Abstract
Objective. The aim of this study was to identify changes in the cervical transcriptome in the human uterine cervix as a function of ripening before the onset of labor.
Study Design. Human cervical tissue was obtained from women at term not in labor with ripe (n = 11) and unripe (n = 11) cervices and profiled using Affymetrix GeneChip® HGU133Plus2.0 arrays. Gene expression was analyzed using a moderated t-test (False Discovery Rate 5%). Gene ontology and pathway analysis were performed. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used for confirmation of selected differentially expressed genes.
Results. (1) Ninety-one genes were differentially expressed between ripe and unripe groups. (2) Cervical ripening was associated with enrichment of specific biological processes (e.g. cell adhesion, regulation of anatomical structure), pathways and 11 molecular functions (e.g. extracelluar matrix (ECM)-structural constituent, protein binding, glycosaminoglycan binding). (3) qRT-PCR confirmed that 9 of 11 tested differentially expressed genes (determined by microarray) were upregulated in a ripe cervix (e.g. MYOCD, VCAN, THBS1, COL5A1). (4) Twenty-three additional genes related to ECM metabolism and adhesion molecules were differentially regulated (by qRT-PCR) in ripe cervices.
Conclusion. (1) This is the first description of the changes in the human cervical transcriptome with ripening before the onset of labor. (2) Biological processes, pathways and molecular functions were identified with the use of this unbiased approach. (3) In contrast to cervical dilation after term labor, inflammation-related genes did not emerge as differentially regulated with cervical ripening. (4) Myocardin was identified as a novel gene upregulated in human cervical ripening.
Keywords:
- Cervix
- versican
- collagen
- ripe cervix
- matrix metalloproteinase
- ADAMTS
- cell adhesion
- regulation of anatomical structure
- regulation of locomotion
- extracellular matrix structural constituent
- structural molecule activity
- integrin binding
- glycosaminoglycan binding
- polysaccharide binding
- heparin binding
- actin filament binding
- cytoskeletal protein binding
- myocardin
Introduction
Cervical ripening is a critical component of the common terminal pathway of parturition, which also includes increased myometrial contractility and membrane/decidual activation [Citation1]. The mechanisms of cervical change in pregnancy have been investigated in animals as well as in humans [Citation2–25]. Critical hypothesis-driven studies of cervical biology have resulted in an improved understanding of the changes that occur in the cervical extracellular matrix during pregnancy and labor and delivery [Citation2,Citation3,Citation5,Citation6,Citation9–11,Citation18–20,Citation23,Citation26–36]. However, the current knowledge of the mechanisms involved in cervical change during pregnancy does not provide a complete understanding of the processes involved in human cervical ripening.
The use of high-dimensional biology techniques allows for the examination of the genome, transcriptome [Citation15] proteome, and metabolome. The study of the transcriptome (changes in gene transcription) in a particular tissue provides a comprehensive, systematic and unbiased description of genes differentially expressed in a specific condition or point in time [Citation15]. The aim of this study was to characterize the cervical transcriptome in patients with a ripe cervix at term before the onset of labor compared to those with an unripe cervix.
Materials and Methods
Study design
A cross-sectional study was performed in patients undergoing elective cesarean section at term with an unripe (n = 11) and ripe cervix (n = 11). As has been used in previous studies of cervical biology in pregnancy, a cervix with a Bishop score of ≥5 was defined as ripe [Citation11]. Patient inclusion criteria were as follows: (1) term gestation (≥37 weeks); (2) no prostaglandin or oxytocin administration; (3) absence of histologic chorioamnionitis; (4) negative Neisseria gonorrhoeae and Chlamydia trachomatis determined by examination of cervical secretions; and (5) a normal Pap smear. Patients were invited to participate in a study which was approved by the Institutional Review Board and provided written, informed consent.
Patients underwent cervical biopsy following elective cesarean section without signs of labor. This procedure has been used extensively by investigators in the United States, Europe and other continents [Citation2,Citation3,Citation6,Citation7,Citation9–11,Citation13,Citation15,Citation16,Citation18,Citation23,Citation25–28,Citation31,Citation35,Citation37–40]. Half centimeter biopsies were obtained transvaginally from the anterior lip of the cervix at the 12 o'clock position and immediately snap frozen in liquid nitrogen or placed in RNAlater® (Ambion, Inc., Austin, TX) and stored at –70°C. No patients experienced complications from the cervical biopsy. The clinical and demographic data, obstetric and gynecological history, as well as pregnancy outcome were extracted from medical records.
