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During normal trophoblast invasion, integrins α6β4 are downregulated, and α1β1 are upregulated in invasive cytotrophoblast cells. In preeclampsia both interstitial and endovascular invasion are shallow and cytotrophoblasts fail to upregulate α1β1 and downregulate α6β4. This study aims to investigate the role of integrins α1β1 and α6β4 on cellular pathways influencing trophoblast integration into endothelial cellular networks in vitro. Red fluorescent-labeled human uterine myometrial microvascular endothelial cells (UtMVECs) were seeded on Matrigel to form endothelial networks. Green fluorescent-labeled trophoblastic HTR-8/SVneo cells pre-incubated with 20 μg/ml of neutralizing antibodies (anti-α1, β1, α6, β4, α1 + β1, or α6 + β4) for 1 h were then co-cultured with endothelial networks with the neutralizing antibodies for 24 h. Fluorescent images were captured, and quantified utilizing Image J. Cells were retrieved to analyze mRNA expression of galectin-1, TIMP-1, and PAI-1 by quantitative PCR. MMP-2, MMP-9, free sFlt-1, and PlGF from conditioned media were measured by ELISA. The integration of trophoblast cells into endothelial cellular networks was inhibited by anti-β1(- 28 ± 3%, p < 0.0001), and increased by anti-α6(+ 19 ± 5%, p < 0.01). Galectin-1 mRNA expression was decreased by anti-α1(- 35 ± 7%, p < 0.001), anti-β1(- 23 ± 5%, p < 0.05), and anti-α1+β1(- 35 ± 5%, p < 0.001). The mRNA expression of TIMP-1 was inhibited by anti-α1(- 59 ± 9%, p < 0.01) and anti-β1(- 63 ± 7%, p < 0.001) while PAI-1 mRNA expression was increased by anti-α1 + β1(+ 285 ± 70%, p < 0.0001). In the conditioned medium, anti-α1 reduced MMP-2(-28 ± 1%, p < 0.001), MMP-9(-27 ± 8%, p < 0.01), and sFlt-1(-27 ± 5%, p < 0.001) production. Anti-β1 reduced MMP-2(- 15 ± 2%, p < 0.05) production. There were no changes in PlGF. Appropriate integrins α1β1 modulate trophoblast cell integration into endothelial cellular networks in vitro through invasive pathways including galectin-1, TIMP-1, PAI-1, MMP-2, and MMP-9 production.

Infection during pregnancy triggers inflammation, which can increase myometrial contractions and the risk of premature labor and delivery. In this study, we assessed the effects of vitamin D, an anti-inflammatory ligand on cytokines, chemokines, toll-like receptors, and contractile-associated proteins on immortalized human myometrial smooth muscle (UtSM) cells stimulated with lipopolysaccharide (LPS), a bacterial endotoxin, or interleukin (IL)-1β and measured Toll-like receptor (TLR)-10 expression in pregnant myometrial tissues. A superarray analysis revealed downregulation of the chemokines monocyte chemoattractant protein (MCP)-1, Chemokine (C-X-C motif) ligand (CXCL)-10, CXCL-11, and chemokine (C-X3-C motif) ligand (CX3CL)-1; the proinflammatory cytokines IL-13 and tumor necrosis factor (TNF)-α; the TLR-4 and -5 and triggering receptor expressed on myeloid cells (TREM)-2 and upregulation of the anti-inflammatory cytokine IL-10, as well as Toll interacting protein (TOLLIP) and TREM-1 in vitamin D-treated UtSM cells. In the presence of LPS, vitamin D caused dose-dependent decreases in the messenger RNA expression of MCP-1, IL-1β, IL-13, TNF-α, TLR-4, and TLR-5, the contractile-associated proteins connexin 43, the oxytocin receptor, and the prostaglandin receptor but caused increases in IL-10 and TLR-10 in UtSM cells. The TLR-10 expression was higher in human myometrial tissue obtained from women at term not in labor compared to labor. Vitamin D also attenuated IL-1β-induced MCP-1, IL-6, connexin 43, cyclooxygenase (COX)-2, and prostaglandin receptor expression. Western analysis showed that vitamin D decreased MCP-1, TLR-4, and connexin 43 in the presence of LPS and decreased connexin 43 in the presence of IL-1β. Our results suggest that vitamin D can potentially decrease infection-induced increases in cytokines and contractile-associated proteins in the myometrium.

