{"paper_id":"df83f497-0740-415e-acf8-3bf5b6f209c1","body_text":"© 2025 Ocktariyana, Nurul Hikmawati, Raden Muharam, Andon Hestiantoro, Muhammad Luky Satria Syahbana Marwali, \nAgus Surur As’adi and Asmarinah. This open-access article is distributed under a Creative Commons Attribution (CC -BY) \n4.0 license.  \nOnLine Journal of Biological Sciences \n  \nOriginal Research Paper \nNerve Growth Factor (NGF) Gene Analysis in Menstrual \nBlood of Endometriosis: DNA Methylation and mRNA \nExpression Levels \n \n1,2Ocktariyana, 3Nurul Hikmawati, 4Raden Muharam , 4Andon Hestiantoro, 5Muhammad Luky Satria \nSyahbana Marwali, 5Agus Surur As’adi and 1,6Asmarinah \n \n1Doctoral of Biomedical Sciences, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia \n2Departement of Midwifery, Politeknik Kesehatan Kemenkes Palembang, Palembang, Indonesia \n3Master Program in Biomedical Sciences, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia \n4Department of Obstetrics and Gynecology, Faculty of Medicine Universitas Indonesia, Cipto Mangunkusumo, Indonesia \n5Department of Obstetrics and Gynecology, Fatmawati Hospital, Jakarta, Indonesia \n6Department of Medical Biology, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia \n \nArticle history \nReceived: 03-09-2024 \nRevised: 21-11-2024 \nAccepted: 03-12-2024 \n \nCorresponding Author:  \nAsmarinah \nDepartment of Medical Biology, \nFaculty of Medicine Universitas \nIndonesia, Jakarta, Indonesia  \nEmail: asmarinah.si@gmail.com \nAbstract: Menstrual blood flow is reversed through the fallopian tubes \nduring the menstrual process and implants outside the uterus, which is the \ntheory of pathogenesis in endometriosis sufferers. This study a ims to \nanalyze the level of DNA methylation and mRNA expression of the \nNerve Growth Factor (NGF) gene in menstrual blood in endometriosis \npatients and analyze the correlation. The study was conducted among 20 \nwomen in each group with and without Endometriosis (controls) of \nreproductive age. Filter paper pads were used to collect the menstrual \nblood sample, while the eutopic endometrium  was collected by biopsy. \nFollowing that, NGF DNA methylation analysis of menstrual blood was \nperformed using Methylation-Specific PCR (MS-PCR) and \nPyrosequencing. Additionally, the study of NGF mRNA expression was \nconducted by RT-qPCR. Data analysis using independent T-test or Mann-\nWhitney, SPSS 22, and p<0.05 was considered statistically significant. \nWe found that the DNA of NGF in the menstrual blood of endometriosis \npatients was hypomethylated; conversely, its mRNA expression was \nincreased compared to that of women without Endometriosis. There was \na statistically significant difference in the level of NGF DNA methylation \nand the relative mRNA expression level of the NGF gene in the menstrual \nblood of endometriosis patients compared to women without \nEndometriosis (p<0.05). However, there was no correlation between \nDNA methylation levels and NGF mRNA expression levels in menstrual \nblood samples of women with Endometriosis (p>0,05). Furthermore, the \nincreased expression of NGF mRNA in the menstrual blood of women \nwith Endometriosis is not driven by  a decrease in DNA methylation. \nHowever, hypomethylation and an increase in mRNA expression of the \nNGF gene in menstrual blood have the potential to be a biomarker for the \nearly diagnosis of non-invasive Endometriosis. \n \nKeywords: Endometriosis, DNA Methylation, mRNA Expression, \nMenstrual Blood, Nerve Growth Factor  \n \nIntroduction \nEndometriosis is a gynecological disorder \ncharacterized by the growth of endometrial-like tissue in \nthe form of glands and stroma that are located outside  \nthe uterine cavity, e specially on the ovaries, pelvic \nperitoneum, rectovaginal septum , and are very sensitive \nto hormones  (Superman, 2012; Machairiotis et al ., \n2013). Suparman (2012); Machairiotis et al . (2013) \nEndometriosis often occurs in women within their  \n\nOcktariyana et al. / OnLine Journal of Biological Sciences 202 5, 25 (2): 408.416 \nDOI: 10.3844/ojbsci.2025.408.416 \n \n409 \nreproductive age by 5-10% incidence, with complaints \nof dysmenorrhea 60 -80%, chronic pelvic pain \ncomplaints 40 -50%, infertility complaints 30 -50% and \nirregular menstruation 10-20%. Bulletti et al. (2010), the \npathophysiology of endometriosis pain is modulated by \ndysregulation of the hormone estrogen, cross -\ncommunication of inflammatory reactions between \nendometriotic cell neurotrophy and peritoneal fluid  and \nhyperinnervation of primary sensory nerve fibres. This \nleads to the formation of nociceptive signals sent to the \nCentral Nervous System (CNS). Nociceptive signals \nare integrated centrally via secondary sensory neurons \nin the spinal cord. Peripheral nerve sensitization \ninvolves a decrease in ion channel thresholds and an \nincrease in nociceptor expression. During \ninflammat ion, NGF molecules secreted from \nmacrophages then stimulate the activation of \nnociception receptors. Neuroimmune interaction \nregulated by estrogen sensitizes peripheral innervation, \ncausing endometriosis pain  (Kobayashi et al., 2014 ). \nEndometriosis is a multifactorial disease caused by the \ninteraction of genetic factors, steroid hormones, the \nimmune system and the environment. Kobayashi et al . \n(2014) Per sophisticated pathogenesis aetiology, it is \ngenerally accepted to include retrograde menstruation, \ncoelomic metaplasia, stem cells, disease induction \nthrough inflammation, oxidative stress  and immune \nsystem dysfunction and dissemination through the \nvascular and lymphatic systems  (Giudice et al ., 2012; \nGupta et al., 2015). \nBulun et al . (2015) emphasized that  ectopic \nendometrial stromal cells of endometriosis women \nexhibited the alteration of characteristics and function \ncompared to normal female eutopic endometrial cells. \nEpigenetic changes in CpG methylation affect several \ngenes' functions that contribute to hormonal and immune \nfactors (Giudice et al., 2012; Monsivais et al., 2015). \nEpigenetics, as a regulator of gene expression, can \nalter malignancy -related characteristics, such as \ngrowth, migration, invasion  and angiogenesis. \nEpigenetic changes in Endometr iosis involve changes \nin the levels of DNA methylation of gene promoters, \nwhich include DNA methylation, histone modification  \nand micro-RNA (miRNA). It is also known that the \npathogenesis of Endometriosis is implied  by \nSteroidogenic Factor  1 (SF-1) and aro matase by \nregulating an increase in DNA methylation \n(hypermethylation) at the progesterone-B (PR-B), HOXA-\n10 and E-cadherin receptors and a decrease in the level of \nDNA methylation (hypomethylation ) of the  estrogen- \nreceptor (Koukoura et al., 2016; Nasu et al., 2011). \nAltered methylation levels may lead to immunological \nand inflammatory disorders. One clear example in the \nresearch of Ocktariyana et al. (2019) was reported the \nDNA methylation status of the P2RX3 nociceptive  \nreceptor gene promoter in endomet riosis samples, \n100% unmethylated band pattern existed on the  gene \npromoter in Endometriosis peritoneal tissue and \ncorrelated with pain severity in endometriosis women  \n(Ocktariyana et al., 2019). Apart from this, Tokushige et al. \n(2006) and Morotti et al . (2014) suggested that the \ndensity of unmyelinated C-type nerve fibres was found in \nendometriotic lesions as well as in the endometrium of \nwomen with Endometriosis and correlated with pain \nseverity and endometriosis stage (Tokushige et al., 2006; \nTokushige et al., 2006a-b). \nOwing to the activation of nociceptive receptors at the \nend of primary sensory nerve fibres, releasing Nerve \nGrowth Factor (NGF) neurotrophin mediator by \nmacrophages leads to nerve fibres' survival, development \nand function (Tokushige et al., 2006b ). Nerve Growth \nFactor (NGF) is released from macrophage cells and \nforms a complex with the high-affinity TrkA receptor on \nneuronal cells, which is redistributed to somatic neuron \ncells (Morotti et al., 2014). \nPeng et al. (2018) investigated the role of NGF with \nits receptor (TrkA/p75NTR) in endometriosis patients \nwith dyspareunia in the posterior pelvis (cul-de-\nsac/uterosacral). Endometriosis was confirmed by \nendovaginal palpation and ultrasound. The results of this \nstudy prove that there is a hi gher immunointensity \nreaction in NGF with TrkA receptors in stromal cells and \nendometrial epithelial cells in endometriosis women with \ndeep dyspareunia compared to women without \ndyspareunia. However, at the p75NTR receptor, the same \nreaction did not occur.  