Microarray Analysis
Microarray analysis was performed using the Affymetrix GeneChip® HGU133Plus2.0 arrays. Microarray statistical analysis included: (1) data preprocessing using the RMA algorithm [Citation41]; (2) calculation of nominal p-values using a moderated t-test and combining nominal p-values of all probesets of the same gene; (3) genes with the false discovery rate (FDR) < 0.05 were considered statistically significant. Pathway analysis was performed on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database using both an enrichment analysis and the Signaling Pathway Impact Analysis (SPIA) [Citation42,Citation43]. Gene ontology analysis was performed using the GOstats package of Bioconductor [Citation44].
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Real-time polymerase chain reaction (PCR)-based human extracellular matrix and adhesion molecules RT2Profiler PCR Array (SA Bioscience Corporation, Frederick, MD) was used to screen the expression of 84 key related genes according to the manufacturer's instructions. The validation of the results of the microarray for the myocardin gene was done individually by qRT-PCR analysis.
Results
Demographic and clinical characteristics of the study population are depicted in .
Table I. Study subjects – patient demographics and clinical characteristics.
Microarray analysis
Microarray analysis revealed that 91 genes were differentially expressed in the cervical tissue of patients not in labor with a ripe cervix when compared to those not in labor with an unripe cervix. Interestingly, 83 of 91 genes were upregulated. The list of differentially expressed genes is presented in , which describes the fold change and FDR.
Table II. Results of Microarray Analysis.
Gene Ontology enrichment analysis [Citation45] was used to gain insight into the biology defined by differential gene expression. This analysis revealed that 11 biological processes and 11 molecular functions were significantly enriched (). The focal adhesion and extracellular matrix–receptor interaction pathways were found to be significant by both SPIA and enrichment analysis (FDR < 0.05).
Table III. Gene ontology analysis.
Quantitative reserve transcription-polymerase chain reaction (qRT-PCR)
Of the 85 genes tested by qRT-PCR (84 in the RT2Profiler PCR Array and myocardin), 32 genes showed differential mRNA expression in patients with a ripe cervix when compared to those with an unripe cervix (). We were able to confirm the findings of microarray for 9 of 11 tested genes whose expression was also upregulated by microarray analysis in patients with a ripe cervix. Myocardin (MYOCD), versican (VCAN), thrombospondin 1 (THBS1), and collagen, type V, alpha1 (COL5A1) were among the genes upregulated in patients with a ripe cervix (p < 0.05) in both the microarray and qRT-PCR assays ( and ). Cadherin 1, type 1, E-cadherin (epithelial) (CDH1) was significantly downregulated in the microarray analysis; this result was confirmed by qRT-PCR. Furthermore, 22 additional genes related to extracellular matrix metabolism and adhesion molecules were upregulated in ripe cervices as demonstrated by the multiplex PCR array (e.g. ADAM metallopeptidase with thrombospondin type I motif, 8 (ADAMTS8), vascular adhesion molecule 1 (VCAM1), collagen types IV, alpha 2 (COL4A2) thrombospondin 1 (THBS1), and TIMP metallopeptidase inhibitor 1 (TIMP1) (). The results of the qRT-PCR for all 32 genes matched the direction of change or demonstrated significance as suggested by the microarray data.
Table IV. Results of qRT-PCR: 32 genes with differential expression between ripe versus unripe cervix in patients at term not in labor.
Figure 1. Results of qRT-PCR assay of versican in cervical tissue in patients at term without labor: unripe cervix versus ripe cervix.
Figure 2. Results of qRT-PCR assay of myocardin in cervical tissue in patients at term without labor: unripe cervix versus ripe cervix.
Comment
Principal findings of the study
(1) This genome-wide study has demonstrated that 91 genes were differentially expressed in patients at term not in labor with a ripe cervix when compared to those with an unripe cervix (83 upregulated and 8 downregulated). (2) Gene Ontology analysis indicated that cervical ripening was associated with enrichment of specific biological processes (e.g. cell adhesion, regulation of anatomical structure, regulation of locomotion and phosphate transport) and 11 molecular functions (e.g. extracellular matrix structural constituent, protein binding, glycosaminoglycan binding, heparin binding). (3) Pathway analysis identified involvement of focal adhesion, extracellular matrix-receptor interaction, cell communication and cell adhesion molecule pathways in the transcriptome differences between ripe and unripe cervices. (4) Genes previously reported to be involved in cervical remodeling (e.g. versican, biglycan, decorin) were upregulated in the cervical tissue of patients with a ripe cervix. (5) This study identifies a new set of genes involved in cervical ripening, such as myocardin, ADAMTS8 and delta-2-catenin. Many other genes not previously known to be differentially regulated with cervical ripening were also identified. (6) In contrast to cervical dilation after term labor, [Citation12,Citation15] inflammation-related genes did not emerge as differentially expressed with cervical ripening.
