Cervical Remodeling Research Articles

Hyaluronan (HA) and H11 Antigen (HABP) Expression in Cervical Cancer Sections and Their Significances as Prominent Markers

This article has been retracted. Background : Cancer is a disease characterized by a population of cells that grow and divide without respect to normal limits, invade and destroy adjacent tissues, and may spread to distant anatomic sites through a process called metastasis . These malignant properties of cancers differentiate them from benign tumors, which are self-limited in their growth and do not invade or metastasize for the development and progression of tumor cells. Cancer is a result of multistep process involving accumulation of genetic alternation, which results in loss of cell-cell interaction, increasing invasive migration, loss of control in cell division and abnormal matrix assembly. Carcinoma of the cervix is one of the most common malignancies. Cervical differentiation requires changes in the composition and structure of extracellular matrix. However, the biological roles of Hyaluronan (HA) in cervical remodeling are still under investigation and HA-receptors are not studied during cervical pathological remodeling. Hence, in the current study we investigated the expression and association of extracellular matrix components namely hyaluronan-hyaluronic acid binding protein (H 11 antigen) (HA-HABP) in normal and invasive lesion in cervical tissue section slides. Methods and Results : Cervical tissues from normal and suspected cancer patients from hospitals were examined. Immunohistochemistry was performed with specific HA probe, b. PG (biotinylated proteoglycan) and anti-HABP antibody H 11 B 2 C 2 for H 11 antigen. HA and H 11 antigen are overexpressed in well-differentiated tumors during progression. Conclusions : We conclude that the expression of HA and H 11 antigen and their interaction are very important in identifying cervical cancers and probably H 11 antigen can be used as a biomarker in identifying the tumor cells in cervical lesions. doi:10.4021/wjon287e

481: Progesterone receptor blockade mediates inflammatory cervical remodeling prompting preterm birth irrespective of systemic progesterone levels in a small animal model

481: Progesterone receptor blockade mediates inflammatory cervical remodeling prompting preterm birth irrespective of systemic progesterone levels in a small animal model

Cervical tissue engineering using silk scaffolds and human cervical cells.

Spontaneous preterm birth is a frequent complication of pregnancy and a common cause of morbidity in childhood. Obstetricians suspect abnormalities of the cervix are implicated in a significant number of preterm births. The cervix is composed of fibrous connective tissue and undergoes significant remodeling in preparation for birth. We hypothesized that a tissue engineering strategy could be used to develop three-dimensional cervical-like tissue constructs that would be suitable for investigating cervical remodeling. Cervical cells were isolated from two premenopausal women undergoing hysterectomy for a benign gynecological condition, and the cells were seeded on porous silk scaffolds in the presence or absence of dynamic culture and with 10% or 20% serum. Morphological, biochemical, and mechanical properties were measured during the 8-week culture period. Cervical cells proliferated in three-dimensions and synthesized an extracellular matrix with biochemical constituents and morphology similar to native tissue. Compared to static culture, dynamic culture was associated with significantly increased collagen deposition (p < 0.05), sulfated glycosaminoglycan synthesis (p < 0.05), and mechanical stiffness (p < 0.05). Serum concentration did not affect measured variables. Relevant human tissue-engineered cervical-like constructs constitute a novel model system for a range of fundamental and applied studies related to cervical remodeling.

Ex vivo assessment of mouse cervical remodeling through pregnancy via 23Na MRS

Preterm birth occurs in 12.5% of births in the United States and can lead to risk of infant death or to lifelong serious health complications. A greater understanding by which the two main processes, uterine contraction and cervical remodeling are regulated is required to reduce rates of preterm birth. The cervix must undergo extensive remodeling through pregnancy in preparation for parturition, the process of labor and delivery of young. One key aspect of this dynamic process is a change in the composition and abundance of glycosaminoglycans (GAGs) and proteoglycans within the extracellular matrix, which influences the loss of tensile strength or stiffness of the cervix during labor. 23Na NMR spectroscopy has previously been validated as a method to quantify GAGs in tissues. In the current study, the Na+ concentration was measured at several time points through pregnancy in mouse cervices using 23Na NMR spectroscopy. The Na+ concentration increased progressively during pregnancy and peaked one day before birth followed by a rapid decline after birth. The same trend was seen in GAGs as measured by a biochemical assay using independent cervix samples over the course of pregnancy. We suggest that monitoring the Na+ concentration via 23Na NMR spectroscopy can serve as an informative physiological marker in evaluating the stages of cervical remodeling ex vivo and warrants further investigation to determine its utility as a diagnostic tool for the identification of women at risk for impending preterm birth.

