{"paper_id":"b4a7588f-7269-437e-81c8-c02d56cc88dd","body_text":"Endometriosis is a chronic and inflammatory\nreproductive disorder, specified by presence of the\nendometrial cells like lesions outside of the uterus ( 1 ).\nThe current understanding of etiology of endometriosis is\nyet uncertain. Retrograde menstruation theory proposed\nby Sampson in 1927 is suggested to contribute to the\nendometrial tissue implantation and endometriotic lesions\ndevelopment at ectopic sites ( 2 ). However, although\napproximately 90% of menstruating women experience\nretrograde menstruation, only 5-10% are at risk of\ndeveloping endometrial lesions ( 3 ). Thus, several factors\nmight be involved in the implantation and growth of this\nectopic endometrium as endometrial cells implantation\nrequires neovascularization to establish, grow and invade\ntissues. From the pathophysiological point of view, it is\nindicated that the factors playing an essential role in the\nsurvival and invasion of ectopic endometrial cells are rich angiogenic factors in these cells, while they work together\nwith metalloproteinases (MMPs) ( 4 ).\nAngiogenesis is an essential step in endometriotic\nlesion formations, since the ectopic survival of the\nendometrium requires formation of new blood vessels.\nVascular endothelial growth factor (VEGF), as a heparinbinding glycoprotein, is an important angiogenic\nfactor that stimulates proliferation and migration of\nendothelial cells and increases vascular permeability\n( 5 ). Several studies reported that VEGF concentration\nwas significantly increased in the peritoneal fluid of\nendometriosis women compared to non-endometriosis ( 6 ,\n 7 ). These results suggested that VEGF is also involved\nin the angiogenesis of endometriosis. In addition, ectopic\nendometrial fragments also require alteration in the\nexpression of molecules responsible for tissue invasion,\nsuch as over-expressed MMPs ( 8 ). These proteolytic\nenzymes are involved in normal endometrial remodeling,\nwhich occurs during the cellular proliferative phase at the\ntime of menstrual bleeding. MMP3 also contributes to the\ninvasion of endometrial cells toward the outside of uterine\nenvironment, as well as tissue remodeling, forming an\nendometriotic lesion ( 9 ).\nRecent studies indicated that dysregulation of microRNAs\n(miRNAs) emerged essential in endometriosis development\n( 10 ). miRNAs are small, noncoding, highly conserved RNA\nmolecules consisting of 19-24 nucleotides. They contribute\nto controlling translation and stability of targeted RNA bases\nto complement sites and promote repression or degradation of\nmessenger RNA transcripts ( 11 ). Burney et al. ( 12 ) evaluated\nmiR-34c-5p, miR-34c-3p, miR-9 and miR-34b expression\nlevels and found that expression of these miRNAs were\ndownregulated in ectopic endometrium from women with\nendometriosis, compared to women without endometriosis.\nThese miRNAs affected stability of the expression of their\ntarget genes and played an essential role in the pathogenesis\nof endometriosis. Furthermore, several miRNAs were also\ncategorized as proangiogenic miRNAs, playing role in the\nregulation of vascular endothelial growth factor A (VEGFA)\n( 13 ,  14 ) and MMP3 translations ( 15 ). In general, VEGFA\nmRNA is targeted by miR-93, resulting in cells proliferation\ninhibition ( 16 ). Additionally, increase of endothelial cell\nmigration and tube formation is also mediated by miR-93.\nHowever, in endometriosis, it is found that low level of miR93 is associated with high level of VEGFA in endometriosis.\nHere, we investigated whether alteration of miR-93 was\ninvolved in overexpression of VEGFA and MMP3 in both\neutopic endometrial and endometriotic tissues of women\nwith endometriosis.\n\nThis was a cross-sectional study at Central Surgical\nInstallasion (Dr. Cipto Mangunkusumo General Hospital;\nJakarta, Indonesia) between October 2020 and November\n2021. Thirty women (mean age 33.89 ± 5.33 years)\ndiagnosed with endometriosis using ultrasonography\nwere recruited as cases to this study. The endometriosis\nstages were classified based on the revised American\nSociety for Reproductive Medicine (ASRM) scoring\nsystem: stages I, II, III, and IV ( 17 ). We surgically\ncollected 30 eutopic endometrial and 30 endometriotic\ntissues, while tissues of the both groups were obtained\nfrom women with endometriosis. Healthy controls (n=30,\nmean age 36.27 ± 6.79 years) were enrolled from women\nwho underwent in vitro fertilization procedure in the same\nhospital and had no gynecologic disorders. None of these\ngroups received hormonal drugs in the last three months.