According to Peng, \nimmunoreactivity in nerve growth factors in stromal cells \nis significantly related to nerve fibre density and pain \nintensity of dyspareunia. In endometrial stromal cell \nculture, NGF was also reported to be significantly \ncorrelated with the increase in PTGS-2/COX-2-2 mRNA \nand PGE2 secretion. This correlation could be mediated \nby high nerve fibre density and COX-2/PGE2 stimulation \nvia the Trk receptor. The NGF/Trk signalling pathway \nmay play a role in endometriosis pain, particularly in \ndyspareunia (Peng et al., 2018). \nMu et al . (2020) analyzed the relationship between \npain levels in patients with deep infiltrating \nEndometriosis (DIE) and the expression of Nerve Growth \nFactor (NGF) through Magnetic Resonance Imaging \n(MRI). The results found  that in patients with deep \ninfiltrating Endometriosis (DIE) in uterine fibula \nligaments, vagina, uterine rectum, rectum  and ureters \ncorrelate to pain. Increased expression of NGF with its \nreceptors manifests as a pain signal. This increase in \nexpression i s essential in the diagnostic and follow -up \nmethods after endometriosis surgery (Mu et al., 2020). \nResearch Out Smart Endometriosis (ROSE) \ndeveloped a method for diagnosing Endometriosis \nthrough menstrual blood. Early diagnosis through  \n\nOcktariyana et al. / OnLine Journal of Biological Sciences 202 5, 25 (2): 408.416 \nDOI: 10.3844/ojbsci.2025.408.416 \n \n410 \nmenstrual blood will help in the treatment at an early stage \nin preventing the spread of endometrial lesions to other \norgans and preventing unnecessary hysterectomy. \nMenstrual blood as DNA samples is used as an initial \nstudy to identify diagnostic biomarkers that are effectiv e \nand efficient in terms of cost and treatment time  \n(MacDonald, 2020).  \nMenstrual blood is a complex biological fluid \nconsisting of blood, vaginal secretions, immune cells , \nand endometrial cells from the uterine wall. Menstrual \nblood was selected as a tiss ue sample in this study to be \na preliminary study to identify biomarkers in the \ndevelopment of early diagnostics. Menstrual blood is \nalso used to identify and evaluate biomarkers through \nspectrometric analysis techniques. Yang et al . (2012) \nstate that many proteins are expressed in the shedding of \nendometrial cells during menstrua tion. Proteins that \nprepare for embryo implantation include integrins, \nMatrix Metallo Proteinases (MMPs), Galectins (GAL), \nGlucose-Regulating Proteins  (GLUT), Interleukins \n(ILs), p roteolytic enzymes, cytokines, apoptotic \nregulatory proteins, several proteins from various \nimmune cells, which is an integral part of menstruation. \nThis protein in menstrual blood can be a non -invasive \nbiomarker in diagnosing endometrial pathology and \ninfertility (Yang et al., 2012).  \nMaterials and Methods \nSamples Collection \nIn this study, samples were taken from research \nsubjects willing to participate by signing an informed \nconsent form at Fatmawati Hospital and Dr Cipto \nMangunkusumo Hospital, RSUPN, J akarta. Menstrual \nblood samples were collected from 20 women with \nEndometriosis and 20 without Endometriosis.  \nThe inclusion criteria for endometriosis cases were \nmarried infertile women aged 20 -45 years. Exclusion \ncriteria included pregnant women, unmarri ed women, \nwomen with endometrial cancer, ovarian cancer , and \nendometritis. Subjects without Endometriosis were \nelected as controls, as well as infertile married women \nbetween 20 -45 years old who underwent endometrial \nmicro curettage examination at Dr. Cipt o \nMangunkusumo, Indonesian Centre Hospital, Jakarta, and \nwithout indication of Endometriosis.  \nDNA Methylation and mRNA Expression were \nMeasured  \nMenstrual blood samples were collected using sanitary \nfilter paper modified with sanitary napkins. Menstrual \nblood is taken on the second to third day of menstruation. \nIf the filter paper has been collected, dried, cut into small \npieces (6 mm in diameter), and weighed 50 mg for nucleic \nacid extraction.  \nDNA isolation from menstrual blood samples was \nperformed usin g the QIAamp® DNA Mini Kit \n(Qiagen) following a dried blood spot DNA \npurification protocol. DNA was also extracted from 20-\n25 mg of eutopic endometrial tissue using the \ngSYNCTM DNA Extraction Kit (Geneaid, Taiwan ). \nThe resulting DNA extraction was then con verted into \na bisulfite solution using the Epitect Bisulfite Kit from \nQiagen, Germany, to examine DNA methylation levels \nusing MSP and pyrosequencing.  \nThe Methylation-Specific PCR (MSP) method. We \nused specially designed primers from MethPrimer as \nfollows: methylated specific primers (M)(F) 5'- \ncgtttcgaagaaaaggagtagtc-3' and (R) 5'-\naaccgactaactaaaactaaacgaa-3', while unmethylated specific \nprimers (U) (F) 5'- ggtgttttgaagaaaaggagtagtt-3' and (R) 5'- \naaccaactaactaaaactaaacaaa-3'. The MSP results were \nsubsequently electrophorized using 2.4% agarose gel with \n100 volts for 45 minutes and analyzed using ImageJ \nsoftware to measure the percentage of band intensity.  \nMeanwhile, DNA methylation level was measured by \npyrosequencing, and we only used 10 menstrual blood \nsamples for endometriosis and non -endometriosis. The \nprimer design software PyroMark Assay Design 2.0. \nThree CpG sites in the NGF gene promoter were \nanalyzed. Briefly, each PCR mixture contained My Taq \nHS Red Mix, 2' (12.5 µL ), 1 µL forward and reverse \nprimers (10 pmol/L each PCR primer), 8.5 µL ddH2O and \n2 µL bisulfite-converted template DNA in a total volume \nof 25 L. PCR conditions were as follows : Initial \ndenaturation at 95°C for 5 min; 45 cycles at 95 °C for 30 \nsec, 59 °C for 30 sec  and 72°C for 30 sec; a nd final \nextension at 72°C for 3 min resulted in an amplicon length \nof 150 bp. \nThe PCR products were visualized with 1 % agarose \ngel electrophoresis. If the results of the electrophoresis \nband show a single solid band and no primary dimer, in \nthat case, the pyrosequencing reaction can be continued \nby making a mixture for Plate 1 with the composition: 37 \nµL Binding Buffer, 3 µL Streptavidin sepharose beads, 25 \nµL ddH 2O and 15 µL PCR product. In Pyrosequencing \nVacuum Preparation Tool (Qiagen, Germany), as much as \n15 µL of biotinylated PCR product was immobilized on \nstreptavidin sepharose beads. Then, sepharose beads were \npurified, rinsed, denatured using 0.2 M NaOH and washed \nagain. The final concentration of 0.3 mM pyrosequencing \nprimer was annealed to single -strand PCR products, and \npyromark Q96 2.5.7 software was used (Qiagen, \nGermany). The pyrosequencing reaction will run \nautomatically. \nTotal RNA from menstrual blood samples was \nextracted using the Quick-RNA Miniprep Plus Kit, Zymo. \n50 ng of RNA was reverse transcribed into cDNA using \nAce qPCR ReverTra RT Master Mix with gDNA \nRemover from Toyobo. The cDNA samples were then \n\nOcktariyana et al. / OnLine Journal of Biological Sciences 202 5, 25 (2): 408.416 \nDOI: 10.3844/ojbsci.2025.408.416 \n \n411 \namplified using Thunderbird SYBR qPCR Mix with \nprimer pairs for the NGF gene. The GAPDH gene was \nused as a reference gene in this study. A nalysis of the \nrelative mRNA expression of the NGF gene using the \nLivak method (2-ΔΔCt), which was carried out by \ncomparing the relative difference between the Cyclic \nthreshold (Ct) of menstrual blood in women with \nEndometriosis and without Endometriosis. \nStatistical Analyses  \nData analysis using SPSS software with the \nShapiro -Wilks t est to determine the normality of the \ndata distribution. DNA methylation and mRNA \nexpression levels were analyzed using independent T -\ntest or Mann -Whitney. Pearson or Spearman' s tests \nwere performed to see the correlation between DNA \nmethylation and mRNA e xpression. The p -value is \nconsidered significant (p<0.05). \nResults  \nCharacteristic of Participants  \nThe characteristics of women in this study included \ndata on age, body mass index, disease history, pain \nsymptoms that commonly occur in Endometriosis , and \nother questions related to the criteria in this study. \nTable (1) summarizes the characteristics of the research \nsubjects from 20 women with Endometriosis and \nwithout Endometriosis.  The endometriosis group found \nan average age  of 37 years old. Meanwhile, the  group \nwithout Endometriosis was 34 years old. Body Mass \nIndex (BMI) of women is dominated by women with \nnormal or ideal BMI, with a percentage of 45 % of \nwomen with Endometriosis and 40 % without \nEndometriosis.  In addition, the m enstrual cycles in the \ngroup of women with Endometriosis were within an \naverage of 28.7±3.802 days with a duration of 7.5±1.02 \ndays and menarche age at 12.8±1.24 years.   \nThe DNA Methylation Level of Human NGF Gene \nAnalyses Used MS-PCR \nThe result of electrophoresis visualization of MS P \nproduct samples of menstrual blood and eutopic \nendometriosis endometrium compared to a group of \nnormal women as controls is shown in Fig. (1). From \nthis figure, the NGF gene showed that partial \nmethylation occurred or that some bands were \nmethylated and some were unmethylated. The size of \nthe NGF gene amplicon is 150 bp. This study also used \na methylated positive control (K+) and an \nunmethylated negative control (K-) from Epitech \nMethylated Human with an amplicon size of 200 bp.  \nTable 1: Characteristics of participants \nVariable  \nEndometriosis \nN = 20  \nWithout \nendometriosis \nN = 20  \nAge, mean ± SD  37,05±6,41  34,55±5,48  \nBody Mass Index, (%)  \n1. Skinny (<18.5)  5%  10%  \n2. Normal (18,5-22,9)  45%  40%  \n3. Overweight (23-24,9)  30%  20%  \n4. Obesity (>25)  20%  30%  \nMenstrual Cycle (days)  28,7±3,80  27,7±1,82  \nMenstruation Length (days)  7,5±1,02  6,9±0,31  \nAverage age at menarche \n(years)  \n12,8±1,24  12,4±1,31  \n \n \n \nFig. 1: Electrophoresis visualization results of the MSP product \nof the NGF gene. K+ = methylation positive control; K- \n= unmethylated negative control; NTC = Non-Template \nControl. M = Methylation; U = Unmethylation \n \nFigure (2) is the result of a quantitative anal ysis of \nNGF gene gel electrophoresis bands. Based on \nmeasuring the surface area of the band using ImageJ \nsoftware, the NGF gene promoter from the \nendometriosis group was 52.05% in menstrual blood and \n50.85% in eutopic endometrium. Meanwhile, in the \nordinary women without endometriosis group, the \naverage DNA methylation percentage was 51.35 % in \nmenstrual blood and 51.2% in eutopic endometrium. The \nstatistical analysis results of the Shapiro -Wilk normality \ntest showed that the data was not normally distribu ted \neven though transformation had been carried out. So, the \nnon-parametric Mann-Whitney test was carried out with \nresults (p = 0.262) for the endometriosis menstrual blood \ngroup compared to non -endometriosis. Likewise, the \nresults of the statistical analy sis of endometriotic \nmenstrual blood compared with eutopic endometrium \nhave a value of p  = 0.095 (p>0.05). This indicates no \nsignificant difference in the level of DNA methylation \nof the NGF gene promoter in either menstrual blood or \neutopic endometrial ti ssue of endometriotic and non -\nendometriotic women using the MS -PCR method.  \n\n\nOcktariyana et al. / OnLine Journal of Biological Sciences 202 5, 25 (2): 408.416 \nDOI: 10.3844/ojbsci.2025.408.416 \n \n412 \n \n \nFig. 2: Differences in DNA methylation of the NGF gene \npromoter in menstrual blood and eutopic Endometriosis \ncompared to a group of women without Endometriosis \nusing the MSP method (ns = p>0.05) \n \nThe DNA Methylation Level of Human NGF Gene \nAnalyses Used Pyrosequencing \nMeasurement of DNA methylation of the NGF gene \npromoter was continued using the quantitative \nPyrosequencing method. This method is widely used in \nresearch explorati on, toxicological testing and \npharmacological approaches. PyroMark CpG Assays \n(Qiagen, Germany; EpigenDX USA ) is a software \nwidely used to analyze DNA methylation of a gene \nbased on specific CpG islands.  \nThe methylation of the NGF gene from \nendometriosis p atients and in women without \nEndometriosis that PyrogramTM showed is depicted in \nFig. (3). The mean percentage of NGF gene DNA \nmethylation levels at CpG sites 1 -3 from 10 \nendometriosis menstrual blood samples were 36.8, 42.4  \nand 38.7%, respectively, with t he mean value of CpG \nsites of NGF gene in the endometriosis group being \n39.3%. Meanwhile, from 10 menstrual blood of women \nwithout Endometriosis, the average percentage of DNA \nmethylation levels at CpG 1 -3 locations was 38.6, 55.2  \nand 50.6%, respectively, with an average CpG location \nin the control group of 48,1 % Fig. (4). \nThe results of statistical analysis of the Shapiro -Wilk \nnormality test show that the data is normally distributed. \nSo, an independent parametric T -test was carried out \nwith significant results (p = 0.332; p = 0.009; p = 0.010 ) \nfor each CpG location, and the average of the 3 CpG \nlocations in the endometriosis group had a significance \nvalue of p = 0.025 (p<0.05). There was a significant \ndifference between DNA methylation of the NGF gene \nin t he menstrual blood of the endometriosis group \ncompared to the group without Endometriosis. DNA \nmethylation of the NGF gene in menstrual blood with \nEndometriosis was lower than in women without \nEndometriosis. This finding was reported for the f