VEGF Induces Cervical Epithelial Proliferation in Mice: A Molecular and Morphological Study

Cervical remodeling is a physiological inflammatory process associated with and possibly regulated by proliferating cervical epithelial. We have previously shown by SEM that administration of VEGF recombinant protein increases cervical epithelial folds in the cervix of pregnant and non‐pregnant rodents. In the present study, we show by SEM that VEGF blocker attenuates the epithelial folds and disfigures the epithelial cells. We also show by real time PCR that levels of PCNA, a marker for cellular proliferation, was up regulated in VEGF‐treated non‐pregnant mice versus control. The proliferating cell types were identified by histology and confocal microscopy. We conclude that VEGF plays a role in cervical remodeling, in part, by promoting proliferation of cervical epithelial cells and endothelial cells during angiogenesis.Grant Funding Source: Appalachian State University

VEGF up‐regulates pro‐inflammatory factors in the cervix of mice

Cervical remodeling (CR) entails a physiological inflammatory response that can be induced by inflammatory agents, such as lipopolysaccharide (LPS), and inflammation is closely associated with vascular remodeling. Recently, we showed that LPS‐induced inflammation up‐regulates mRNA levels of vascular endothelial growth factor (VEGF) and its receptors, KDR and Flt‐1. Here, we sought to investigate whether VEGF can modulate inflammation by characterizing the expression pattern of pro‐inflammatory factors in VEGF‐treated mice using real time PCR. Our data revealed that VEGF up‐regulates a group of pro‐inflammatory factors in the cervix, including IL‐6. Collectively, our studies show a possible existence of a positive feed forward loop between inflammatory and vascular factors in the remodeling cervix.Grant Funding Source: Appalachian State University

Second harmonic generation imaging as a potential tool for staging pregnancy and predicting preterm birth

We use second harmonic generation (SHG) microscopy to assess changes in collagen structure of murine cervix during cervical remodeling of normal pregnancy and in a preterm birth model. Visual inspection of SHG images revealed substantial changes in collagen morphology throughout normal gestation. SHG images collected in both the forward and backward directions were analyzed quantitatively for changes in overall mean intensity, forward to backward intensity ratio, collagen fiber size, and porosity. Changes in mean SHG intensity and intensity ratio take place in early pregnancy, suggesting that submicroscopic changes in collagen fibril size and arrangement occur before macroscopic changes become evident. Fiber size progressively increased from early to late pregnancy, while pores between collagen fibers became larger and farther apart. Analysis of collagen features in premature cervical remodeling show that changes in collagen structure are dissimilar from normal remodeling. The ability to quantify multiple morphological features of collagen that characterize normal cervical remodeling and distinguish abnormal remodeling in preterm birth models supports future studies aimed at development of SHG endoscopic devices for clinical assessment of collagen changes during pregnancy in women and for predicting risk of preterm labor which occurs in 12.5% of all pregnancies.

Cervical tissue changes in women with miscarriage: A morphological and biochemical investigation

