Evaluation of Plasma MMP-9 and VEGF-A mRNAs as Non-invasive Diagnostic Biomarkers in Women with Endometriosis

Background: Endometriosis is one of the common gynecological diseases and can lead to pelvic pain, dysmenorrhea, dyspareunia, and infertility in women. Thus, accurate and early diagnosis is a pivotal issue and an essential need for managing this disorder. At the present, the gold standard diagnostic method for endometriosis is laparoscopic surgery that is an invasive method and can lead to delay in diagnosis. Thus, there is an immediate necessity to search for non-invasive diagnostic biomarkers, such as blood-based ones.

Matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor-A (VEGF-A) have essential roles in the patho- genesis of endometriosis. Therefore, in this study , we evaluated the plasma mRNA levels of MMP-9 and VEGF-A, as potential non- invasive diagnostic biomarkers for endometriosis.

This study included 48 women (24 cases and 24 controls) who underwent laparoscopy for suspected endometriosis. Pre- operative plasma samples were collected, and after RNA extraction, the levels of MMP-9 and VEGF-A mRNAs were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

Plasma MMP-9 mRNA level was statistically higher in endometriosis patients compared with the control group (P value = 0.01). However, plasma VEGF-A mRNA level did not show a significant difference between the two groups (P value =0.5).

It seems that the plasma level of MMP-9 mRNA in endometriosis patients is significantly higher than in non- endometriosis women. This finding can provide new insights regarding this mRNA’s applicability as a non-invasive diagnostic biomarker for discovering new cases of endometriosis (newly diagnosed). According to our results, despite the suggested role of VEGF-A in endometriosis pathogenesis, it seems that the plasma level of VEGF-A mRNA does not have the potential to be used as a non-invasive diagnostic biomarker.