Previously, we found that myometrial tumor necrosis factor-α (TNF-α) messenger RNA (mRNA) expression did not increase with preterm or term labor. To further investigate the role of TNF-α in human labor, we studied TNF-α receptor (TNFR1A and B) expression, regulation, and associated intracellular signaling pathways in human myometrial samples obtained both before and after the onset of labor and in primary cultures of uterine smooth muscle cells (USMCs). We found that the mRNA expression of both receptors increased with advancing gestation and labor and protein levels of TNFR1B were significantly higher in term laboring myometrial samples than in nonlabor controls. Tumor necrosis factor- treatment of USMCs activated all mitogen-activated protein kinase (MAPK) subtypes and nuclear factor κ-B (NF-κB). The TNF-α induced increases in the expression of TNFR1B and prostaglandin H synthase type 2 were reduced by inhibitors of NF-κB and MAPKs, respectively. The TNF-α induced increase in interleukin 8 (IL-8) appeared to be independent of MAPK and NF-κB pathway. These data suggest that the uterus may become more sensitive to the action of TNF-α with advancing gestation and labor and that TNF-α acts via MAPK and NF-κB to promote labor-associated gene expression.

An important action of progesterone during pregnancy is to maintain the uterus in a quiescent state and thereby prevent preterm labor. The causes of preterm labor are not well understood, so progesterone action on the myometrium can provide clues about the processes that keep the uterus from contracting prematurely. Accordingly, we have carried out Affymetrix GeneChip analysis of progesterone effects on gene expression in immortalized human myometrial cells cultured from a patient near the end of pregnancy. Progesterone appears to inhibit uterine excitability by a number of mechanisms, including increased expression of calcium and voltage-operated K(+) channels, which dampens the electrical activity of the myometrial cell, downregulation of agents, and receptors involved in myometrial contraction, reduction in cell signal components that lead to increased intracellular Ca(2+) concentrations in response to contractile stimuli, and downregulation of proteins involved in the cross-linking of actin and myosin filaments to produce uterine contractions.

Maternal obesity is associated with increased rates of labor induction, dysfunctional labor requiring intrapartum cesarean delivery, and postpartum hemorrhage, implying that maternal obesity has an inhibitory effect on myometrial function and its ability to respond to oxytocin. This study aimed to use an in vitro model to investigate the relationship between maternal body mass index (BMI) and the ability of myometrium to contract spontaneously and in response to oxytocin. Linear mixed effects regression modeling was applied to contractile data from 609 strips from 85 women. No correlation was found between maternal BMI and any indices of spontaneous myometrial activity. A single addition of oxytocin increased contractility, however, this was not related to maternal BMI. Similarly, oxytocin concentration-response curves were unrelated to BMI. Overall, the results from this in vitro study suggest that the observed implications of obesity on parturition in vivo cannot be explained by a direct effect on myometrial contractile mechanisms per se.

Uterine leiomyomas (also known as uterine fibroids) are the most common benign tumors of female reproductive tract and are the single most common indication for hysterectomies. Despite their high prevalence, the exact pathogenesis of these benign tumors is still unknown. One possible mechanism for leiomyoma formation is dysregulation of mesenchymal stem cell activity. Mesenchymal stem cells have been identified in both human and murine uteri and cancer stem cells have been identified in female reproductive malignancies. We compared stem/progenitor cell characteristics in both normal myometrium and the corresponding leiomyoma of patient's undergoing hysterectomies. We found that leiomyoma cells form fewer mesenchymal stem cell colonies and exhibit less Hoechst dye-excluding side population (SP) activity, which is a function associated with progenitor cells in other tissues, than cells isolated from normal myometrium. Whereas in normal myometrium, we observed heterogeneous expression of CD90, a cell surface marker associated the with differentiation potential of uterine fibroblasts, in leiomyomas, we observed homogenous expression of CD90, suggesting leiomyoma cells are more terminally differentiated. Furthermore, we found that while leiomyoma cells could only produce CD90 expressing cells, both CD90+ and CD90- myometrial cells could reestablish their original heterogeneous CD90 profile when expanded in vitro. These results suggest that normal myometrium contains cells with stem/progenitor cell activities that are absent in leiomyomas.

