Abstract
Endometriosis affects approximately 12% of reproductive-age women and is currently diagnosed using invasive laparoscopic surgery. Differences in gene expression in the eutopic endometrium between women with and without endometriosis have been reported, and determining the reproducibility of these genetic differences in the endocervical epithelium would represent an important step toward developing novel diagnostic strategies. In this study, we analyzed gene expression in the endocervical epithelium in women with and without moderate or severe endometriosis. Using RT2 Profiler PCR Arrays, we analyzed gene expression in endocervical epithelial cells from women with deep endometriosis (n = 4) and healthy women (n =6). Nine genes were identified as being upregulated: 5 cell cycle genes (cyclin B1 [CCNB1], cyclin G1 [CCNG1], cullin 1 [CUL1], general transcription factor IIH, polypeptide 1 [GTF2H1], and proliferating cell nuclear antigen [PCNA]), 3 cytokine genes (C3, chemokine (C-C motif) ligand 21 [CCL21], and chemokine (C-X-C motif) ligand 14 [CXCL14]) and 1 gene related to dendritic cell pathways (ICAM2), showing that differential gene expression is present in the endocervical epithelium of women with deep endometriosis.
Introduction
The multifactorial chronic inflammatory characteristic of endometriosis, which is characterized by the implantation and development of endometrial tissue outside the uterus, is known. 1 Studies on the pathogenesis of this condition have revealed phenotypic differences between the eutopic endometrium of women with endometriosis and that of healthy women, and these differences include a higher capacity for cell adhesion, increased levels of the enzymes responsible for extracellular matrix degradation (matrix metalloproteinases), increased expression of angiogenic factors (ie, vascular endothelial growth factor), and disturbances in the cell cycle. 2 –5
The origin of these phenotypic differences in the endometrium of women with endometriosis has been extensively investigated. Previous studies on endometriosis-linked genes, different pathways associated with inflammatory cytokines, 6,7 the cell cycle, 8 and dendritic cells (DCs) 9,10 have been conducted. Moreover, focused attention has been given to the endometrial action of progesterone due to its key influence on the structure of endometrial cells. The endometrium of women with endometriosis possesses a degree of progesterone resistance 11 which may explain the described phenotypic changes in these women. An in vitro study evaluated a culture of endometrial stromal cells from women with endometriosis and healthy women treated with progesterone and revealed a number of differentially expressed genes, supporting the effect of progesterone in the endometriosis phenotype. 12 Additionally, significant differences in the expression of genes, including genes regulated by progesterone, 13 have been detected in the eutopic endometrium of women with endometriosis. 14
The endocervical epithelium is Mullerian in terms of embryological origin, similar to the endometrium, 15 and contains progesterone receptors that cause it to be susceptible to progesterone action during the menstrual cycle. 16 –18 Since differentially expressed genes have already been described as altered in endometrium of women with endometriosis, we hypothesized that the endocervix could also present some gene expression changes representative of these endometrial modifications observed in patients with endometriosis. It is worth of noting that this approach has not been studied in endocervix of women with endometriosis, and therefore, the aim of the current study is to investigate the gene expression profile in the endocervical epithelium of women with deep endometriosis, allowing the identification of differentially expressed genes in women with endometriosis.
Materials and Methods
This is a transversal study conducted between January 2012 and September 2014 which included 10 patients presenting with endometriosis (n = 4) or not (n = 6) who attended the Gynecology Department of a public tertiary hospital in Brazil. The study was approved by the Ethics Committee (Ref. n 1360/10), and informed consent was obtained from all of the participants.
Study Participants and Sample Collection
Women diagnosed with deep infiltrating endometriosis by magnetic resonance imaging, whose diagnosis was confirmed by histology of an endometriotic lesion obtained by laparoscopy, who had no other pelvic pathology and were receiving follow-up care for endometriosis in a pelvic pain unit of a public tertiary hospital in Brazil were eligible for this study. The healthy women included those who underwent laparoscopy for tubal ligation during the same period. The absence of endometriosis or other pelvic pathologies was confirmed by observations during laparoscopy.