Endometriosis, Matrix Metalloproteinase 9, Diagnosis, Biomarkers, VEGF-A, Plasma 1. Background Endometriosis is a debilitating and chronic disease among women. In this disorder, the endometrial tissue grows in ectopic locations. It is diagnosed in approxi- mately 10-15% of reproductive-aged women and 35-50% of infertile women. In addition, it is observed in 50-70% of women with pelvic pain (1). The common signs of en- dometriosis comprise pelvic pain, dysmenorrhea, dyspare- unia, and infertility . Thus, it can influence the quality of life and impose health-related costs (2). The gold standard diagnostic method for endometrio- sis is laparoscopic surgery and biopsy (3). Laparoscopic surgery is an invasive and costly procedure. In addition, it harbors the risks associated with general anesthesia and post-procedure adhesions. Thus, a non-invasive diagnostic test for endometriosis can lead to timely diagnosis and bet- ter disease management (4). Blood biomarkers have been considered as non-invasive diagnostic parameters for this disease. Blood-based diagnostic markers for endometrio- sis can lead to cost-effective, less invasive, and easily accept- able procedures for patients and provide quick results (5). Despite many efforts, no non-invasive biomarker has been approved for the diagnosis of endometriosis yet. Extracellular circulating RNAs in biological fluids such as plasma, urine, and saliva are becoming optimistic non- invasive and cost-effective diagnostic tools (6). A previous Copyright © 2021, Gene, Cell and Tissue. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited. Abdollahi S et al. study showed the capacity of some miRNA panels as blood- based non-invasive biomarkers for endometriosis (7), and some studies have suggested the usefulness of plasma mR- NAs as biomarkers for different cancers (8-11). Accordingly , circulating mRNAs in serum may be helpful non-invasive tools for the diagnosis of endometriosis. According to previous investigations, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor-A (VEGF-A) make an essential contribution to the pathogenesis of endometriosis (12, 13). Also, the mRNAs of these genes are overexpressed in endometrial lesions in women with endometriosis (14, 15). The MMP-9 gene encodes a matrix metalloproteinase involved in extracellular matrix (ECM) degradation (16). This feature is vital for the invasion of the endometrial tissue to ectopic locations (17). The VEGF-A gene encodes a factor essential for angiogenesis; a process that is necessary for the sur- vival and propagation of ectopic endometrial lesions (18). Some studies have detected the mRNAs of these genes in the peripheral blood of endometriosis patients (19, 20). However, the diagnostic potential of these mRNAs circulating in the plasma of endometriosis patients is uncertain. 2. Objectives The aim of this study was to quantify the mRNA levels of MMP-9 and VEGF-A in endometriosis patients’ plasma samples by RT-qPCR. 3. Methods 3.1. Patients and Sample Collection The study population consisted of 48 women with suspected endometriosis referred to a gynecologist with complications such as dysmenorrhea or pelvic pain. All the patients were scheduled to undergo la- paroscopy and signed written informed consent before surgery . This study was approved by the Research Ethi- cal Committee of Tehran University of Medical Sciences (IR.TUMS.MEDICINE.REC.1398.579). Before surgery , 5 mL of blood was obtained from each participant and transferred into a tube containing EDTA as an anticoagulant. Then blood samples, without delay , were transferred to the lab- oratory for subsequent processing and plasma separation. According to the gold standard for endometriosis diagno- sis (direct visualization of lesions in laparoscopic surgery and confirmation by pathological assessment of the tis- sue), the patients were categorized into two groups: 24 patients with a confirmed diagnosis of endometriosis (as the case group) and 24 patients without endometriosis (as the control group). Endometriosis stage was defined based on the guidelines of the American Society of Reproductive Medicine (ASRM). The characteristics of study participants, including the menstrual cycle phase and medical background, were recorded in a clinical questionnaire based on their medi- cal records. Moreover, none of the patients received pre- operative treatments, such as hormone therapy , which was regarded as one of important inclusion criteria due to the possible effects of drugs on gene expression. In addition, all the participants were in reproductive age and referred to a gynecologist with endometriosis symptoms. Patients with a previous diagnosis of diseases such as cancer, au- toimmune diseases, and endocrine disturbances (polycys- tic ovarian syndrome, etc.) were excluded from this study . 3.2. Plasma Separation and RNA Extraction By centrifugation (at 5000 rpm and then 1500 rpm for 15 min at 4 ºC), plasma samples were initially separated from whole blood. Thermo Scientific Nanodrop 2000 was used to measure plasma quality at 414 nm for detecting hemolysis in samples. Then the plasma samples that had no hemolysis were stored at -80 °C. The TRIzol reagent (Ri- boEx LS, GeneAll, Korea) was used to isolate total RNA from 400 uL plasma. Afterward, the RNA samples were sub- jected to standard ethanol precipitation, and the pellet was resuspended in 10 µL of RNAase-free water. Finally , the quality and quantity of the extracted RNAs were appraised by a spectrophotometer. 