Overexpression of human telomerase reverse transcriptase (hTERT) has facilitated establishing in vitro model systems for biological research. The plasmid containing hTERT gene was stably transfected into ULTR cells, a retroviral transformed human uterine leiomyomatous smooth-muscle cell line. Cells that express hTERT, termed as ULTR-hT, shared the morphological characteristics of the parental proliferative ULTR cells. They expressed a set of smooth-muscle-specific genes and had increased proliferation rate and prolonged lifespan. Quantitative real-time polymerase chain reaction (PCR) analysis revealed a correlation of proliferation rates of ULTR-hT clonal cells with the level of hTERT expression. ULTRhT cells also preserved expression of estrogen, progesterone, and oxytocin receptor genes, confirming a myometrial phenotype. Expression of angiotensin II receptors and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase isoforms were also preserved. Our finding suggests that ULTR-hT cells can be a useful in vitro model for studying human myometrium differentiation both in pregnancy and pathological growth.

The mechanisms underlying the preparation of the uterus for labor are not fully understood. We have previously found a significant increase in the expression of messenger RNA (mRNAs) encoding extracellular basement membrane (BM) proteins of the smooth muscle cells (SMCs) in late pregnant rat myometrium. At term, the myometrium is stretched by growing fetuses and these mechanical signals are transmitted from extracellular matrix into SMCs through focal adhesions (FA). The aim of this study was to investigate the effect of gravidity on the expression and spatiotemporal distribution of major BM proteins, laminin-gamma2 and collagen IV, as well as typical FA constituents, vinculin and paxillin, in the myometrium during gestation and parturition, using a unilaterally pregnant rat model. We found that the expression of laminin-gamma2 and collagen IV proteins increased significantly with gestational age (P < .05) and was dependent on gravidity whereas vinculin and paxillin proteins were not affected. Near term, BM proteins from gravid horn myometrium demonstrated increased extracellular immunostaining and major rearrangement from sporadic protein distribution to organized, continuous, and regular structures surrounding the plasma membrane of each myocyte. Examination of FA proteins revealed that paxillin was translocated from the cytoplasm to the cell periphery, while vinculin was sequestered specifically to FAs. At labor, BM and FA proteins, organized in similar bead-like structures, were localized on opposing sides of SMC plasma membrane into 2 different compartments. We suggest that these stretch-induced changes facilitate formation of stable cell-matrix adhesions and provide the molecular basis for optimal force transduction during labor contractions.

Objective: To define the role of Toll-like receptors (TLR-2, TLR-4) and triggering receptor expressed on myeloid cells-1 (TREM-1) in pregnant human myometrium., Study Design: Expression of TLR-2 and -4 mRNA and protein and TREM-1 mRNA was quantified in human myometrial samples. Subsequently, we investigated the effect of medroxyprogesterone acetate (MPA) as a functional inhibitor of the TLR agonist lipopolysaccharide (LPS)., Results: Messenger RNA expression of TLR-2 and -4 was significantly higher in myometrium at term compared with preterm (P = .009 and .016, respectively). Toll-like receptor-2 protein expression was significantly higher during labor (P = .002) compared with nonlaboring samples. Triggering receptor expressed on myeloid cells-1 mRNA expression was increased in both myometrium and cervix after labor at term (P < .05). Medroxyprogesterone acetate significantly suppressed LPS-induced production of interleukin 1b (IL-1b), IL-6, and IL-8 in vitro (P < .05)., Conclusion: Toll-like receptors 2and 4 and TREM-1 are expressed in human myometrium and may play a role in the mechanism of labor at term, and their functional effects are inhibited by MPA.

