Abstract
Background/Aim: The activity and expression of matrix metalloproteinase-7 (MMP7) have been found to be upregulated in the late stages of endometriosis. However, the contribution of MMP7 genotype to endometriosis has seldom been examined. This study aimed to investigate the role of MMP7 promoter A-181G (rs11568818) and C-153T (rs11568819) genotypes in determining personal susceptibility to endometriosis in a Taiwanese cohort. Patients and Methods: In this hospital-based case–control study, MMP7 genotypes were analyzed in 153 endometriosis and 636 individuals without endometriosis using typical polymerase chain reaction-restriction fragment length polymorphism methodology. Results: The statistical analysis revealed that MMP7 rs11568818 genotypes were differentially distributed between the endometriosis and control groups (p for trend=0.0048). Specifically, the MMP7 rs11568818 homozygous variant GG was associated with endometriosis risk compared to the wild-type AA genotype (OR=4.59, 95% CI=1.46-14.48, p=0.0136). However, the MMP7 rs11568818 heterozygous variant AG was not associated with endometriosis risk (OR=1.57, 95% CI=0.97-2.53, p=0.0854). The frequency of than variant allele G of MMP7 rs11568818 was 12.7% in the endometriosis group, significantly higher than the 7.2% observed in the control group (OR=1.90, 95% CI=1.27-2.82, p=0.0021). Conclusion: MMP7 rs11568818 GG genotype was found to be a novel marker for endometriosis risk in Taiwanese.
Endometriosis is a hormone-dependent, inflammatory, benign gynecological disease characterized by the growth of endometrial cells outside the uterus (1, 2). It is a significant health concern affecting approximately 10% of women of reproductive age globally (3). In Taiwan, the prevalence of endometriosis has been observed to have increased in recent years, with estimated rates ranging from 1.5% to 30.8% among women of reproductive age (4-6). Women with endometriosis have an elevated risk of developing ovarian, breast, endocrine, and colorectal cancer (7, 8). Clinical features include dysmenorrhea, chronic pelvic pain, painful intercourse, and infertility (2). Although the etiology of endometriosis remains incompletely understood, accumulating evidence suggests that it is a multifactorial disease involving inflammation, hormonal dysregulation, and genetic factors (9-11).
The matrix metalloproteinases (MMPs) are a group of peptidases that play a critical role in inflammation, carcinogenesis, and cancer cell migration through the regulation of extracellular matrix (ECM) components (12, 13). MMP7 is normally expressed in bronchial, ductal, skin glandular, urogenital, gastrointestinal, and especially endometrial tissues (14). Conversely, low levels of MMP7 expression are reported in lung, gallbladder, and bladder tissues, and normally ultra-low levels of MMP7 are upregulated in abnormal conditions, such as malignant tumorigenesis (15-17). As evidenced by the literature, MMP7 is responsible for cleaving a variety of ECM proteins, including collagen IV, fibronectin, laminin, and tenascin-C, as well as non-ECM proteins, such as E-cadherin, tumor necrosis factor-α, and other MMP family members (14, 18-22). Thus, MMP7 plays a crucial role in maintaining the balance of many cellular processes, including cell growth, inflammation, wound healing, cell remodeling, carcinogenesis, and angiogenesis (23-27). Moreover, MMP7 is specifically expressed in multiple tumor types, such as digestive (28), urinary (29, 30), and reproductive (31) system tumors. By inhibiting apoptosis of cancer cells (32), reducing cell adhesion (33), and inducing angiogenesis (34), MMP7 promotes tumor progression and functions as an oncogenic protein that regulates the occurrence and development of various tumors.
Among these MMPs, the gene for MMP7 is located on human chromosome 11 q22.3 and consists of 13 exons (35).