3.3. Reverse Transcription-quantitative Polymerase Chain Reac- tion The extracted RNA samples were converted to cDNA us- ing a Reverse Transcription kit (Geneall, Korea) according to the manufacturer’s protocol. The mRNA levels of MMP- 9 and VEGF-A were measured by SYBR Green RealQ Plus 2x Master Mix Green (Ampliqon, Denmark) using Roche Light- Cycler® 96 System. Normalization was carried out employ- ing β-Actin as a reference gene. The reactions included an initial step at 95°C for 30 sec, followed by 40 cycles of am- plification; 95 °C for 10 s (denaturation), 60 °C for 15 s (an- nealing), and 72 °C for 30 s (extension). The qRT-PCR reac- tion mixture consisted of 5 µL SYBR Green master mix, 1µL of each of forward and reverse primers (primer concentra- tion: 5 pmol), 1 µL of target cDNA, and 2 µL sterile water in a total volume of 10 µL. Each reaction was performed in duplicate. The specificity of RT-qPCR amplification was confirmed by melting curve analysis and observing a sin- gle peak. The 2 -∆∆Cq method was used for calculating the relative expression of each target mRNA. The sequences of the primers used in this study have been represented in Ta- ble 1. 2 Gene Cell Tissue. 2022; 9(2):e118656. Abdollahi S et al. Table 1. The Sequences of the Primers Used for Gene Expression Analysis Primers Sequences MMP-9 Forward 5’- CAGGCAGCTGGCAGAGGAAT-3’ Reverse 5’- TTCGACTCTCCACGCATCTC-3’ VEGF-A Forward 5’- CTCCACCATGCCAAGTGGT-3’ Reverse 5’- TCTCGATTGGATGGCAGTAGC-3’ β-Actin Forward 5’- CCCAGCACAATGAAGATCAAGATCAT-3’ Reverse 5’- ATCTGCTGGAAGGTGGACAGCGA-3’ Abbreviations: MMP-9, matrix metalloproteinase-9; VEGF-A, vascular endothe- lial growth factor-A. 3.4. Statistical Analysis The Kolmogorov-Smirnov test showed that the data had a non-normal distribution (P < 0.05). Thus, a non- parametric test, the Mann-Whitney U test, was used to com- pare the studied variables between the groups. All statisti- cal tests were performed in IBM SPSS Statistics 22 (SPSS Inc., Chicago, IL, USA), and P < 0.05 denoted a statistically sig- nificant difference between the two groups. 4. Results 4.1. Participants’ Descriptive Parameters The participants’ clinicopathological information has been briefed in Table 2. A total of 24 endometriosis patients with stage III (n = 10) and IV (n = 14) were enrolled in this study (mean age: 33.5 ± 5.1 years). The control group in- cluded 21 women with benign gynecological diseases and three healthy women (mean age: 37.5 ± 9.1 years). A def- inite diagnosis in all the patients was made by histolog- ical analysis. The menstrual cycle of all the participants was determined; there was no significant difference in the menstrual phase between patients with endometriosis and control subjects (P value > 0.05). 4.2. MMP-9 and VEGF-A mRNAs’ Plasma Levels The relative levels of the mRNAs of MMP-9 and VEGF- A were measured in plasma samples using RT-qPCR (Fig- ure 1). The plasma mRNA level of MMP-9 was significantly higher in endometriosis patients compared with the con- trol group (P value = 0.01). In contrast, no statistically sig- nificant difference was observed between the two groups regarding VEGF-A mRNA plasma level (P value = 0.5). 20 15 10 5 0 mRNA Fold Change N-EMS EMS MMP-9 VEGF-A Groups NS Figure 1. The mRNA fold-change of MMP-9 and VEGF-A in the plasma of endometrio- sis (EMS) patients compared with non-endometriosis (N-EMS) counterparts. (*: P value < 0.05, NS: non-significant). 4.3. Association of mRNA Levels of MMP-9 and VEGF-A with Men- strual Cycle Phase and Disease Stage There was no significant association between the mRNA level of MMP-9 and the menstrual cycle (P value > 0.05). However, there was a significant positive association between VEGF-A mRNA level and the proliferative phase of the menstrual cycle in the study participants (P value 0.05). 5. Discussion Endometriosis is a common and chronic gynecologi- cal disease that affects women’s reproductive health and quality of life. Endometriosis can have complications such as pelvic pain, dysmenorrhea, dyspareunia, and infertil- ity (21), and its diagnosis requires a surgical intervention that is accompanied by risks and complications for women (22). The lack of non-invasive diagnostic tests for en- dometriosis leads to a considerable delay in disease detec- tion (3). So, developing non-invasive diagnostic biomark- ers for endometriosis is an urgent need. In this context, blood-based biomarkers can be helpful. In recent years, most efforts for finding non-invasive diagnostic biomark- ers for endometriosis have been directed towards blood miRNAs. Also, it seems that circulating mRNAs can be use- ful as potential biomarkers (5, 7). Recently , it has been indicated that diffusion RNAs are protected by lipopro- tein complexes or phospholipids. Thus, these molecules Gene Cell Tissue. 2022; 9(2):e118656. 