Cellular function is modulated by the interaction with the extracellular matrix within the myometrium. We formed the hypothesis that the cytokine-stimulated pro-labour gene expression by human uterine smooth muscle cells would be increased by growing the cells on collagen-coated plates. Primary cultures of human uterine smooth muscle cells grown on uncoated plates and on plates coated with collagen were exposed to the inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta and interleukin-6) and assessed the messenger RNA expression of oxytocin receptor, interleukin-8, prostaglandin H synthase type-2 and prostaglandin F(2) alpha receptor. Basal pro-labour gene expression was unaffected by collagen coating and the response to the inflammatory cytokines was similar for oxytocin receptor and prostaglandin H synthase type-2, but appeared to be reduced for interleukin-8 and enhanced for FP. Collagen coating made no significant impact on basal integrin expression and interleukin-1beta induced phosphorylation of extracellular-regulated-kinase1/2 and RelA subunit of nuclear factor-kappa B (p65). We conclude that growing human uterine smooth muscle cells on collagen-coated plates may modulate the pro-labour gene response to the inflammatory cytokines.

The regulatory mechanisms underlying myometrial smooth muscle contractility during labor are poorly understood. The authors therefore investigated the transcriptional profile of the changes that occur in the human myometrium at term pregnancy when compared with that at labor. Microarray technology was used to identify differentially expressed genes in human myometrium at labor. Real-time fluorescence reversetranscriptase polymerase chain reaction (RT-PCR) was subsequently performed to verify the microarray data. Semiquantitative RT-PCR, Western blotting, and microscopy methodologies were also used. Certain novel genes were found to be upregulated in human myometrium at labor. Of these, PSCDBP, TLR2, TWIST1 , FLJ35382, andRGS12 have not been previously characterized or identified in human myometrium. EDNRB is the other novel labor-associated gene whose reported expression is also upregulated at labor. All 6 genes were expressed on human myometrial smooth muscle cells. These novel upregulated genes are involved in multiple pathways that may be associated with a variety of cellular processes including inflammation, transcriptional regulation, and intracellular signaling.

Observations from the authors' laboratory suggest a physiological role for increased cyclin G1 protein levels in human uterine leiomyoma. The hypothesis of the present study is that the strategic modulation of cyclin G1 by antisense technology will inhibit the survival of in vitro-grown uterine leiomyoma cells. Cultured uterine leiomyoma cells were transfected with cyclin G1 ribbon-type antisense oligonucleotide (cyclin G1 RiAS) to effectively reduce cyclin G1 expression. Cell viability, in situ terminal deoxyuridine nick end-labeling (TUNEL) assay, flow cytometry, DNA fragmentation, and expression of cell cycle regulatory-related proteins were evaluated by Western blot. Antisense oligonucleotides compromised uterine leiomyoma cell viability and inducted apoptosis in a caspase-independent mechanism. In situ TUNEL and DNA fragmentation revealed apoptosis induction, and fluorescent-activated cell sorting analysis showed increased sub-G1-phase cells. Furthermore, abrogation of cyclin G1 enhanced p53 accumulation, phosphorylation of p53 at Ser-15 residue, and increased expression of cyclin-dependent kinase inhibitors p21 and p27. These data imply that cyclin G1 expression is associated with growth promotion and the potential utility and novelty of using ribbon-type antisense oligonucleotides as a gene therapy strategy to treat human uterine leiomyoma.

At term, amniotic fluid contents may mediate the onset of labor through the activation of amniotic fluid macrophages and their migration into the myometrium. To test this concept, the authors examine the histological changes that occur in myometrial biopsies at term prior to (n = 53) and during (n = 15) labor. Biopsies were stained with an antimacrophage antibody, anti-CD34 (endothelial cells), and anti-caspase 3 (apoptotic cells). The samples showed a variable inflammatory infiltrate of neutrophils and macrophages, with a greater infiltrate in the samples obtained during labor (P < .001, Fisher exact test). Prior to labor, there were prominent changes in the myometrial fibers that reflected shearing, shrinkage, edema, and particularly apoptosis; endothelial cells of thin-walled vessels prominent in the biopsies displayed marked nuclear biotinylation, and the vascular lumen contained fibrin and platelet thrombi, microparticles, desquamated endothelial cells, amniotic squamous cells, and mucoid material. These changes were also present in samples obtained during labor. In an additional 10 patients in labor with male fetuses, myometrial samples were examined for the presence of macrophages carrying a Y chromosome indicative of a fetal origin, but none were observed. These findings suggest that endothelial cell damage and amniotic fluid embolism are very common at term prior to clinical labor and provide a mechanism by which surfactant protein A and phospholipids present in the amniotic fluid may access myometrial cells and provoke the inflammatory response that occurs during parturition. The authors' studies give no support to the suggestion that fetal macrophages might invade the human myometrium at term.