Importance of the study
Disorders of cervical ripening complicate term (e.g. arrest of dilatation or protracted dilatation) and preterm (e.g. premature cervical dilation in the midtrimester) pregnancies. Human cervical biopsy specimens have been obtained from patients in preterm and term labor, preterm and term nonlabor and nonpregnant patients after a hysterectomy to describe the cervical extracellular matrix and its relationship to abnormal labor [Citation2–13,Citation15,Citation16,Citation18,Citation25,Citation28,Citation46–48]. Animal studies have also provided insight into the changes in extracellular matrix that occur in the uterine cervix during pregnancy [Citation17,Citation19–22,Citation28,Citation33,Citation34,Citation36,Citation49–54]. Studies of cervical biopsy tissue have been conducted in pregnant women as early as 1960 [Citation18]. Yet, the precise mechanism of cervical ripening in human pregnancy has not been fully elucidated. The current study represents the first description of the changes in the human cervical transcriptome in unripe versus ripe cervices. Some of our results confirm differential expression of genes previously implicated in cervical ripening, such as those involved in extracellular matrix metabolism and cell adhesion molecules. Interestingly, the novel finding of increased expression of myocardin in patients with a ripe cervix when compared to those with an unripe cervix was demonstrated. In addition, inflammation-related genes were not differentially expressed in patients with cervical ripening. The results reported herein characterize the processes involved in cervical ripening in humans before the onset of labor. An unbiased microarray analysis of the cervical tissue was carried out, followed by confirmation of selected genes by the use of qRT-PCR. A separate study must be conducted to confirm our results with an independent set of samples. Such studies are not easy to conduct because of the difficulties in obtaining these samples.
The cervix is comprised of smooth muscle and extracellular matrix, which consists of collagen, elastin, proteoglycans, and glycoproteins such as fibronectins [Citation55,Citation56]. The proteoglycans found in the cervix include decorin, fibromodulin, biglycan, versican, aggrecan and heparan sulfate proteoglycan [Citation35,Citation38,Citation57,Citation58]. Examination of cervical biopsies from nonpregnant women with a history of cervical insufficiency suggests that increased distensibility of the cervix during pregnancy can be a result of prepregnancy decreased collagen concentration and perhaps a prepregnancy increased smooth muscle content, i.e. a congenital abnormality of the cervix [Citation27,Citation59,Citation60]. Some investigators have suggested that the period of cervical ripening can be divided into a ‘slow’ and a ‘fast’ phase [Citation35]. The current study suggests that the ‘slow’ ripening process is upregulated in women with a Bishop score of 5 or more before the start of labor, whereas there is no indication of activation of the ‘fast’ ripening process involving inflammatory mediators. Furthermore, the upregulation of myocardin suggests that high muscle content in the cervix should be considered as a possible etiology for a ripe cervix.
Known and novel processes involved in cervical ripening during pregnancy
Collagen types IV, V and VI
Our study demonstrated an increased mRNA expression (both in microarray and qRT-PCR) of collagen types IV (alpha 2), V (alpha1), and VI (alpha 2) in patients with a ripe cervix. Collagens type IV, V, and VI have not previously been studied during cervical ripening. Collagen type IV, alpha 2 is the major structural component of basement membranes and interacts with laminin and proteoglycans [Citation32,Citation61–63]. Collagen type V has been implicated as a critical determinant of fibril structure and matrix organization [Citation64], while collagen type VI is found in most tissues and interacts with type IV collagens and the basement membrane and the surrounding matrix [Citation65]. The major structural collagens, types I and III, that dominate the cervix quantitatively, were not differentially regulated in this study, despite the fact that there is a known decrease in their concentrations in the cervix during pregnancy at term [Citation31].
Proteoglycans
Versican, a large extracellular matrix proteoglycan, was upregulated 3-fold by both microarray and qRT-PCR analysis in patients with a ripe cervix. Biglycan (1.45-fold) and decorin (1.39-fold) were also upregulated in patients with a ripe cervix as demonstrated by microarray analysis. These proteoglycans have many functions within the extracellular matrix which include effects upon collagen disorganization, cell adhesion, migration and proliferation [Citation66].