All of the patients included in the study reported regular menstrual cycles (length of 24-38 days) and a normal cervical screening with a Papanicolaou test at least 6 months prior to sample collection. None of the women had received hormonal preparations in the 3 months preceding the sample collection for this study, and endocervical swabs from all of the participants were obtained during the proliferative phase of their menstrual cycle, as identified by the menstrual cycle length. An endocervical brush was used to collect the samples at most 30 days prior to surgery. After collection, the brushes were immediately placed into microtubes and stored at −80°C until analysis.
Polymerase Chain Reaction Array
The total RNA from each sample was extracted using the TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, California), following the manufacturer’s instructions. The RNA concentration and quality (260:280 ratio) were assessed using a NanoDrop instrument (Thermo Scientific, Wilmington, Delaware). Reverse transcription was performed with 0.5 µg of total RNA to generate double-stranded complementary DNA using an RT2 First Strand Kit (SA Biosciences/Qiagen, Frederick, MD, United States).
Real-time quantitative polymerase chain reaction (PCR) assays were performed to evaluate gene expression. We used PCR arrays for the following signaling pathways: the human cell cycle (PAHS-020, RT2 Profiler PCR Array; SA Biosciences/Qiagen), human inflammatory cytokines and receptors (PAHS-011, RT2 Profiler PCR Array; SA Biosciences/Qiagen), and human dendritic and antigen-presenting cells (PAHS-406, RT2 Profiler PCR Array; SA Biosciences/Qiagen). The 96-well PCR array plates were assayed using a Step One Plus Real-Time PCR System (Applied Biosystems). We obtained cycle threshold (Ct) values for the genes under investigation, and the ΔΔCt method was utilized for gene expression analysis using the PCR Array Data Analysis Web Portal (SA Biosciences).
Statistical Analyses
Student t test was used to compare the levels of each of the evaluated genes between the healthy and the endometriosis groups. Genes that presented expression levels with a fold change of at least ≥2 and P ≤ .05 were considered to be differentially expressed. The differentially expressed genes are discussed based on their function, as determined using an online database.
Results
The age of the patients was similar between the groups (endometriosis, 36.2 ± 1.71 years; healthy, 37.8 ± 2.64 years; P = .333). Among the 3 assessed pathways, patients with endometriosis had 9 genes that were upregulated in the endocervix compared to the levels observed in the healthy participants. Five of these upregulated genes are found in cell cycle signaling pathways (CCNB1, CCNG1, CUL1, GTF2H1, and PCNA), and 3 of the upregulated genes are found in cytokine signaling pathways (C3, CCL21 and CXCL14). One of the genes related to dendritic cell pathways (ICAM2) presented substantially higher expression (Figure 1). Additionally, another gene (CXCL12, P = .07) in the dendritic cell pathway as well as 4 genes in the cell cycle and cytokines group (CUL2, P = .06; CCR5, P = .08; ATP-binding cassette, sub-family F (GCN20), member 1 [ABCF1], P = .06; and CCL13, P = .06) did not reach statistical significance; however, as the difference was remarkable, these genes will be analyzed in subsequent study.
Significant fold changes in the endocervix of women with deep endometriosis compared to healthy women. *P ≤ .05 and **P > .05 and <.1. Quantitative real-time polymerase chain reaction (qRT-PCR) array analysis of the gene expression profile in the endocervix of women with deep endometriosis and healthy women.
Three hierarchical clustering analyses, one for each signaling pathway studied, were performed using genes that were upregulated in the patients with deep infiltrating endometriosis compared to the controls (Figure 2). The clustering analysis of the cytokine pathway showed a perfect separation of the control and the endometriosis groups (Figure 2A); however, the cell cycle and dendritic cell pathways presented a partial clustering between the groups (Figures 2B and C).
Hierarchical clustering diagrams of the differentially expressed genes belonging to the 3 signaling pathway studied in the endocervical epithelium of patients with deep endometriosis (fold change ≥2.5 and P < .1). (A) cytokine pathway; (B) cell cycle pathway; (C) dendritic cell pathway.