In terms of the genotype–phenotype correlation, increased MMP7 activity was observed in promoter constructs containing the MMP7 rs11568818 and rs11568819 variant alleles (36). Previous literature has examined the association of MMP7 genotypes with various cancer types, such as oral, esophageal, gastric, colorectal, gallbladder, lung, breast, bladder and prostate, astrocytoma, renal cell carcinoma, and childhood leukemia (37-43). However, the investigation of MMP7 genotypes in relation to endometriosis is extremely limited. At the time of writing, there were only four reports (44-47).
Based on the aforementioned information, our study aimed to assess the potential correlation between MMP7 rs11568818 and rs11568819 genotypes and the risk of developing endometriosis in a Taiwanese cohort consisting of 153 patients with endometriosis and 636 healthy controls. The selected MMP7 polymorphic sites are illustrated in Figure 1.
The polymorphic sites of matrix metalloproteinase-7 (MMP7) rs11568818 and rs11568819 on chromosome 11.
Patients and Methods
Recruitment of patients with endometriosis and non-endometriosis control groups. One hundred and fifty-three individuals diagnosed with endometriosis were enrolled at China Medical University Hospital between 2000 and 2010. The doctors confirmed the diagnosis of endometriosis and classified the cases according to the guidelines established by the American Society for Reproductive Medicine (48). Participants were excluded if they had leiomyoma, adenomyosis, or any uterine, cervical, or ovarian cancer, and if they had received hormone therapy within the preceding 12 months. The basal follicle-stimulating hormone level was 7.2±1.4 IU/l. All patients provided written informed consent and donated 5 mL of peripheral blood for DNA extraction and genotyping analyses. Additionally, 636 healthy individuals without endometriosis were recruited as controls. To minimize the likelihood of including individuals with endometriosis in the control group, potential controls who reported any symptoms (such as pelvic pain) or had suspicion of endometriosis during our questionnaire interview were recommended for pelvic exams, ultrasound, or magnetic resonance imaging and excluded from the control group. However, we acknowledge that some controls may still have had undiagnosed endometriosis, as up to 16-20% of patients with endometriosis may be asymptomatic (4-6). Regarding the questionnaire, we collected and kept confidential information about participants’ personal smoking and drinking habits, age of menarche, and pregnancy history. We defined smokers as individuals who had smoked at least five packs of cigarettes in their lifetime and reported smoking on a daily or almost daily basis. Smokers were also asked about the age at which they began smoking, whether they continued smoking or had quit, and if so, when they quit. Furthermore, they were asked to report their average daily cigarette consumption. Non-drinkers were classified as individuals who consumed less than 200 mL of alcohol per week and consumed alcohol less than twice per month for social purposes.
MMP7 genotyping methodology. The DNA extraction procedure utilized in this study involved the use of the QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan, ROC) to extract peripheral blood leukocytes from each participant, as described in prior publications (49, 50). The primer design, selection of corresponding restriction endonucleases, and polymerase chain reaction conditions for genotyping of MMP7 were consistent with those employed in our previous publication (51). The genotyping procedure was conducted independently and in a double-blind manner by at least two well-trained researchers, with each genotypic analysis being repeated multiple times. The results of all repeated genotyping analyses were found to be 100% concordant with one another.
Statistical analyses. Age indices between the endometriosis case and control groups were compared using Student’s t-test. Distribution of the MMP7 genotypes among analyzed subgroups was assessed using Pearson’s chi-square test. The contribution of MMP7 genotypes to endometriosis risk was evaluated using odds ratios (ORs) and their associated 95% confidence intervals (CIs). Statistical significance was defined as a p-value of less than 0.05.
Results
Demographic characteristics of the investigated population. Table I presents the frequency distributions of age, age at menarche, full-term pregnancy, smoking and alcohol consumption status, and clinical stages of endometriosis in 153 patients with endometriosis and 636 healthy controls without endometriosis. The cases had a mean age of 40.3±4.9 years, and 55 (35.9%) of them had not experienced a full-term pregnancy. Among the patients, 32 (20.9%) had minimal or mild endometriosis (stage I or II), while 121 (79.1%) had moderate or severe endometriosis (stage III or IV) according to the revised American Fertility Society classification (48). No significant difference was observed in age at menarche and smoking and alcohol consumption status between cases and controls (p>0.05). However, the percentage of full-term pregnancies was significantly lower (p=0.0041) among patients with endometriosis (64.1%) than among women without endometriosis (75.9%) (Table I).