irst \ntime that DNA methylation of the NGF gene in menstrual \nblood samples of subjects with Endometriosis was \nhypomethylated compared to those without \nEndometriosis (p<0.05). \n \n \n(a) PyrogramTM of a menstrual blood sample of case \n \n (b) PyrogramTM of a menstrual blood sample of control \n \nFig. 3: The DNA Methylation of human NGF gene analyses in \nmenstrual blood of Endometriosis and without \nendometriosis, used by Pyrosequencing \n \n \n \nFig. 4: The differences in DNA methylation levels at 3 CpG \nsites of the NGF gene by Pyrosequenci ng and the \naverage methylation levels of the three CpG sites in \nmenstrual blood samples of endometriosis women \ncompared to a group without Endometriosis. (ns = \np>0.05; *p<0.05)  \n39% 50%\n% \n49%\n% \n39% 56% 52% \n\nOcktariyana et al. / OnLine Journal of Biological Sciences 202 5, 25 (2): 408.416 \nDOI: 10.3844/ojbsci.2025.408.416 \n \n413 \nThe mRNA Expression Level of the Human NGF \nGene in Endometriosis \nThe differ ences in mRNA expression levels of the \nNerve Growth Factor (NGF) gene as a growth factor for \nnerve fibres in the menstrual blood of endometriosis \npatients and women without Endometriosis were \nmeasured using the quantitative real -time PCR method. \nThe quanti tative result is an amplification curve that \nshows the Cycle threshold (Ct) value. Owing to the Cycle \nCt value, the number of cycles captured by the fluorescent \nsubstance was subsequently analyzed using the Livak \nmethod to determine the relative mRNA expression value \nof the NGF gene. \nBased on the analysis of the Livak method, the average \nvalue of the relative expression of NGF gene mRNA \nincreased 4.29 times in the menstrual blood of women \nwith Endometriosis compared to women without \nEndometriosis. The resul ts of the Mann -Whitney test \nstatistical analysis showed that there was a significant \ndifference (p = 0.021) (Fig. 5).  \nCorrelation between DNA Methylation Level and \nmRNA Expressions of NGF Gene in Menstrual \nBlood of Endometriosis Patients  \nOwing to Spearman's rho statistical correlation \nanalysis for DNA methylation levels and relative \nexpression of NGF mRNA in menstrual blood samples in \nthe endometriosis group, a negative and no significant \ncorrelation was found (r = -0.073 and p = 0.841).  \n \n \n \nFig. 5: The differences in the relative expression levels of NGF \ngene mRNA in menstrual blood of women with \nEndometriosis compared to m enstrual blood without \nendometriosis *p<0.05, there is a significant difference \nin the relative expression level of NGF gene mRNA in \nmenstrual blood \nDiscussion \nEndometriosis is considered a form of malignancy in \ngynaecology. In which the development is often \nassociated with genetic and epigenetic alteration. It is well \nknown that DNA methylation in Endometriosis can drive \nthe expression changes of genes involved in Estrogen \nReceptor (ER) and Progesterone Receptor (PR) signalling \npathways. Alterations in these signalling pathways result \nin molecular changes in the endometriosis \nmicroenvironment, such as the increased activity of NF -\nB, PGE2 , E2  and ER-β, leading to inflammation, local \ndysregulation of hormonal pathways  and pain \nsensitization (Monsivais et al., 2015). \nThis study reports that women with Endometriosis \nhave an average age of 37.05±6.41 with an age range \nbetween 29 -45 years as th e reproductive age \n(premenopause). At the same time, the Body Mass Index \n(BMI) patients with Endometriosis exhibited BMI \ndifferences such as a normal BMI of 45, 30 overweight  \nand 20% obese. Although studies of endometriosis \npopulations in Asia are still minimal, research by Liu and \nZhang (2017) reported that the epidemiological factors of \nbody mass index in East Asian women influence the \nseverity of Endometriosis. Women with a higher BMI \ntend to be prone to early or mild symptoms of \nEndometriosis. At the sa me time, other factors are \ninfluential, such as genetics, menstrual and environmental \nfactors (Liu and Zhang, 2017; Yan et al., 2017). Darrow \nalso investigated the increased risk of Endometriosis by \nemphasizing six days of longer menstrual flow by \nenlisting specific factors: Heavy flow, severe cramps, and \nthose aged more than 30 years have a susceptibility to \nEndometriosis (Darrow et al., 1993). Abnormal menstrual \nbleeding is the basis for markers of endometrial pathology \n(Yang