{"paper_id":"088740e3-a1c0-4ce3-aee0-95300e4041e4","body_text":"American Journal of Molecular Biology, 2026, 16(1), 1-18 \nhttps://www.scirp.org/journal/ajmb \nISSN Online: 2161-6663 \nISSN Print: 2161-6620 \n \nDOI: 10.4236/ajmb.2026.161001  Dec.  5, 2025 1 American Journal of Molecular Biology \n \n \n \n \nDifferential Expression of EMT-Related \nTranscription Factors and Mitochondrial \nDynamics Genes across Endometriosis Stages \nRoya Rozati1*, Aleem Ahmed Khan2, Wajeeda Tabasum3, Taalia Nazeer Ahmed3,  \nAyapati Gautam Mehdi3, Vikram Aiman Ayapati3, Habeeba Sultana4, Syeda Asma Jabeen4, \nCharulata Chatterjee3 \n1Department of Obst & Gynecology, Shadan Institute of Medical Sciences, Medical Health and Research Institute (MHRI), \nHyderabad, India \n2Centre for Liver Research and Diagnostics-DCMS, CMH Research and Innovation, Hyderabad, India \n3Medical Health and Research Institute (MHRI), Hyderabad, India \n4Shadan Institute of Medical Sciences, Hyderabad, India \n \n \n \nAbstract \nBackground:  Endometriosis is a chronic, debilitating gynecological disorder \ncharacterized by the ectopic presence of endometrial-like tissue, often leading \nto pain and infertility. However, the role of mitochondrial dynamics, mitoph-\nagy, and mitochondrial-related complexes in the development of endometri-\nosis— and their relationship with epithelial-mesenchymal transition (EMT)—\nremains unclear. Objective:  This study aims to investigate the role of mito-\nchondrial oxidative phosphorylation (OXPHOS) dysfunction and EMT in the \npathogenesis and progression of endometriosis, and to explore how these mo-\nlecular alterations correlate with disease severity. Materials  and Methods:  A \ntotal of 180 women with surgically confirmed endometriosis, categorized into \nminimal-mild (Stage I-II) and moderate-severe (Stage III-IV) groups, along \nwith 180 healthy controls, were included. Clinical, hormonal, and demographic \ndata were recorded. Endometrial tissue samples were analyzed using quanti-\ntative real-time PCR, flow cytometry, and phase-contrast microscopy. Gene \nexpression profiling focused on OXPHOS components, mitochondrial dy-\nnamics (fission/fusion), mitophagy-related genes, hypoxia markers, EMT tran-\nscription factors, and mesenchymal cell surface markers. In vitro cultures of \nendometrial stromal cells were evaluated morphologically and molecularly to \nassess EMT progression. Results:  Quantitative PCR and in vitro analyses re-\nHow to cite this paper: Rozati, R., Khan, \nA.A., Tabasum, W., Ahmed, T.N., Mehdi, \nA.G., Ayapati, V.A., Sultana, H., Jabeen, \nS.A. and Chatterjee, C. (2026) Differential \nExpression of EMT-Related Transcription \nFactors and Mitochondrial Dynamics \nGenes across Endometriosis Stages. Ameri-\ncan Journal of Molecular Biology, 16, 1-18. \nhttps://doi.org/10.4236/ajmb.2026.161001 \n \nReceived:  July 3, 2025 \nAccepted: December 2, 2025 \nPublished: December 5, 2025 \n \nCopyright © 2026 by author(s) and  \nScientific Research Publishing Inc. \nThis work is licensed under the Creative \nCommons Attribution International  \nLicense (CC BY 4.0). \nhttp://creativecommons.org/licenses/by/4.0/   \n  \nOpen Access\n\nR. Rozati et al. \n \n \nDOI: 10.4236/ajmb.2026.161001 2 American Journal of Molecular Biology \n \nvealed that advanced endometriosis is associated with significant downregu-\nlation of OXPHOS genes (ND1, ND6, CYTB, CO2, CO3, ATP6) and mito-\nchondrial dynamics genes (DRP1, OPA1, MFN1/2, LCLAT1), along with de-\ncreased mitophagy markers (PINK1, PARKIN) and elevated hypoxia marker \nHIF-1α. EMT activation was confirmed by morphological transitions in cul-\ntured stromal cells, reduced E-cadherin, increased N-cadherin, and elevated \nexpression of mesenchymal and EMT-related markers (CD73, CD90, CD105, \nTWIST, SNAIL, SLUG). These molecular alterations were most pronounced \nin advanced (Stage III-IV) disease, minimally evident in Stage I-II, and not sig-\nnificantly different from controls. Conclusion:  Our findings demonstrate that \nmitochondrial dysfunction and EMT activation are interrelated processes con-\ntributing to the severity and persistence of endometriosis. The distinct altera-\ntions in gene expression associated with mitochondrial bioenergetics and cel-\nlular plasticity in advanced disease highlight their potential as biomarkers for \ndiagnosis, disease staging, and as targets for novel therapeutic