Demographics of the 153 endometriosis and 636 healthy controls.
MMP7 genotyping outcomes. Table II presents the distribution of MMP7 rs11568818 and rs11568819 genotypes among 153 endometriosis cases and 636 healthy controls. The results revealed that the MMP7 rs11568818 genotypes were differentially distributed between the endometriosis and control groups (p for trend=0.0048) (Table II). Specifically, the MMP7 rs11568818 homozygous GG variant was found to be associated with an increased risk of endometriosis compared to the wild-type AA genotype (OR=4.59, 95% CI=1.46-14.48, p=0.0136, Table II). However, the MMP7 rs11568819 heterozygous AG variant was not associated with endometriosis risk (OR=1.57, 95% CI=0.97-2.53, p=0.0854, Table II). In the dominant model, the data demonstrated that the frequency of MMP7 rs11568818 G allele-bearing genotypes was significantly higher in the endometriosis group than in the healthy control group (21.6% versus 13.4%, respectively; OR=1.78, 95% CI=1.14-2.79, p=0.0001, Table II). It is worth noting that all individuals had the CC genotype at the rs11568819 polymorphic site, without any CT or TT genotype carriers (Table II).
Genotypic frequency distributions of matrix metalloproteinase-7 rs11568818 and rs11568819 among the 153 endometriosis cases and 636 healthy controls.
MMP7 allelic frequency distribution. Consistent with the results in Table II, the frequency of the MMP7 rs11568818 variant allele G was 12.7% in the endometriosis group, which was significantly higher than the 7.2% observed in the control group (OR=1.90, 95% CI=1.27-2.82, p=0.0021, Table III). Furthermore, the allelic frequency of MMP7 rs11568819 was 100% for the C allele in both the case and control groups (Table III).
Allelic frequencies for matrix metalloproteinase-7 rs11568818 and rs11568819 polymorphisms among the endometriosis cases and healthy controls.
Discussion
MMP7 was initially reported to be expressed primarily in epithelial endometrial cells in 1993 (52). Subsequently, accumulating evidence has demonstrated that the levels of various MMPs, such as MMP2 and MMP9, are increased in the ectopic tissues and peritoneal fluids of patients with endometriosis (53). Nevertheless, only a limited number of studies have investigated the involvement of MMP7 in the pathogenesis of endometriosis, and its genetic contribution has not yet to be thoroughly examined (54).
In this case–control study, we investigated MMP7 genotype profiles in a representative Taiwanese population of 789 individuals, comprising 153 endometriosis cases and 636 non-endometriosis controls (as shown in Table I). Our study is the first to evaluate the contribution of MMP7 genotypes to endometriosis risk among Taiwanese. One of the significant findings is that MMP7 rs11568818 genotypes were differentially distributed between the endometriosis and control groups (as presented in Table II), suggesting that the GG and AG genotypes of MMP7 rs11568818 could potentially serve as novel diagnostic predictors for endometriosis. Notably, the G allele of MMP7 rs11568818 has been identified as an effective biomarker for endometriosis worldwide. In 2006, Shan and colleagues reported that variant (GG and AG) genotypes of MMP7 rs11568818 were a risk factor for endometriosis in a Chinese population containing 160 controls and 143 patients with endometriosis (44). There was only one endometriosis case with GG genotype at MMP7 rs11568818. In 2008, the contribution of MMP7 rs11568818 genotype to endometriosis was examined in a Caucasian population containing 241 controls and 227 endometriosis cases. The authors found a negative association (45). In 2016, Yarmolinskaya and colleagues provided another negative finding in an extremely small cohort with only 45 controls and 78 cases (46). Thus, although Yang and colleagues analyzed Shan et al.’s and Yarmolinskaya et al.’s work in a meta-analysis discussing the contribution of MMP7 rs11568818 genotypes to endometriosis, the knowledge about the impacts of MMP7 genotypes on endometriosis is still controversial and far from satisfying (47).
