal exosomes to carry inflammatory mediators that can result in cell type dependent fetal inflammatory response.

Parity associates with chromosomal damage in uterine leiomyomas

Mechanical forces in a constrained cellular environment were recently established as a facilitator of chromosomal damage. Whether this could contribute to tumorigenesis is not known. Uterine leiomyomas are common neoplasms that display relatively few chromosomal aberrations. We hypothesized that if mechanical forces contribute to chromosomal damage, signs of this could be seen in uterine leiomyomas from parous women. We examined the karyotypes of 1946 tumors, and found a striking overrepresentation of chromosomal damage associated with parity. We then subjected myometrial cells to physiological forces similar to those encountered during pregnancy, and found this to cause DNA breaks and a DNA repair response. While mechanical forces acting in constrained cellular environments may thus contribute to neoplastic degeneration, and genesis of uterine leiomyoma, further studies are needed to prove possible causality of the observed association. No evidence for progression to malignancy was found.

Extracellular Vesicles from Maternal Uterine Cells Exposed to Risk Factors cause Fetal Inflammatory Response

BackgroundFetal cell-derived exosomes (extracellular vesicles, 40–160 nm) are communication channels that can signal parturition by inducing inflammatory changes in maternal decidua and myometrium. Little is known about maternal cell-derived exosomes and their functional roles on the fetal side. This study isolated and characterized exosomes from decidual and myometrial cells grown under normal and inflammatory/oxidative stress conditions and determined their impact on fetal membrane cells. MethodsDecidual and myometrial cells were grown under standard culture conditions (control) or exposed for 48 hours to cigarette smoke extract or tumor necrosis factor-α, as proxies for oxidative stress and inflammation, respectively. Exosomes were isolated from media (differential ultra-centrifugation followed by size exclusion chromatography), quantified (nano particle tracking analysis), and characterized in terms of their size and morphology (cryo-electron microscopy), markers (dot blot), and cargo contents (proteomics followed by bioinformatics analysis). Maternal exosomes (109/mL) were used to treat amnion epithelial cells and chorion trophoblast cells for 24 hrs. The exosome uptake by fetal cells (confocal microscopy) and the cytokine response (enzyme-linked immunosorbent assays for IL-6, IL-10, and TNF-α) was determined.ResultsExosomes from both decidual and myometrial cells were round and expressed tetraspanins and endosomal sorting complexes required for transport (ESCRT) protein markers. The size and quantity was not different between control and treated cell exosomes. Proteomic analysis identified several common proteins in exosomes, as well as unique proteins based on cell type and treatment. Compared to control exosomes, pro-inflammatory cytokine release was higher in both amnion epithelial cell and chorion trophoblast cell media when the cells had been exposed to exosomes from decidual or myometrial cells treated with either cigarette smoke extract or tumor necrosis factor-α. In chorion trophoblast cells, anti-inflammatory IL-10 was increased by exosomes from both decidual and myometrial cells.ConclusionVarious pathophysiological conditions cause maternal exosomes to carry inflammatory mediators that can result in cell type dependent fetal inflammatory response.

Understanding the Impact of Uterine Fibroids on Human Endometrium Function

Uterine fibroids (leiomyomas) are the most common benign gynecological tumors in women of reproductive age worldwide. They cause heavy menstrual bleeding, usually leading to severe anemia, pelvic pain/pressure, infertility, and other debilitating morbidities. Fibroids are believed to be monoclonal tumors arising from the myometrium, and recent studies have demonstrated that fibroids actively influence the endometrium globally. Studies suggest a direct relationship between the number of fibroids removed and fertility problems. In this review, our objective was to provide a complete overview of the origin of uterine fibroids and the molecular pathways and processes implicated in their development and growth, which can directly affect the function of a healthy endometrium. One of the most common characteristics of fibroids is the excessive production of extracellular matrix (ECM) components, which contributes to the stiffness and expansion of fibroids. ECM may serve as a reservoir of profibrotic growth factors such as the transforming growth factor β (TGF-β) and a modulator of their availability and actions. Fibroids also elicit mechanotransduction changes that result in decreased uterine wall contractility and increased myometrium rigidity, which affect normal biological uterine functions such as menstrual bleeding, receptivity, and implantation. Changes in the microRNA (miRNA) expression in fibroids and myometrial cells appear to modulate the TGF-β pathways and the expression of regulators of ECM production. Taken together, these findings demonstrate an interaction among the ECM components, TGF-β family signaling, miRNAs, and the endometrial vascular system. Targeting these components will be fundamental to developing novel pharmacotherapies that not only treat uterine fibroids but also restore normal endometrial function.