et al., 2012). \nEndometriosis-associated pain with dyspareunia has \nbeen reported by Peng et al. (2018), which is stimulated \nby the nerve fibre growth factor (NGF) signalling \npathway with the TrkA receptor. High levels of NGF \nincrease the secretion of cyclooxygenase-2 (COX-2) and \nprostaglandins (PGE2) (Peng et al ., 2018 ). The same \ncorrelation results were reported by Mu et al. (2020); it is \nknown that patients with Deep Infiltrating Endometriosis \n(DIE) in the uterine fibular ligament, vagina, uterine \nrectum, rectum, ureter are closely rela ted to pain during \nmenstruation. This pain signal is modulated by elevated \nlevels of expression of the NGF protein with its receptor  \n(Mu et al., 2020). NGF has been associated with several \ndifferent persistent pain conditions, including \nOsteoarthritis (OA), low back pain, diabetic peripheral \nneuropathy, bladder pain syndrome, bone cancer pain and \nEndometriosis (Kelleher et al., 2017). \n\n\nOcktariyana et al. / OnLine Journal of Biological Sciences 202 5, 25 (2): 408.416 \nDOI: 10.3844/ojbsci.2025.408.416 \n \n414 \nOcktariyana (2019) also reported the simultaneous \nincrease in NGF mRNA expression and nociceptive \nP2RX3 receptor in eutopic endometrial tissue of women \nwith Endometriosis that is correlated with the intensity of \npain severity and the increase in the incidence of \nEndometriosis. The higher the expression of NGF mRNA \nand P2RX3 receptors, the higher the intensity of pain felt \nby endometriosis subjects (Ocktariyana, 2019). Owing to \nthe findings, the researchers aimed to prove the expression \nof NGF mRNA and DNA level methylation in the \nmenstrual blood of patients with Endometriosis. \nMenstrual blood is considered a reliable sample DNA \nsource for exploring and investigating endometrial cells. \nMenstrual blood was selected as a sample for a \npreliminary study to identify biomarkers in the \ndevelopment of early diagnostics. Research using  \nmenstrual blood has also been carried out by Madjid et al. \n(2020) in detecting matrix metalloproteinase (MMP-9) \nprotein and inhibitors of metalloproteinase-1 (TIMP-1) in \nwomen with Endometriosis and evaluated by \nimmunohistochemistry. As a result, MMP -9 expression \nwas more robust , and  TIMP-1 expression was low er in \nwomen with endometriosis than in the control group, which \nhad statistically significant results (Madjid et al., 2020). \nGene expression is the consecutive transformation of \ngenetic information of a gene into functional ones. \nMessenger RNA (mRNA) is a product of gene \nexpression at the transcriptional level, while protein is a \nproduct of gene expression at the translational level. \nAnalysis of gene expression can be detected at the \nmRNA and protein levels. Dysregulation of gene \nexpression plays a crucial role in disease development. \nDefining the characteristics of each gene is the first step \ntoward developing new therapeutic strategies (Ping et al., \n2016). Our study has shown overexpression of NGF \nmRNA in menstrual blood patients with Endometriosis. \nThis f inding is important for the advancement of the \ndetection of Endometriosis  (Ping et al., 2016). \nManconi et al . (2018) proved an increase in NGF \nlevels in endometrial lesions and intense \nimmunoreactivity in endometriotic glands, which was \nassumed to stimulate neurogenesis (Manconi et al., 2018). \nBarcena de Arellano  et al. (2011) also reported that the \noverexpression of NGF in peritoneal fluids of \nendometriosis patients was analyzed using Western \nmethods, such as blot and cell culture staining (Barcena \nde Arellano et al., 2011). High levels of NGF were able to \npromote the expression of nociceptors and neurogenesis \nof sensory neurons, which influence pain by nociceptive \nmechanisms (Manconi et al ., 2018 ). The mRNA \nexpression of the NGF gene in eutopic endometrium  has \nbeen reported to exhibit a positive correlation with \nincreased expression by the nociceptor P2RX3 \n(Ocktariyana, 2019). \nAlterations in gene expression are often associated \nwith epigenetic changes, such as DNA methylation in \nEndometriosis, which is often associated with pathological \nconditions. Hypermethylation and hypomethylation in the \npromoter region of the CpG islands result in aberration of \ngene expression. Hypermethylation results in decreased \nexpression, and  hypomethylation results in increased \nexpression (Monsivais et al., 2015). \nNaqvi et al. (2014) stated that a certain amount of DNA \nmethylation is unknown to affect gene expression. \nSubsequently, it is correlated to several additional regulatory \nfactors and the study extension of cells and tissue s. The \ncorrelation analysis of DNA methylation and gene \nexpression varies according to the corresponding region of \nthe genome (Liang et al., 2018; Naqvi et al., 2014). \nBorghese et al. (2017) stated that the mechanism of \nepigenetic alteration does not only act on each mechanism \nin regulating transcription of gene expression. But also \ntheir frequent interaction with each other as intra -\nepigenetic. For example, DNA methylation and histone \nmodification can increase expression (Borghese et al ., \n2017; Yan et al., 2015). \nThe results of pyrosequencing method analysis, DNA \nmethylation of the NGF gene showed a significant \ndifference with a decrease in DNA methylation or \nhypomethylation in menstrual blood in the group of \nwomen with Endometriosis compared to the group \nwithout Endometriosis as control. Yuan et al. (2020) also \ninvestigated the role of NGF in chronic inflammatory \npain, which proved the existence of hypomethylation of \nCpG islands in the NGF gene promoter by administering \nthe Freund Adjuvant Complex (CFA). CF A not only \ncauses hypomethylation but also induces upregulation of \nNGF mRNA, as well as dorsal root ganglion protein levels \nin mice (Yuan et al., 2020). \nBased on this research, an increase in NGF gene \nmRNA expression also occurred in both menstrual blood \nand eutopic endometrial tissue of Endometriosis \ncompared to controls. Meanwhile, the results of the \ncorrelation test between DNA methylation and mRNA \nexpression of the NGF gene did not show a significant \nrelationship (p>0.05). The results of this study cann ot \nprove that changes in hypomethylation of the NGF gene \nin menstrual blood affect the increase in its mRNA \nexpression. However, hypomethylation and an increase in \nNGF gene mRNA expression in endometriotic menstrual \nblood were shown to be much higher than in controls. \nHence, we propose that NGF hypomethylation and \nmRNA expression can be used as non-invasive diagnostic \nbiomarkers for early detection of Endometriosis.  \nThe limitation of this research is that it uses a small \nsample size. This research is a pil ot study to identify \nbiomarkers in menstrual blood that represent endometrial \ntissue in reproductive women. In addition, it is also \ndifficult to obtain eutopic endometrial tissue in the same \nphase. So, proper protocols are required for sampling. \n\nOcktariyana et al. / OnLine Journal of Biological Sciences 202 5, 25 (2): 408.416 \nDOI: 10.3844/ojbsci.2025.408.416 \n \n415 \nConclusion \nThe increased mRNA expression of the NGF gene in \nmenstrual blood in women with Endometriosis \ncompared to menstrual blood in women without \nEndometriosis was not due to decreased DNA \nmethylation. However, hypomethylation of the NGF \ngene has the potential to  be a biomarker for the early \ndiagnosis of non-invasive Endometriosis. \nAcknowledgment \nThank you to the Indonesian Ministry of Research, \nTechnology and Higher Education, the Universitas \nIndonesia, and  the Ministry of Health of Indonesia \nsupported this study. \nFunding Information \nThe authors would like to thank The Leading Basic \nResearch of Higher Education Institutions (PDUPT) \nresearch grant from the Indonesian Ministry for \nResearch, Technology and Higher Education as support \nfor this research . \nAuthor’s Contributions \nOcktariyana: Collected subjects, performed analysis, \nand edited manuscript revision. \nNurul Hikmawati : Collected subjects, took  \nmeasurements, and prepared original draft of the manuscript. \nRaden Muharam  and Andon Hestiantoro: \nSupervised work, collected samples, interpreted the results. \nMuhammad Luky Satria Syahbana Marwali  and \nAgus Surur As’adi: Supervised work collected samples.  \nAsmarinah: Idea, design study, revised the  \nmanuscript, and gave the final approval. \nEthics \nThe Ethics Committee of the Faculty of Medicine, \nUniversity of Indonesia, has approved the study with the \nnumber 0126/UN2.F1/ETIK/2018 and addendum number \nKET-284/UN2.F1/ETIK/PPM.00.02/2020, and  the \npatient signed the consent for analysis. \nReference \nBarcena de Arellano, M. 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