To evaluate morphological and inflammatory events in the uterine cervix in first trimester miscarriages. Experimental study. Sahlgrenska University Hospital, Gothenburg, Sweden. Nulliparous women with first trimester symptomatic (n = 7) or silent (n = 11) miscarriage scheduled for surgical evacuation and nulliparous women scheduled for surgical termination of first trimester pregnancies (control group n = 11). Before evacuation, biopsies were obtained from the cervix. The specimens were either fixed in glutaraldehyde for electron microscopy or snap-frozen in liquid nitrogen and stored at -70 degrees C until analyses of interleukin-8 (IL-8) and matrix metalloproteinases (MMPs). Ultrastructure of cervical tissue, cervical tissue levels of IL-8, immunohistochemistry of MMP-1, MMP-8, MMP-9 and IL-8. The organization of the cervical collagen framework was deranged, the fibroblasts were reactive and the number of activated mast cells appeared to be increased in specimens from women with miscarriage compared with controls. IL-8 was significantly increased in women with miscarriage. Immunohistochemistry of MMP-1 and MMP-8 did not demonstrate any significant difference between the groups. MMP-9 was significantly lower in specimens from women with symptomatic miscarriage compared to women with silent miscarriage and women in the control group. An inflammatory-like response takes place in the cervix both in women with symptomatic and silent miscarriage. The intensity of the inflammatory response seems to be similar in the two groups. Therefore, inadequate cervical remodeling does not seem to be the reason why some miscarriages remain silent.

Matrix metalloproteinases in the cervical mucus plug in relation to gestational age, plug compartment, and preterm labor

BackgroundHigh concentrations of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been identified in the cervical mucus plug (CMP) at term of pregnancy. Their physiological and pathophysiological implications, however, remain to be elucidated, and CMPs from preterm labor have never been examined. This study was therefore conducted to describe the concentrations of MMP-2, TIMP-1, MMP-8 and MMP-9 in the CMP in relation to gestational age, IL-8 as an indicator of inflammation, compartment of the CMP, and preterm labor.MethodsAn aliquot of the distal plug compartment facing the vaginal microflora (CMP-dist) was collected from non-pregnant (n = 15), early pregnant (n = 15) and term pregnant women (n = 15). Whole CMPs shed during active vaginal term (n = 15) and preterm (n = 4) labor were also included. Protein concentrations were determined by enzyme-linked immunosorbent assay (ELISA).ResultsMMP-2 was not detectable in the non-pregnant CMP-dists whereas high concentrations were found in early pregnancy followed by an 85% decline at term. High concentrations of TIMP-1 were found in both the non-pregnant and early pregnant CMP-dists with a 90% decline at term. Consequently, the molar TIMP/MMP ratio was 40 in the non-pregnant state and 0.2 at term. The MMP-2 and TIMP-1 concentrations were alike in the CMP-dists and the whole CMPs.MMP-8, MMP-9, and IL-8 were mainly found in the distal CMP compartment. MMP-8 and MMP-9 concentrations were several fold increased in this compartment during pregnancy compared to the non-pregnant state. In the preterm whole CMPs, MMP-8, MMP-9 and IL-8 were 2 to 5 fold increased compared to term whole CMPs.ConclusionsThese results suggest that CMP MMP-2 reflects the non-leukocyte dependent cervical remodeling that occurs in early pregnancy, whereas MMP-8 and MMP-9 are involved in the defense against ascending infections primarily located to the distal compartment of the CMP. The upregulation of MMP-8, MMP-9 and IL-8 in whole CMPs from preterm labor may indicate the involvement of an intrauterine infection.

464: Targeting genetic variations in gene pathways associated with cervical remodeling

464: Targeting genetic variations in gene pathways associated with cervical remodeling

MicroRNA expression profiling of the human uterine cervix after term labor and delivery

MicroRNAs (miRNAs) are noncoding RNAs involved in posttranscriptional regulation of target genes. The objective of this study was to determine the miRNA expression profile of the human uterine cervix after spontaneous term labor (TL). The miRNA expression pattern of cervical tissue was characterized using microarrays. Samples were collected at term from patients with (n = 8) and without (n = 9) TL. Moderated t tests and false discovery rate correction were applied. Results were confirmed using quantitative reverse transcriptase-polymerase chain reaction. A total of 226 miRNAs were expressed in human cervical tissue. miR-223, miR-34b, and miR-34c were overexpressed in cervical tissue of patients with TL compared to those without. Quantitative reverse transcriptase-polymerase chain reaction assays confirmed these findings (miR-223 [fold change {FC} = 5.7], miR-34b [FC = 4.5], miR-34c [FC = 6.2]; P < .001). This is the first report of miRNA expression in the human uterine cervix in pregnancy. Cervical remodeling after TL and delivery was associated with changes in miR-223, miR-34b, and miR-34c.