Discussion
Although endometriosis significantly impairs women’s quality of life due to pelvic pain or infertility, it takes an average of 7 years to be diagnosed. 19 This period is sufficient to prompt a mild version of disease progression, characterized by deep lesions. A recent study used genomic data derived from 148 archived endometrial samples of women with or without endometriosis across the menstrual cycle and evaluated the expression of specific genes. The authors described that best classifiers could identify endometriosis with 90% to 100% accuracy and can therefore detect pelvic endometriosis with high accuracy. 20 Motivated by the identification of several genes that are differentially expressed in the eutopic endometrium of women with endometriosis, we chose to further investigate the endometriosis-related gene expression profile in endocervical epithelial cells as a new tool for the possible identification of endometriosis. We evaluated the expression of 3 different PCR array signaling pathways, namely, the inflammatory cytokine, cell cycle, and dendritic cell pathways, resulting in a total of 252 genes, in patients with deep infiltrating endometriosis and healthy controls.
We found genes that were upregulated in the endocervical epithelium of the endometriosis group compared to the healthy control for all 3 signaling pathways, and most of the identified genes were associated with inflammatory mediators, cytokines, and cell cycle regulators. The association between endometriosis and immune system disorders has been established for many years. 21 The inflammatory response appears to play a fundamental role in endometriosis by causing an impaired clearance of endometrial cells. 22,23 Additionally, several studies have reported cell cycle disturbances in the eutopic endometrium of women with endometriosis. 6,7 Increased levels of cell proliferation, decreased apoptosis, and alterations in cell cycle control have emerged as important mechanisms responsible for the development of the disease. 24
Some of the genes found to be differentially expressed in our study were previously related to endometriosis pathogenesis. Human and animal studies have shown that complement factor C3 is irregularly expressed in women with endometriosis, corroborating our results, which showed that the C3 expression level in women with endometriosis was 12.6-fold higher than that in healthy women. Infertile women with endometriosis have higher C3 levels in their peritoneal fluid compared to healthy women, 25 and rats with experimentally induced endometriosis had 7.8-fold higher levels of C3 expression than those without the disease. 26
Similarly, the expression of the CCL21 gene varies across the menstrual cycle and is relatively higher in women with endometriosis. The activity of this gene is linked to immune surveillance and leads to the attraction of B cells and DCs, 27 reinforcing our finding that CCL21 is highly expressed (11.8-fold higher) in the endocervical epithelium. Additionally, the CXCL14 gene is a potent inducer of cancer-associated fibroblasts, 28 and considering the invasiveness of endometrial foci, this finding suggests a possible connection between CXCL14 and the etiopathology of endometriosis because we demonstrated that the expression of CXCL14 in the endometriosis group was 12.5-fold higher than that in the control group.
The CCNB1 gene has been previously studied in women with endometriosis because its expression is higher in endometriotic lesions and mediates ectopic endometrial cell proliferation under the regulation of ovarian hormones. 29 In the present study, CCNB1 was also very highly expressed. The PCNA gene is a marker of cell proliferation that has been studied in the context of endometriosis and endometrial cancer. Expression of PCNA correlates with aggressive growth in specific cell types and has been found to be higher in patients with deep endometriosis compared to superficial endometriosis and even higher in endometrial adenocarcinoma. 30 We also demonstrated that PCNA expression in the endocervical epithelium is 7.0-fold higher in the endometriosis group compared to the control group. The CCNG1, CUL1, and GTF2H1 genes were found to be significantly upregulated in patients with endometriosis in our study, but these genes have not been previously directly linked to endometriosis.
The DCs constitute a population of antigen-presenting cells that are directly involved in the modulation of the immune response, and altered gene expression may lead to a disruption of the normal endometrial cytokine profile, which may contribute to the development of endometriosis or endometriosis-associated infertility. 31 In the endocervical epithelium of women with endometriosis, we found 1 gene belonging to the DC pathway that was overexpressed (ICAM2, 2.7-fold higher).
The present study constitutes the first investigation of differentially expressed endometriosis-related genes with a focus on the endocervical epithelium using a simple sample collection method similar to that used to collect cells for Papanicolaou smears. This study consist of a primary screening of 252 genes and hence included a restricted number of patients with deep infiltrating endometriosis. This method could represent a tool for characterizing patients with endometriosis based on a profile of highlight expressed genes; nevertheless, the overexpressed genes identified in this study must be validated in a higher number of patients to assess the potential use of this method.
Footnotes
Authors’ Note
This work was conducted at Pelvic Pain and Endometriosis Unit, Gynecology Department, Paulista School of Medicine, Federal University of Sao Paulo (EPM-UNIFESP).
Acknowledgment
The authors would like to thank FAPESP and CNPQ for their financial support.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.