Of interest, the molecular function term ‘heparin binding’ was significant after Gene Ontology analysis of the differentially regulated genes. Recently, the role of heparin in cervical remodeling has been examined. Ekman-Ordeberg and colleagues. [Citation67] have demonstrated that low molecular weight heparin increased IL-8 secretion in cervical fibroblasts. This area of research has great potential for targeting the mechanisms involved in cervical change during pregnancy.
Metalloproteinases
Matrix metalloproteinases (MMPs) are major regulators of the extracellular matrix [Citation68] and have been implicated as possible mediators in the cervical remodeling process by cleaving one or more constituents of the extracellular matrix [Citation50,Citation69]. In ripe cervices, matrix metalloproteinase-3 (MMP-3, stromelysin) was upregulated 5-fold by qRT-PCR but not by microarray analysis. MMP-3 degrades fibrillin, a glycoprotein that is critical for the formation of elastic fibers in connective tissue [Citation70]. In addition, administration of antiprogesterone in a rabbit model results in augmentation of MMP-3 in the uterine cervix [Citation71]. The finding reported here, of a 5-fold increase in MMP-3 mRNA expression in the cervical tissue of patients with a ripe cervix when compared to patients with an unripe cervix, suggests a role for MMP-3 in human cervical ripening.
Furthermore, MMP-3 has been shown to cleave fibrinogen, cross-linked fibrin, the cell adhesion molecule E-cadherin and exhibits proteolytic activity on laminin, alpha-2-macroglobulin, fibronectin, casein, and alpha-1-antitrypsin [Citation72–74]. In this study, E-cadherin (CDH1), a calcium dependent cell–cell adhesion molecule, was significantly downregulated (by qRT-PCR) while laminin and fibronectin (by both the microarray and qRT-PCR analysis) were upregulated in patients with a ripe cervix when compared to those with an unripe cervix in.
In addition, the molecular function categories of actin filament binding, actin binding, and cytoskeletal binding were among those that were significant in this study. The interaction of E-cadherin with the actin cytoskeleton has been shown to be directly regulated by the epidermal growth factor receptor in a breast cancer cell line [Citation75]. Further study is required to elucidate the mechanism of action of E-cadherin as it relates to MMP-3, fibronectin, laminin, and actin in human cervical ripening.
Delta-2-catenin (CTNND2) mRNA was increased by 5-fold in patients with a ripe cervix when compared to those with an unripe cervix. Delta-2-catenin is involved in cell adhesion and movement [Citation76] and has not previously been described as playing a key role in cervical ripening or remodeling in pregnancy. In addition, delta-catenin interacts with E-cadherin and beta-catenin and has been implicated in the organization of cell–cell junctions [Citation77]. The precise role of delta-2-catenin in cervical ripening is unknown, and future research in this area is warranted.
We found that ADAMTS8 and ADAMTS1 mRNA expression were increased in the cervical tissue of patients with a ripe cervix when compared to those with an unripe cervix. ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) is thought to participate in the degradation of extracellular matrix [Citation78,Citation79]. Members of the ADAMTS family are secreted enzymes, and several of these bind to the extracellular matrix. The proteases ADAMTS-4, -5, and -8 degrade aggrecans and hence have been designated as ‘aggrecanases’ [Citation79]. ADAMTS8 cleaves aggrecan at the aggrecanase-susceptible Glu373-Ala374 peptide bond [Citation80]. In addition, ADAMTS-1 and -4 cleave versican [Citation81,Citation82]. Ruscheinsky [Citation83] demonstrated an upregulation of ADAMTS1 in the cervix before birth in a mouse model. Thus, the cleavage of aggrecan and versican by ADAMTS-1 and -8 might contribute to the changes in proteoglycans observed during the process of cervical ripening before the onset of labor in humans.