Low molecular weight heparin stimulates myometrial contractility and cervical remodeling in vitro

The low molecular weight heparin, Dalteparin, shortens human labor time. The aim of this study was to investigate if the mechanism behind this effect involves myometrial contractility and cervical ripening and if the anticoagulative activity is necessary for its effect. Experimental in vitro study. Lund University and Karolinska Institute, Sweden. The effect of low molecular weight heparins with or without anticoagulative properties on myometrial contractility was measured in vitro on smooth muscle strips from biopsies obtained at elective cesarean sections. The effects on cervical ripening were assessed in cervical fibroblasts cultured from explants of cervical biopsies obtained at delivery. Mean force and number of contractions in uterine smooth muscle strips and interleukin-8 (IL-8) secretion in cervical fibroblasts. Myometrial smooth muscle strips pretreated with low molecular weight heparins showed increased contractile activity compared to untreated smooth muscle strips. Secretion of IL-8 from cultured cervical fibroblasts was significantly increased after treatment with low molecular weight heparin. Both these effects were independent of anticoagulative activity of the low molecular weight heparin. A possible underlying mechanism for the shortened labor time after low molecular weight heparin treatment is enhanced myometrial contractility and an increased IL-8 secretion in cervical fibroblast, mimicking the final cervical ripening in vivo. Our data support the notion that anticoagulant activity is not required to promote labor.

The role of transforming growth factor β in cervical remodeling within the rat cervix

Transforming growth factor beta (TGFbeta) plays a central role in extracellular matrix remodeling. We hypothesized that TGFbeta signaling is involved in cervical remodeling. This study evaluated patterns within this signaling pathway. The cervices of nonpregnant and timed pregnant rats were obtained. Messenger ribonucleic acid (mRNA) expression of TGFbeta1, TGFbeta receptor 1 (TbetaR1), TbetaR2, and TbetaR3 was evaluated. Four animals were euthanized for each time point. Western blotting was performed for protein expression. Phosphorylated mothers against decapentaplegic (Smad)-2 and -3 phosphorylation was assessed to evaluate TGFbeta activation. TGFbeta1 mRNA increased through day 21 and declined on day 22 (analysis of variance, P = .001). TbetaR1 expression was unchanged. TbetaR2 and TbetaR3 mRNA expression was similar to TGFbeta1. TbetaR3 protein expression was similar to mRNA. Smad2 phosphorylation paralleled changes in TbetaR3. Components of the TGFbeta signaling pathway increase during pregnancy along with Smad2 activation. The decline on day 22 correlates with a transition to the ripening phase supporting a role in cervical remodeling.

Milk and Relaxin Affect Expression of Proteins Important for Growth, Remodeling, and Apoptosis of the Neonatal Porcine Cervix.