\nEndometriosis tissues were collected using laparoscopy\nmethod. Meanwhile, normal endometrial tissues were\ncollected using a sterile pipelle cannula CCD (Pipelle de\nCornier, Laboratoire CCD; Paris, France).\nAll participants signed the informed consent prior to the\ntissue collection. Ethical approval was obtained from the\nEthics Committee of the Faculty of Medicine, University\nof Indonesia - Cipto Mangunkusumo Hospital (KET-972/\nUN2.F1/ETIK/PPM.00.02/2020).\nTotal RNA was extracted from all tissues using Tissue Total RNA Mini Kit (Geneaid,\nTaiwan) according to the manufacturer’s protocol. Quality and quantity of RNA was measured\nby NanoPhotometer Implen P300 (Implen GmbH, Germany). ReverTra Ace TM  qPCR RT\nMaster Mix (Toyobo Co., Japan) was used for complementary DNA (cDNA) synthesis. A suitable\namount of RNA template (6 μl, 50 ng/ μl) was prepared and followed by denaturation at 65°C\nfor 5 minutes. RNA template was mixed with 2 μl of 4x DN Master Mix and incubated at 37°C\nfor 5 minutes. Then, 5x RT Master Mix II was added to the mixture and placed in the\nthermal cycler with the following condition: 37°C for 15 minutes, 50°C for 5 minutes and\n98°C for 5 minutes.\nPairs of primer targeting the selected ( VEGFA  and  MMP3 )\nand reference (actin beta;  ACTB ) genes were designed using the software\nplatform Primer3Plus (https://www.bioinformatics.nl/cgi-bin/primer3plus/ primer3plus.cgi)\n( Table 1 ). SensiFAST TM  SYBR ®  NoROX Kit (Meridian Bioscience, USA)\nwas used for performing quantitative reveres transcription polymerase chain reaction\n(qRT-PCR). Each reaction contains 10 μl of 2x master mix, 0.5 μl (20 μM) of each primer, 3\nμl of cDNA template and 6 μl nuclease free water. An initial denaturation step was run at\n95°C for 2 minutes, continued by 40 cycles of amplification with denaturation at 95°C for\n5 seconds, annealing at 55°C for 10 seconds and extension at 72°C for 15 seconds. qRT-PCR\nwas carried out using Prime Pro 48 Real-Time PCR (Techne Cole-Parmer, UK). Melt curve\nanalysis was used to assess whether the qRT-PCR assays have generated a single specific\nproduct. The experiment was carried out twice for each sample.\nPrimers sequence used for mRNA expression\nSpecific miRNA sequences for hsa-mir-93 and U6 small nuclear (snRNA) were obtained from\nthe microRNA database (https://www.mirbase.org). Mature miRNA was reverse transcribed to\nsingle-stranded cDNA using the Taqman ®  MicroRNA Reverse Transcription Kit and\nTaqman TM  MicroRNA Assays has-mir-93 (Applied Biosystems, USA). The cDNA was\namplified using Taqman ®  Fast Advanced Master Mix (Applied Biosystems). Reaction\nmixture included cDNA template (1:10 dilution), miRNA assay (20x), master mix (2x) and\nnuclease-free water, for final volume of 10 μl. The cycling condition was initiated by\npolymerase activation at 95°C for 20 seconds and then 40 cycles at 95°C for 3 seconds and\nannealing at 60°C for 30 seconds. Expression of target miR-93 was normalized using\nexpression of the reference miRNA, U6 snRNA. The experiment was carried out twice for each\nsample.\nData were collected with Pro Study Software (Techne Cole-Parmer, UK) using linear\nbaseline correlation method and global auto cycle threshold (Ct) method. Then, Ct values\nwere normalized using mean expression of the housekeeping genes selected. Relative mRNA\nand miRNA expression were presented using the formula 2 -ΔΔCt  (Livak method)\n( 18 ). Fold change was calculated to determine upregulation and downregulation of the\ntarget genes. The Prism 5 software (GraphPad software, USA) was used for all statistical\nanalysis. One-way ANOVA test was used to determine difference of  VEGFA \nand  MMP3  mRNA with miRNA-93 expressions between ectopic and eutopic\ntissues from women suffering endometriosis and normal endometrial tissues. The correlation\nof miRNA-93 with mRNA (VEGFA and MMP3) expressions in the endometriotic tissues were\nassessed using Pearson’s correlation. The level of significant was set as 5%.