interventions. \n \nKeywords \nEndometriosis, Mitochondrial Oxidative Phosphorylation, Epithelial to \nMesenchymal Cell Transitioning \n \n1. Introduction \nEndometriosis is a chronic, estrogen -dependent inflammatory condition charac-\nterized by the presence of endometrial-like tissue outside the uterine cavity [1]. It \naffects approximately 10% - 15% of women of reproductive age and is frequently \nassociated with pelvic pain, dysmenorrhea, and infertility [2]. Despite its preva-\nlence and significant impact on quality of life, the pathogenesis of endometriosis \nremains incompletely understood [3]. \nEndometriosis is a multifactorial disorder influenced by immunological, mo-\nlecular, and environmental factors that contribute to its development [4]. Current \ntheories, including retrograde menstruation, coelomic metaplasia, and stem cell \nimplantation, fail to fully explain the cellular and molecular dynamics underlying \nlesion establishment and progression. Increasing evidence suggests that a complex \ninterplay of immune dysregulation, oxidative stress, and hormonal imbalances \ncontributes to the pathophysiological landscape of the disease [5] [6]. \nMitochondria, the powerhouses of the cell, play a central role not only in energy \nproduction via oxidative phosphorylation (OXPHOS) but also in regulating redox \nhomeostasis, apoptosis, and cellular metabolism [7] . Recent studies suggest that \nmitochondrial dysfunction is a critical contributor to the pathophysiology of en-\ndometriosis. Impairments in OXPHOS compromise ATP generation, leading to \nbioenergetic stress and excess reactive oxygen species (ROS), which can ex acer-\nbate inflammation and tissue injury [8] . Disruptions in mitochondrial dynamics, \nincluding altered fission and fusion processes and impaired mitophagy, may fur-\n\nR. Rozati et al. \n \n \nDOI: 10.4236/ajmb.2026.161001 3 American Journal of Molecular Biology \n \nther aggravate mitochondrial damage and cellular stress [9] . These dysfunctions \ncould not only facilitate the survival of ectopic endometrial cells but also promote \ntheir resistance to apoptosis and metabolic reprogramming [10] [11]. \nPathogenic mutations in mitochondrial DNA (mtDNA), particularly those af-\nfecting genes encoding subunits of the mitochondrial respiratory chain complexes, \nmay further exacerbate mitochondrial dysfunction. Such mutations can disrupt \nOXPHOS efficiency and elevate ROS production, potentially contributing to on-\ncogenic processes. The functional impact of these mutations can be assessed by  \nexamining the expression of OXPHOS -related genes in endometrial tissues [12] . \nMoreover, dysregulated mitochondrial quality control mechanisms, including mi-\ntophagy, may facilitate disease progression through sustained oxidative and met-\nabolic stress [13]. Despite these insights, the role of OXPHOS gene expression in \nthe pathophysiology of endometriosis, particularly among Indian patients, re-\nmains inadequately characterized. \nEpithelial-mesenchymal transition (EMT) is a dynamic process in which epi-\nthelial cells transform into motile and invasive mesenchymal cells such as en-\nhanced motility and invasiveness. This transition enables individual epithe lial \ncells to migrate, invade surrounding tissues, and facilitate metastasis through in-\ntravasation and extravasation [14] [15]. \nMolecularly, EMT is driven by transcription factors such as TWIST, SNAIL, \nand SLUG, and is characterized by a cadherin switch— the downregulation of ep-\nithelial markers like E -cadherin and the upregulation of mesenchymal markers \nincluding N -cadherin, CD73, CD90, and CD105 [16]  [17]. These mesenchymal \ntraits enhance cellular plasticity, invasiveness, and resistance to apoptosis, hall-\nmarks of more aggressive or treatment-resistant forms of the disease. \n2. Materials and Methods \n2.1. Study Design \nThe study protocol was approved by the Institutional Ethics Committee with eth-\nics registration number ECR/1609/Inst/TG/2021, and written informed consent \nwas obtained from all participants prior to their enrollment in the study.  \n2.2. Study Population \nA total of 300 women presenting with symptoms suggestive of endometriosis were \ninitially recruited from 1 st March 2023 to 31 st March 2025 in the Department of \nObstetrics and Gynecology, MHRT Hospital & Research Centre, Hyderabad, In-\ndia as per the inclusion and exclusion criteria. Of these, 240 underwent diagnostic \nlaparoscopy. Endometriosis was confirmed in 180 cases based on surgical and his-\ntopathological criteria. These patients were stratified into two groups: Stage I- II \n(minimal to mild disease, n = 90) and Stage III-IV (moderate to severe disease, n \n= 90) according to the revised American Society for Reproductive Medicine  \n(rASRM) classification. Additionally, 180 healthy women without clinical or lap-\naroscopic evidence of endometriosis were included as controls. Demographic data, \n\nR. Rozati et al. \n \n \nDOI: 10.4236/ajmb.2026.161001 4 American Journal of Molecular Biology \n \nreproductive history, body mass index (BMI), and infertility duration were rec-\norded. Serum hormone levels including FSH, LH, AMH, and estradiol were meas-\nured using standard immunoassay techniques. \n2.3. Tissue Collection and Cell Culture \nCell viability of isolated cells from endometriotic tissue biopsies was evaluated us-\ning the Trypan Blue Exclusion Assay and Fluorescein Diacetate (FDA) staining. \nEndometrial biopsy samples were obtained from all participants during the pro-\nliferative phase of the menstrual cycle. Single-cell suspensions were prepared from \nthe tissue samples, and cell viability was assessed using FDA staining, while total \nand viable cell counts were performed using a hemocytometer. Viable cells were \nsubsequently cultured unde r appropriate conditions to evaluate adherence and \nmorphological characteristics over a five -day period. Phase -contrast microscopy \nwas employed to monitor morphological changes associated with epithelial -mes-\nenchymal transition (EMT). \n2.4. Collection of Peripheral Blood Samples \nPeripheral blood samples (4 mL) were collected from all study participants, in-\ncluding individuals with endometriosis and healthy controls, into EDTA-coated \ntubes. Samples were anonymized using coded identifiers and stored at 4˚C until \nfurther processing for RNA extraction. \n2.5. RNA Isolation and Quantitative Real-Time PCR \nTotal RNA was extracted from endometrial tissues and cultured cells using a com-\nmercial RNA isolation kit. Complementary DNA (cDNA) synthesis was performed \nusing reverse transcriptase. Five nanograms of complementary DNA (cD NA) \nwere used for gene expression analysis. Conventional PCR was employed for the \ndetection of selected gene transcripts, while quantitative real-time PCR (RT-qPCR) \nwas performed to quantify transcript levels relative to endogenous reference genes. \nQuantitative real -time PCR (RT -qPCR) was  used to evaluate the expression of \ngenes associated with mitochondrial oxidative phosphorylation (ND1, ND6, CO2, \nCO3, CYTB, ATP6, ATP8), mitochondrial dynamics (DRP1, OPA1, MFN1, MFN2, \nLCLAT1), mitophagy (PINK1, PARKIN), hypoxia (HIF-1α), mesenchymal mark-\ners (CD73, CD90, CD105), EMT transcription factors (TWIST, SNAIL, SLUG ), \nand cadherins (E -cadherin, N -cadherin). Gene expression levels were quanti-\nfied relative to those in control subjects, using the endogenous housekeeping gene \nglyceraldehyde -3-phosphate dehydrogenase (GAPDH) as an int ernal refer ence \nfor normalization. Relative gene expression was calculated usi ng the 2^–ΔΔCt \nmethod, and fold-change values were determined with the aid of StepOne ™ Soft-\nware (Applied Biosystems). \n2.6. Flow Cytometry \nMesenchymal stem/stromal cell surface markers CD73, CD90, and CD105 were \n\nR. Rozati et al. \n \n \nDOI: 10.4236/ajmb.2026.161001 5 American Journal of Molecular Biology \n \nanalyzed using flow cytometry on day 5 of in vitro culture. Cells were stained with \nfluorochrome -conjugated antibodies and analyzed using a flow cytometer t o \ndetermine the expression profiles. \n2.7. Statistical Analysis \nData were analyzed using appropriate statistical software. Group comparisons \nwere performed using one-way ANOVA followed by post hoc tests. A p -value of \nless than 0.05 was considered statistically significant. Results were expressed as \nmean ± standard error of the mean (SEM) or percentages where applicable. \n3. Results \nA total of 300 subjects were initially enrolled in the study. Out of these, 24 0 \npatients underwent laparoscopic examination. Endometriosis was confirmed in \n180 of the laparoscopically evaluated patients. Among the confirmed cases, 90 \npatients were classified with Stage I -II endometriosis, representing minimal t o \nmild disease, while the remaining 90 patients were diagnosed with Stage III -IV \nendometriosis, corresponding to moderate to severe disease severity (\nTable 1 ). \n \nTable 1.  