Cervical ripening and lack of differential expression of inflammation-related genes
Liggins was the first to propose that cervical ripening can be likened to an inflammatory process [Citation3]. This concept was based largely upon histological observations of the uterine cervix after cervical ripening. However, recently, the role of inflammation in cervical ripening has been debated [Citation14,Citation84,Citation7,Citation10,Citation13]. Sakamoto [Citation10] reported that there was no correlation between the degree of clinical cervical ripening and IL-8 concentrations in cervical tissue. In contrast, IL-8 concentrations in the cervical tissue increased after labor and delivery. In a mouse model with a transgene insertion on chromosome 6, Word et al. [Citation14] reported that parturition did not occur despite uterine contractions because of a rigid nonelastic cervix at term. Unexpectedly, cervical ripening was not observed following the infiltration with neutrophils and macrophages in the cervical tissue [Citation14]. Similarly, Timmons and Mahendroo [Citation84] have challenged the importance of the influx of inflammatory cells as a major regulatory event of cervical ripening using steroid 5 alpha-reductase type 1 null mice (Srd5a1–/–). The investigators concluded that cervical ripening does not require activation of a typical inflammatory response, because macrophages, eosinophils, and myloperoxidase activity did not increase during cervical ripening. Moreover, depletion of neutrophil numbers (after injection of a rat anti-mouse monoclonal antibody directed against Ly6G (GR1), an antigen on the surface of mature murine neutrophils) before birth has no effect on the timing or success of parturition [Citation84]. Of importance, experiments conducted in tissues collected before the onset of labor and after vaginal delivery demonstrated overexpression of genes involved in neutrophil chemotaxis, apoptosis, and extracellular matrix regulation [Citation12,Citation15]. However, these studies should not be interpreted to represent the biology of cervical ripening because the observed alterations in gene expression may be due to the process of parturition (dilatation, remodeling, etc.) rather than the events that prepare the cervix for the onset of labor.
In the present study, increased mRNA expression (based upon microarray and qRT-PCR analysis) of fibronectin 1 (FN1), laminin, gamma 1, laminin alpha 2, collagen type IV alpha 2, collagen type V alpha 1 and collagen type VI alpha 2 was demonstrated in patients with a ripe cervix when compared with those with an unripe cervix. These genes are known to be involved in the focal adhesion, extracellular matrix interaction, and cell communication pathways. In addition, the novel genes encoding for delta-2-catenin and myocardin were upregulated in patients with a ripe cervix. In contrast, genes involved in the inflammatory pathway were not differentially regulated based upon microarray analysis. It is possible that the changes represented in this study are those of early cervical ripening.
A novel gene involved in cervical ripening – myocardin
Myocardin mRNA expression was upregulated in both microarray and qRT-PCR analysis (3.6-fold) in the cervical tissue of patients with a ripe cervix. This is a new factor possibly involved in cervical ripening that has never been described before as playing a role in this process. Myocardin, expressed in smooth and cardiac muscle lineages, has been named as a serum response factor transcriptional coactivator [Citation85–87]. Myocardin activates smooth muscle differentiation, can carry out this function in nonmuscle cells, and has been described as a ‘master regulator’ of smooth muscle gene expression [Citation88]. Although the uterine cervix only contains 10–15% smooth muscle [Citation26], cervical ripening may not only include changes in the extracellular matrix, but also alterations in the smooth muscle component of the cervix. These findings require further investigation into the mechanism, localization, and significance of myocardin in cervical change in the pregnant uterine cervix.
Human cervical ripening
The traditional view is that cervical ripening occurs during the last few weeks of pregnancy prior to the onset of labor. Indeed, the Bishop score, which is widely used to assess the state of cervical ripening, was first introduced as a method to predict the likelihood that a patient would go into spontaneous labor based upon digital examination of the cervix (effacement, dilatation, consistency and position). Although attempts have been made to generate an objective definition of cervical ripening, clinical examination remains the standard (Bishop score or modification of this system). The clinical diagnosis of cervical ripening in animals presents challenges. The conduction of this study in pregnant women in which the Bishop score has been determined allows examination of the relationship between cervical ripening in the human and the transcriptome.
More importantly, the mechanism of action of specific treatments (e.g. prostaglandins or mechanical devices) is not known. Cervical ripening is likely the result of several processes that may involve more than changes in the extracellular matrix. Deciphering the mechanisms of normal cervical ripening is a first step to understand premature or protracted cervical change during pregnancy.
Strengths, limitations and future investigations
This study represents the first description of the changes in the human cervical transcriptome in ripe versus unripe cervices. Contrary to former hypothesis driven studies on cervical ripening, this method is unbiased thus allowing for discovery of new pathways. Some of our results confirm differential expression of genes previously implicated in cervical ripening before the onset of labor. Furthermore, novel genes including myocardin were suggested.
Our results provide several hypotheses for future exploration. The validation of the reported results by the use of a second set of samples will be required. In addition, the analysis of a larger sample size would be optimal. The role of myocardin in cervical ripening must be validated and further characterized by the performance of studies of immunohistochemistry and protein expression. In addition, analysis of the cervical tissue will allow for examination of smooth muscle staining. Furthermore, histological examination of the tissue of patients with a ripe cervix should be analyzed for the presence of infiltration of neutrophils and macrophages to further elucidate the role of inflammation in cervical ripening. Of importance, follow-up studies must include a demonstration of changes in protein expression for those genes that have been reported to be significantly altered in patients with a ripe cervix.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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