\n\nWe performed qRT-PCR to determine alteration of  VEGFA  and\n MMP3  expression in the eutopic endometrium and ectopic endometriosis\ntissues collected from women suffering endometriosis in comparison with normal endometrium\nfrom healthy controls. As shown in Figure 1, no significant difference was seen in the\nexpression levels of  VEGFA  and  MMP3  between eutopic and\nectopic endometriosis tissues of women with endometriosis compared to the heathy\nendometrium. However, expression of VEGFA was increased by 0.66 fold in the eutopic\nendometrium and 1.14 fold in the endometriotic lesion from endometriosis women compared to\nthe normal endometrium. In addition, an increased expression level of\n MMP3  was observed in the endometriotic lesion from endometriosis women\ncompared to the normal endometrium, by 2 fold and 1.99 fold, respectively.\nRelative mRNA expression levels of  VEGFA  and  MMP3  in the\nectopic endometriosis lesions, eutopic endometrium and normal endometrium. The y-axis\nrepresents fold change in expression level as determine by quantitative\nreverse-transcription polymerase chain reaction (qRT-PCR), while it is expressed as\nmean ± SE. The difference of  VEGFA  and  MMP3  mRNA\nexpressions in three groups was analyzed using One-way ANOVA test. ns; Not\nsignificant.\nSimilar to  VEGFA  and  MMP3  expressions, we examined\nexpression level of  miR-93  in three sample groups using qRT-PCR. We\nobserved that expression of  miRNA-93  was significantly decreased in the\neutopic endometrium (16.7 fold) and ectopic endometriotic lesion (20 fold) compared to the\nnormal endometrium (P<0.001,  Fig .2 ).\nRelative expression level of  miRNA-93  in the ectopic lesions, eutopic\nendometrium and normal endometrium. The y-axis represents fold change in expression\nlevel, as determine by quantitative reversetranscription polymerase chain reaction\n(qRT-PCR), while it is expressed as mean ± SE. The difference of\n miRNA-93  expression levels in three groups was analyzed using\nOne-way ANOVA test.  ** ; P<0.01 was considered statistically\nsignificant.\nWe also analyzed  VEGFA  and  MMP3  mRNA expression levels\nin the eutopic endometrium and endometriotic lesions obtained from women suffering\nendometriosis based on the menstrual phase: the proliferative and secretory phases. The\nresults showed that there was no difference in the mRNA expression levels of\n VEGFA  and  MMP3  in the eutopic endometrium and\nendometriotic lesions of both menstrual cycle phases ( Table 2 )\nVEGFA  and  MMP3  expressions in eutopic and ectopic of women with\nendometriosis in menstrual cycle\nThe values are expressed as mean ± SE. T-independent test.\nIn this study, endometriosis was laparoscopically confirmed in 30 women of revised ASRM\nstage II (n=3), stage III (n=9) and stage IV (n=18). However, we found no difference in\nthe  VEGFA  and  MMP3  mRNA expression levels of both\neutopic and ectopic tissues based on the stages of endometriosis ( Table 3 ).\nVEGFA  and  MMP3  expression levels in eutopic endometrium and\nectopic tissues based on endometriosis stages\nThe values are expressed as mean ± SE. One-way ANOVA test. P<0.05 was considered to\nindicate statistically significance.\nWe also analyzed expression of  miR-93  in different stages of\nendometriosis. According to the statistical analysis, we found no difference in the\nexpression levels of  miR-93  based on the stages of endometriosis in both\neutopic and ectopic tissues ( Table 4 ).\nmiR-93  expression in eutopic endometrium and ectopic tissue based on\nendometriosis stages\nThe value are expressed in mean ± SE. One-way ANOVA test. P<0.05 was considered to\nindicate statistically significance.\nCorrelation analysis was also performed to determine whether  VEGFA  and\n MMP3  mRNA expression levels was upregulated by  miR-93 .\nWe found that there was significantly negative correlation between the expression levels\nof  miR-93  and  MMP3  in endometrial cells and\nendometriotic lesions collected from endometriosis women (r=-0.544, P<0.05,\nr=-0.749, P<0.01, respectively). In addition, significantly negative correlation\nwas observed in ectopic endometrium (r=-0.539, P<0.05), but not eutopic lesions of\nwomen with endometriosis.