Nutritional and immunological benefits of colostrum (herein referred to as milk) for neonatal growth and survival are well known. However, the role of milk as a conduit for bioactive factors that can affect neonatal development is not well understood. Immunoreactive relaxin (RLX) was detected in porcine milk and in the systemic circulation of newborn pigs only if they were allowed to nurse. In addition, the neonatal cervix is sensitive to trophic effects of RLX by postnatal day (PND) 2. Thus, the lactocrine hypothesis was proposed as a mechanism whereby milk-borne growth factors are delivered to nursing offspring where they can affect target tissues. Whether milk and/or RLX are necessary for neonatal porcine cervical development is unknown. Here, objectives were to determine effects of: 1) age and consumption of milk on expression of molecular markers of cervical growth, remodeling and apoptosis at birth (PND 0; before nursing) and on PND 2; and 2) exogenous RLX on cervical expression of targeted markers in gilts allowed to nurse versus those fed hormone-free milk replacer. Targeted proteins included estrogen receptor-alpha (ESR1) and vascular endothelial growth factor (VEGFA), both mediators of porcine cervical growth, matrix metalloproteinase 2 (MMP2), involved in tissue remodeling, and the anti- and pro-apoptotic markers BCL2 and caspase 3. Gilts (n = 4-7/group) were assigned randomly to one of four treatment groups at birth, in which they were: 1) allowed to nurse ad libitum; 2) fed milk replacer; 3) nursed and given either RLX (20 µg/kg BW, im) or vehicle every 6 h for 48 h; or 4) fed replacer and treated with exogenous RLX/vehicle for the first 48 h of life. Cervices were collected on PND 2, approximately 3 h after the last treatment. Additionally, cervices were collected from a fifth group at birth, before gilts were allowed to nurse. Protein expression was evaluated by immunoblotting and quantified by densitometry. Expression of cervical ESR1, VEGFA and BCL2 proteins, undetectable at PND 0, was induced in gilts allowed to nurse for two days, but remained undetectable in replacer-fed gilts. In contrast, MMP2 expression, also undetectable at PND 0, was induced in both nursing and replacer-fed gilts. RLX increased ESR1, VEGFA and BCL2 expression in nursing (p < 0.05) but not in replacer-fed gilts. MMP2 expression was increased by RLX treatment in both nursing and replacer groups (p < 0.01). Caspase 3 expression was undetectable at both PND 0 and 2, regardless of treatment. Data support the lactocrine hypothesis for maternal programming of neonatal development, showing that milk-borne factors are important for induction of growth and anti-apoptotic proteins in the cervix at PND 2. Data support the idea that lactocrine-acting factors may be cooperative in facilitating the actions of RLX. In the absence of milk, altered expression patterns for morphoregulatory proteins, as observed here, could affect neonatal cervical development with long-term consequences for reproductive performance and health. Data complement earlier results indicating that the neonatal porcine cervix is sensitive to RLX as reflected by increased expression of specific growth, remodeling and anti-apoptotic proteins. Thus, lactocrine-driven programming must be considered a potentially critical component of the maternal continuum of factors affecting female reproductive tract development. (Support: USDA-NRI 2003-35203-1357 and 2007-35203-18098; NSF-EPS 0814103) (poster)

VEGF Regulates Gene Expression of Matrix Factors During Cervical Remodeling in Rodents

Cervical remodeling is a physiological inflammatory process regulated by various factors including vascular factors. We have previously shown that VEGF regulates multiple genes involved in cervical remodeling using DNA microarray analysis, such as growth factors, inflammatory factors, neuronal factors and matrix proteins. Among the matrix factors regulated by VEGF are biglycan, collagen type I, II, IV and V, decorin, fibronectin and others. Here, we validate the DNA microarray data using real time PCR and found that in the gene expression of a selected number of genes were down‐regulated such as decorin. We conclude that VEGF plays a role in cervical remodeling by regulating various genes including matrix factors.

VEGF Regulates Gene Expression of Oxytocin during Pregnancy in the Cervix of Rodents

Cervical remodeling is a physiological inflammatory process regulated by various factors including vascular factors. We have previously shown that VEGF regulates multiple genes involved in cervical remodeling using DNA microarray analysis, including oxytocin. Here, we validate the DNA microarray data using real time PCR. Consistent with DNA microarray data, we found that levels of oxytocin mRNA and its receptor were up‐regulated by VEGF treatment. We conclude that VEGF plays a role in regulating levels of VEGF in the cervix of rodents.

VEGF Regulates Cervical Remodeling By Increasing Epithelial Folds During Pregnancy in Rodents

Cervical remodeling is a physiological inflammatory process which is associated and possibly regulated by proliferating cervical epithelial. We have previously shown that levels of VEGF increase with advancing pregnancy in rodents (mice and rats). Here, we show that as VEGF increases, the folds in the surface epithelium of the cervix increase as pregnancy advances in untreated rodents, which we interpreted as increased epithelial growth. We also show that ovariectomized nonpregnant rodents treated with VEGF have increased epithelial folds versus control using SEM.. We conclude that VEGF plays a role in cervical remodeling as pregnancy progresses by an increase in epithelial proliferation.