TBX2, a Novel Regulator of Labour

Background and Objectives: Therapeutic interventions targeting molecular factors involved in the transition from uterine quiescence to overt labour are not substantially reducing the rate of spontaneous preterm labour. The identification of novel rational therapeutic targets are essential to prevent the most common cause of neonatal mortality. Based on our previous work showing that Tbx2 (T-Box transcription factor 2) is a putative upstream regulator preceding progesterone withdrawal in mouse myometrium, we now investigate the role of TBX2 in human myometrium. Materials and Methods: RNA microarray analysis of (A) preterm human myometrium samples and (B) myometrial cells overexpressing TBX2 in vitro, combined with subsequent analysis of the two publicly available datasets of (C) Chan et al. and (D) Sharp et al. The effect of TBX2 overexpression on cytokines/chemokines secreted to the myometrium cell culture medium were determined by Luminex assay. Results: Analysis shows that overexpression of TBX2 in myometrial cells results in downregulation of TNFα- and interferon signalling. This downregulation is consistent with the decreased expression of cytokines and chemokines of which a subset has been previously associated with the inflammatory pathways relevant for human labour. In contrast, CXCL5 (C-X-C motif chemokine ligand 5), CCL21 and IL-6 (Interleukin 6), previously reported in relation to parturition, do not seem to be under TBX2 control. The combined bioinformatical analysis of the four mRNA datasets identifies a subset of upstream regulators common to both preterm and term labour under control of TBX2. Surprisingly, TBX2 mRNA levels are increased in preterm contractile myometrium. Conclusions: We identified a subset of upstream regulators common to both preterm and term labour that are activated in labour and repressed by TBX2. The increased TBX2 mRNA expression in myometrium collected during a preterm caesarean section while in spontaneous preterm labour compared to tissue harvested during iatrogenic preterm delivery does not fit the bioinformatical model. We can only explain this by speculating that the in vivo activity of TBX2 in human myometrium depends not only on the TBX2 expression levels but also on levels of the accessory proteins necessary for TBX2 activity.

Identification of mundulone and mundulone acetate as natural products with tocolytic efficacy in mono- and combination-therapy with current tocolytics

Currently, there are a lack of FDA-approved tocolytics for the management of preterm labor. We previously observed that the isoflavones mundulone and mundulone acetate (MA) inhibit intracellular Ca2+-regulated myometrial contractility. Here, we further probed the potential of these natural products to be small molecule leads for discovery of novel tocolytics by: (1) examining uterine-selectivity by comparing concentration-response between human primary myometrial cells and a major off-target site, aortic vascular smooth muscle cells (VSMCs), (2) identifying synergistic combinations with current clinical tocolytics to increase efficacy or and reduce off-target side effects, (3) determining cytotoxic effects and (4) investigating the efficacy, potency and tissue-selectivity between myometrial contractility and constriction of fetal ductus arteriosus (DA), a major off-target of current tocolytics. Mundulone displayed significantly greater efficacy (Emax = 80.5% vs. 44.5%, p=0.0005) and potency (IC50 = 27 μM and 14 μM, p=0.007) compared to MA in the inhibition of intracellular-Ca2+ from myometrial cells. MA showed greater uterine-selectivity, compared to mundulone, based on greater differences in the IC50 (4.3 vs. 2.3 fold) and Emax (70% vs. 0%) between myometrial cells compared to aorta VSMCs. Moreover, MA demonstrated a favorable in vitro therapeutic index of 8.8, compared to TI = 0.8 of mundulone, due to its significantly (p<0.0005) smaller effect on the viability of myometrial (hTERT-HM), liver (HepG2) and kidney (RPTEC) cells. However, mundulone exhibited synergism with two current tocolytics (atosiban and nifedipine), while MA only displayed synergistic efficacy with only nifedipine. Of these synergistic combinations, only mundulone + atosiban demonstrated a favorable TI = 10 compared to TI=0.8 for mundulone alone. While only mundulone showed concentration-dependent inhibition of ex vivo mouse myometrial contractions, neither mundulone or MA affected mouse fetal DA vasoreactivity. The combination of mundulone and atosiban yielded greater tocolytic efficacy and potency on term pregnant mouse and human myometrial tissue compared to single-drugs. Collectively, these data highlight the difference in uterine‐selectivity of Ca2+‐mobilization, effects on cell viability and tocolytic efficacy between mundulone and MA. These natural products could benefit from medicinal chemistry efforts to study the structural activity relationship for further development into a promising single- and/or combination-tocolytic therapy for management of preterm labor.