\n\nIn the present study, we compared mRNA expression levels of  VEGFA  and\n MMP3  in endometrial cells and endometriotic lesions between women with\nendometriosis and those without endometriosis using qRT-PCR. The result showed that mRNA of\n VEGFA  was enhanced in both endometrial cells and endometriotic lesions\nfrom endometriosis women than normal endometrial tissue of non-endometriosis women although\nwe observed no significant difference. In agreement with our study, Yerlikaya et al. ( 19 )\nreported no significant difference in  VEGFA  expression levels of eutopic\nand ectopic endometriums. However, it was seen an increased trend of  VEGFA \nexpression in ectopic lesions. In contrast to our study, a study by performed by Takehara et\nal. ( 20 ) showed that  VEGFA  gene expression was significantly higher in\nendometriotic lesions obtained from peritoneal of women with endometriosis, than those\nwithout endometriosis.\nThe invasiveness of endometrial cells is similar to cancer cells ( 21 ). One of these\ninvasive properties is related to the increased proteolytic activity of the tissue,\nresulting in the development of advanced endometriosis. In this case, overexpression of MMPs\nis believed to be related to the aggressive behavior of the endometrial cells ( 22 ) .\nHowever, we found no significant difference in the mRNA expression level of\n MMP3  between women with endometriosis and those without endometriosis.\nThe increasing  MMP3  mRNA expression level was also observed in this study,\nwhere  MMP3  mRNA expression level was two times higher in endometriotic\nlesions and endometrium of endometriosis patients than normal endometrium. Luddi et al. ( 9 )\nstudied both pro- and active  MMP3 . It was determined that their expression\nlevels were significantly increased in endometriotic lesions compared to the normal\nendometrial cells. Consistent with these findings, Lv et al. ( 23 ) showed MMP3 expression\nlevels were significantly higher in endometriosis compared to non-endometriosis. These\nresults indicated that overexpression of  MMP3  is involved in the invasion\nof endometrial tissue to develop endometriotic lesions outside the uterine environment.\nAccording to the implantation theory, eutopic endometrium can survive and develop into\nendometrial lesions in the peritoneal cavity due to retrograde menstruation ( 24 ). Hence,\nthis study also examined differences in  VEGFA  and  MMP3 \nexpression level of the both eutopic and ectopic endometrial tissues of women with\nendometriosis, based on the menstrual phase and stage of endometriosis. Our results showed\nno difference in the  VEGFA  and  MMP3  expression levels\nbetween eutopic and ectopic endometrial based on menstrual cycle and endometriosis stage. In\nline with our results, Yerlikaya et al. ( 19 ) revealed no difference in the expression of\n VEGFA  with the menstrual cycle. In addition, Augoulea et al. ( 25 ) showed\nthat expression level of  VEGFA  was not changed in all stages of\nendometriosis.\nSeveral studies have focused on the expression profiling of miRNAs in endometriosis,\nfurther to their coordination in angiogenesis and invasion of endometrial cells to the\nperitoneal environment ( 10 ). In the present study, we also measured miR-93 expression and\nrevealed that miR-93 expression level in both eutopic and ectopic endometrial tissues from\nendometriosis women was significantly decreased in comparison with healthy controls. Some\nstudies also reported that the expression of  miR-93  was downregulated in\nhuman cancer cells. Beside, high expression of  miR-93  suppressed\nproliferation and inhibited tumorigenesis in cancer ( 26 ,  27 ). Furthermore, we analyzed\ncorrelation of  miR-93  with  VEGFA  and\n MMP3 . A negative correlation was observed between miR-93 expression level\nand  VEGFA  in endometriotic lesions. In addition,  miR-93 \nexpression was inversely correlated with  MMP3  expression in both eutopic\nand ectopic endometrial cells of women suffering endometriosis. Previous studies performed\nby Lv et al. ( 23 ) was in line with our findings that indicated the downregulation of\n miR-93  was negatively correlated with the increased expression levels of\n VEGFA  and  MMP3  in endometriosis.\n\nIncreased expression levels of  VEGFA  and  MMP3  were\nobserved in endometriosis compared to nonendometriosis. Additionally, decreased expression\nlevel of  miR-93  was negatively correlated with high expression of\n VEGFA  and  MMP3  in both eutopic and ectopic endometrial\ncells of women with endometriosis. These findings could affect expression of angiogenic\nfactors and play a role in the pathogenesis of endometriosis. However, further\ninvestigations are required to examine the role of  miR-93  in the\npost-transcriptional regulation of target mRNA in endometriosis.","source_license":"CC0","license_restricted":false}