Delineation of VEGF‐regulated genes and functions in the cervix of pregnant rodents by DNA microarray analysis

VEGF‐regulated genes in the cervices of pregnant and non‐pregnant rodents were delineated by DNA microarray and Real Time PCR, after locally altering levels of or action of VEGF using VEGF agents, namely siRNA, VEGF receptor antagonist and mouse VEGF recombinant protein. Tissues were analyzed by genome‐wide DNA microarray analysis, Real‐time and gel‐based PCR, and SEM, to decipher VEGF function during cervical remodeling. Data were analyzed by EASE score (microarray) and ANOVA (Real Time PCR) followed by Scheffe's F‐test for multiple comparisons. Of the 30,000 genes analyzed, about 4,200 genes were altered in expression by VEGF. Based on EASE score, i.e., grouping of genes according to their biological process, cell component and molecular functions, a number of vascular‐ and non‐vascular‐related processes were found to be regulated by VEGF in the cervix, including immune response, cell proliferation, protein kinase activity, and cell adhesion molecule activity. Of interest, mRNA levels of a select group of genes were altered. VEGF agents also altered mRNA levels of decorin, which is involved in cervical collagen fibrillogenesis, and expression of eNO, PLC and PKC mRNA, critical downstream mediators of VEGF. We show that VEGF may regulate cervical epithelial proliferation, as revealed by SEM. These data are important in that they shed new insights in VEGF's possible roles and mechanisms in cervical events near‐term, including cervical remodeling.

Medroxyprogesterone Acetate Modulates Remodeling, Immune Cell Census, and Nerve Fibers in the Cervix of a Mouse Model for Inflammation-induced Preterm Birth

To determine whether a progestational agent can modify inflammation-induced preterm cervical ripening, mice on day 15 of gestation were given an intrauterine injection of (1) saline, (2) lipopolysaccharide, (3) an intramuscular injection of medroxyprogesterone acetate alone prior to lipopolysaccharide, or (4) medroxyprogesterone acetate alone. Cervices were obtained 6 hours later, then fixed, sectioned, and processed to stain collagen structure or to identify immune cells or nerve fibers. Cervical remodeling was induced by lipopolysaccharide treatment compared with that in saline controls, an effect blocked by medroxyprogesterone acetate pretreatment. Moreover, lipopolysaccharide reduced macrophages and enhanced neutrophils in the cervix, effects also forestalled by medroxyprogesterone acetate pretreatment. Although the density of nerve fibers was not altered by lipopolysaccharide, medroxyprogesterone acetate reduced innervation in the cervix. Thus, progestational treatment forestalls the inflammation-induced reduction in collagen structure and immune cell traffic through a mechanism that is independent of nerve fiber density. These findings raise the possibility that progestational treatment may regulate ripening of the cervix early in the process leading to preterm birth.

Temporal Changes in Myeloid Cells in the Cervix during Pregnancy and Parturition

Preterm birth occurs at a rate of 12.7% in the U.S. and is the primary cause of fetal morbidity in the first year of life as well as the cause of later health problems. Elucidation of mechanisms controlling cervical remodeling is critical for development of therapies to reduce the incidence of prematurity. The cervical extracellular matrix must be disorganized during labor to allow birth, followed by a rapid repair postpartum. Leukocytes infiltrate the cervix before and after birth and are proposed to regulate matrix remodeling during cervical ripening via release of proteolytic enzymes. In the current study, flow cytometry and cell sorting were used to determine the role of immune cells in cervical matrix remodeling before, during, and after parturition. Markers of myeloid cell differentiation and activation were assessed to define phenotype and function. Tissue monocytes and eosinophils increased in the cervix before birth in a progesterone-regulated fashion, whereas macrophage numbers were unchanged. Neutrophils increased in the postpartum period. Increased mRNA expression of Csfr1 and markers of alternatively activated M2 macrophages during labor or shortly postpartum suggest a function of M2 macrophages in postpartum tissue repair. Changes in cervical myeloid cell numbers are not reflected in the peripheral blood. These data along with our previous studies suggest that myeloid-derived cells do not orchestrate processes required for initiation of cervical ripening before birth. Additionally, macrophages with diverse phenotypes (M1 and M2) are present in the cervix and are most likely involved in the postpartum repair of tissue.