The pathogenesis of leiomyoma may be related to an imbalance in the interaction of sex steroids with paracrine growth factors that may control the modulation of mitogenesis and local immunity. The authors investigate the temporal and spatial expression of proliferative and preapoptotic molecules that may participate in the modulation of myometrial function and leiomyoma pathogenesis. Immunohistochemistry and Western blot analysis are used to investigate Fas ligand (FasL), phosphatase and tensin homolog deletion on chromosome 10 (PTEN), and proliferating cell nuclear antigen (PCNA) expression in myometrium and leiomyoma. Western blot results show that in the secretory phase, FasL expression is 1.8-fold and 2.3-fold higher compared with the proliferative phase in the myometrium and leiomyoma, respectively (P = .022 and .047, respectively). A paired comparison between myometrium and leiomyoma reveals higher FasL expression in the leiomyoma (P = .003). On the contrary, when compared with the secretory phase, PCNA expression during the proliferative phase is 4.6-fold and 3.7-fold higher in the myometrium and leiomyoma, respectively (P = .041 and .034, respectively). A paired comparison between myometrium and leiomyoma reveals higher PCNA expression in the leiomyoma. Furthermore, lower PTEN expression is detected in the leiomyoma compared with the myometrium (P < .032). Immunohistochemistry results reveal that FasL, PTEN, and PCNA are expressed in the myometrium and leiomyoma, consistent with the results from the Western blot analysis. The results suggest that FasL, PTEN, and PCNA may be involved in the pathophysiology of leiomyoma. A higher FasL level in the leiomyoma is likely to correspond to suppression of local immunity by inducing apoptosis of immune cells, while a higher level of PCNA and a lower level of PTEN may be related to increased mitogenesis and decreased apoptosis in leiomyoma.

It has been suggested that corticotropin-releasing hormone (CRH) receptors CRH-R1 and CRH-R2 might be involved in the modulation of uterine activity during pregnancy. The authors determine the localization, concentrations, and variants of CRH-R1 and CRH-R2 in the human pregnant myometrium from patients undergoing labor and patients not undergoing labor. Biopsies were taken from 40 patients undergoing either elective or emergency cesarean delivery after spontaneous labor. The localization of CRH-R1 and CRH-R2 was examined by immunohistochemistry. The mRNA and protein levels of CRH-R1 and CRH-R2 were measured by quantitative reverse-transcriptase polymerase chain reaction (PCR) and Western blot analysis, respectively. The variants of CRH-R1 and CRH-R2 were determined by PCR analysis followed by sequencing. Both CRH-R1 and CRH-R2 were found by immunohistochemistry to be expressed by smooth muscle cells in the pregnant myometrium. There was no significant difference in mRNA and protein levels of CRH-R1 and CRH-R2 in myometria between the labor and nonlabor groups. Levels of CRH-R1 alpha , R1 beta, R1c, R1e, R1f, and R2 alpha were identified in the pregnant myometrium, and levels of CRH-R1 alpha, R1c, and R2 alpha were detected in both the term labor and nonlabor myometrium. A heterogeneous distribution of other CRH-R1 variants in term labor and nonlabor myometrium was observed. Human myometrium expresses both CRH-R1 and CRH-R2 during pregnancy. A heterogeneous distribution of CRH-R1 variants in term labor and nonlabor myometrium might be related to the effects of CRH on contractile phenotype of myometrium at the term.