As shown in Table I, a notable finding is that the proportion of full-term pregnancy cases was significantly lower in the endometriosis group than in the control group (64.1% versus 75.9%). On the other hand, there were no significant differences observed between the two groups with regards to smoking and alcohol consumption behaviors. A previous study by Kim and colleagues reported that women with a family history of endometriosis and either smoking or early menarche had a significantly increased risk of developing endometriosis compared to the general population in Korea (55). However, due to incomplete family history records of various cancer types or endometriosis, we were unable to investigate the interaction between MMP7 rs11568818 genotypes and family history. Stratification analysis based on these factors (first full-term pregnancy, smoking, alcohol drinking, and first menarche status) did not reveal any genotypic preferences in our population (all p>0.05, data not shown). These findings are consistent with the meta-analysis conducted by Bravi and colleagues, which reported no association between tobacco smoking and endometriosis risk (56). We previously found that human 8-oxoG DNA glycosylase 1 (hOGG1) codon 326 genotype may have a joint effect with smoking on the development of endometriosis (57).
MMP7 is primarily responsible for the degradation of fibronectin, type IV collagen, laminin, nidogen, elastin, and β4-integrin (14). These extracellular matrix components are produced by various cancer cells, including breast, gastrointestinal, prostate, and endometrial tumor cells (58). MMP7 has been reported to be expressed by several types of cancer cells, such as lung (59), breast (60), head and neck (61), gastric (62), liver (63), pancreatic (64), colorectal (65) and most noticeable endometrial cancer (65). Thus, MMP7 is considered a practical marker and a potential therapeutic target for intervention. However, the role of MMP7 genotype may vary significantly among different types of cancer, and the conclusions are still uncertain. In this study, we revealed the significant association of MMP7 genotype with endometriosis in a representative population in Taiwan for the first time. Although the genotypic findings require validation in diverse populations, the phenotypic aspects proposed by Matsuzaki and colleagues must also be examined for more detailed mechanisms. Currently, it is only suggested that the epidermal growth factor receptor–MMP7 signaling pathway may be involved in the regulation of epithelial-mesenchymal transition during endometriosis progression (66).
Our results have provided an indication that MMP7 rs11568818 genotype might serve as a novel diagnosis predictor for endometriosis. More studies are urgently needed to reveal the possible critical regulation of ECM by MMPs, especially MMP7, in regard to endometriosis etiology.
Acknowledgements
The Authors are grateful to Yu-Ting Chin and Hou-Yu Shih for their excellent technical assistance. All the participants including those who were not selected for the control group of the study are appreciated. This study was supported by Asia University and China Medical University Hospital (grant number: ASIA-112-CMUH-10) and Chung Shan Medical University and Changhua Christian Hospital, Changhua, Taiwan (CSMU-CCH-111-01). The funders had no role in study design, patient collection, experiment conduction, statistical analysis, data annotation, or decision to publish or preparation of the article.
Footnotes
Authors’ Contributions
Research design: Chien HJ, Bau DT, and Tsai CW; patient and questionnaire summaries: Chien HJ, and Liu YF; experimental work: Tsai CW, Chen JC, Wang YC and Yang YC; statistical analysis: Hsieh YH, Yang YC and Tsai CW; article writing: Chang WS, Tsai CW, and Bau DT; article checking and discussion: Chien HJ, Wang YC, Chang WS, Hsieh YH, Liu YF, Yang YC, Chen JC, Bau DT and Tsai CW.
Conflicts of Interest
The Authors declare no conflicts of interest regarding this study.
- Received April 8, 2024.
- Revision received May 6, 2024.
- Accepted May 8, 2024.
- Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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