3 Abdollahi S et al. Table 2. Participants’ Characteristics in This Study . Characteristics Endometriosis Women (n = 24) Non-endometriosis Women (n = 24) Age (y , mean± SD) 33.5 ± 5.1 37.5 ± 9.1 Cycle phase, No. (%) Follicular 20 (83) 19 (79) Luteal 4 (17) 5 (21) ARSM stage, No. (%) III 10 (42) NA IV 14 (58) NA Other diagnoses, No. (%) Healthy NA 3 (13) Ovarian cysts NA 8 (33) Myoma NA 11 (46) Other NA 2 (8) Abbreviations: ARSM, American Society of Reproductive Medicine; NA, Not Available. are stable in circulation despite the high amounts of the RNases present in blood (23). It has been shown that various kinds of RNAs, including mRNAs and non-coding RNAs, can be extracted and detected in body fluids such as plasma and serum (24, 25). Indeed, several investigations have shown that cell-free mRNAs in plasma can be promis- ing non-invasive diagnostic tools for cancer (26, 27). Hence, in this study , the plasma levels of MMP-9 and VEGF-A mR- NAs, as the genes relevant to endometriosis pathogenesis, were evaluated by RT-qPCR. In this study , for the first time, we measured plasma MMP-9 mRNA level via qRT-PCR in women with en- dometriosis. Our results showed a significant increase in serum MMP-9 mRNA in endometriosis compared with non-endometriosis women. Moreover, this study showed that the expression of MMP-9 was independent of the menstrual phase. Interestingly , endometriosis, as a be- nign disease, shares a common feature with malignancies, and that is its invasive behavior (28). It has been shown that MMP-9, as a matrix metalloproteinase, is an essential fac- tor for the degradation of the extracellular matrix (ECM) and the basement membrane and a crucial factor for cells’ invasive behaviors (29). Several studies have reported that MMP-9 can induce invasiveness in the endometrial tissue, leading to the degradation of ECM, invading other locations, and creating ectopic endometriosis lesions (30, 31). There are pieces of evidence suggesting that the MMP-9 gene is significantly overexpressed in the endometriosis tissue, which can be expected according to its role in endometriosis pathogenesis (13, 27). The elevation of MMP-9 mRNA in the plasma of endometriosis patients, observed in this study , was consistent with the results of previous investigations on the expression MMP-9 in the endometriosis tissue, suggesting its potential as a non-invasive diagnostic biomarker for endometriosis. This study showed no significant difference in plasma VEGF-A mRNA level between endometriosis and non- endometriosis women. Moreover, we showed that VEGF- A mRNA level positively correlated with the proliferative phase of the participants’ menstrual cycles. This molecule is an essential factor for physiological and pathological an- giogenesis and induces vascular endothelial cells’ prolifer- ation and migration (18). Angiogenesis plays a significant role in endometriosis progression because, similar to tu- mors’ behaviors and their metastatic spread, endometrio- sis lesions require neovascularization to survive in ectopic locations. Indeed, the establishment, proliferation, and survival of endometriosis lesions depend on an adequate blood supply (12). Thus, regarding the role of VEGF-A in en- dometriosis pathogenesis, the overexpression of its gene in the endometriosis tissue can be expected, as shown in a previous study (12). According to our results, however, the plasma level of VEGF-A did not show a significant dif- ference between endometriosis patients and controls. It seems that the plasma level of VEGF-A mRNA, in contrast to its expected role in endometriosis pathogenesis, cannot be a useful blood-based biomarker for discriminating en- dometriosis in women. Furthermore, our finding regard- ing the comparable levels of VEGF-A mRNA in plasma sam- ples from endometriosis and non-endometriosis women was inconsistent with that of a previous study , indicating a significantly higher level of VEGF-A mRNA in endometrio- sis patients (32). This discrepancy may be due to the limited number of control subjects in the mentioned study (i.e., 4 Gene Cell Tissue. 2022; 9(2):e118656. Abdollahi S et al. only 10 women, mostly infertile) and different samples in the two studies (serum vs. plasma). We must address some limitations of the present study . Our sample size was relatively small, and the patients mainly presented stage III and IV endometriosis. Hence, large-scale studies should be performed to clarify the ap- plicability of MMP-9 and VEGF-A mRNAs as non-invasive di- agnostic biomarkers for endometriosis. 5.1. Conclusion In the present study , the levels of MMP-9 and VEGF- A mRNAs, as non-invasive diagnostic biomarkers, were evaluated by RT-qPCR in the plasma samples of women endometriosis. A significant difference was detected in plasma MMP-9, but not VEGF-A, mRNA level between en- dometriosis and non-endometriosis women. In conclu- sion, it can be suggested that MMP-9 mRNA has the po- tential for being used as a diagnostic biomarker for en- dometriosis, and it can be considered for further investi- gations along with other mRNAs. However, VEGF-A seemed to be inapplicable as a non-invasive diagnostic biomarker despite its role in endometriosis pathogenesis. Footnotes Authors’ Contribution: Concept and design: S. A. and P. I.; Collecting and interpreting clinical data and revising the manuscript: S. A., P. I., S. CH., and SH. N.; Statistical analysis: S. Y., S. A., and P. I.; Drafting the manuscript: S. A. and P. I. All authors read and approved the final manuscript. Conflict of Interests: No author has any potential conflict of interest. Ethical Approval: This study was approved by the Re- search Ethical Committee of Tehran University of Medical Sciences (IR.TUMS.MEDICINE.REC.1398.579). Funding/Support: This study was a part of a M.Sc. the- sis supported by Tehran University of Medical Sciences (Tehran, Iran; grant No: 43115). Informed Consent: All patients signed written informed consent before surgery .

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