Summary of subjects with surgically confirmed endometriosis. \nNo of  \nsubjects \nNo of patients undergoing  \nlaparoscopy \nNo of the patients confirmed diagnosis of \nEndometriosis \n300 240 180 \n  Stage I-II (Minimal to mild): 90 \n  Stage III-IV (Moderate to severe): 90 \n \nThe study included 180 women diagnosed with surgically confirmed endome-\ntriosis, evenly divided into minimal to mild (Stage I -II) and moderate to seve re \n(Stage III-IV) groups, alongside 180 healthy control subjects. The mean age was \nnotably lower in the minimal to mild group (26 ± 0.8 years) compared to the mod-\nerate to severe group (33 ± 0.4 years) and controls (35.3 ± 0.7 years). Body mass \nindex (BMI) values were similar between the endometriosis groups (23.2 ± 0.7 and \n23.5 ± 0.4, respectively), but markedly lower than in controls (35.3 ± 0.4). \nHormonal profiles showed minor variations, with follicle-stimulating hormone \n(FSH) levels slightly higher in the moderate to severe group (7.13 ± 0.4 mIU/mL) \ncompared to controls (7.02 ± 0.2 mIU/mL) and the mild group (6.13 ± 0.5 mIU/mL). \nAnti-Müllerian hormone (AMH) levels were somewhat reduced in both endome-\ntriosis groups relative to controls. Luteinizing hormone (LH) and estradiol con-\ncentrations were comparable across all groups. \nRegarding reproductive and demographic factors, the majority of participants \nin all groups were married. A higher proportion of women with endometriosis \nwere nulliparous (72 and 77 in mild and severe groups, respectively) compared to \n\nR. Rozati et al. \n \n \nDOI: 10.4236/ajmb.2026.161001 6 American Journal of Molecular Biology \n \ncontrols (13). Duration of infertility was longer in women with endometriosis, \naveraging 7 ± 1.8 years in the minimal to mild group and 9 ± 2.5 years in the \nmoderate to severe group, compared to 5 ± 1.1 years in controls. Primary infertil-\nity was more prevalent than secondary infertility among women with endometri-\nosis (Table 2 ). \n \nTable 2 . Demographic, hormonal, and clinical outcome measures in individuals with surgically confirmed endometriosis. \nBaseline  parameter  \nSurgical  endometriosis  (n = 180) Controls  (n = 180) \nMinimal  to mild (n = 90) Moderate  to severe  (n = 90)  \nAge (mean ± SEM) 26 ± 0.8 33 ± 0.4 35.3 ± 0.7 \nBMI in kg/m2 (median  [IQR]) 23.2 ± 0.7 23.5 ± 0.4 35.3 ± 0.4 \nFSH (mean ± SEM) 6.13 ± 0.5 7.13 ± 0.4 7.02 ± 0.2 \nAMH (ng/mL)  1.82 ± 3.02 1.78 ± 2.02 2.78 ± 3.02 \nLH (mIU/mL)  4.18± 1.11 4.56± 2.58 3.12 ± 1.72 \nEstradiol  (pg/mL)  42.16 ±3.18  44.73 ± 5.92  40.26 ±1.62 \nMarital  status  (n)    \nSingle 12 16 13 \nMarried 74 71 165 \nDivorced 4 3 2 \nParity (n)    \n0 72 77 13 \n1 18 13 87 \nDuration  of infertility  (years)  7 ± 1.8 9 ± 2.5 5 ± 1.1 \nInfertility  type (n)    \nPrimary 65 72 NA \nSecondary 25 18 NA \nTotal number of subjects  90 90 180 \n3.1. In Vitro Culture of Cells Isolated from Different Forms of  \nEndometrial Tissue Biopsies \nSingle cells were isolated from endometrial tissue biopsy samples and assessed for \nviability using fluorescein diacetate (FDA) staining, which selectively labels live \ncells with intact membranes. Following confirmation of viability, the cells were \ncultured under appropriate in vitro conditions to support adhesion and prolifer-\nation. Over time, the adherent cells exhibited morphological transitions from a \nspherical to a spindle -shaped phenotype, indicative of epithelial -mesenchymal \ntransition (EMT). These morphological changes provided the basis for subsequent \ninvestigations into EMT at both the cellular and molecular levels (\nFigure 1 ). \n\nR. Rozati et al. \n \n \nDOI: 10.4236/ajmb.2026.161001 7 American Journal of Molecular Biology \n \n \nFigure 1.  Assessment of membrane integrity. Panel (A) shows unstained cells, while Panel \n(B) shows cells stained with fluorescein diacetate (FDA), indicating viable cells with intact \nmembranes. Images were captured at 40× magnification. Scale bar = 20 µm. \n3.2. Identification of EMT-Related Morphological Alterations in  \nEndometrial Biopsies \nMorphological evaluation was performed using phase -contrast microscopy from \nday 0 to day 5 of in vitro culture. Initially, cells from both mild and severe endo-\nmetriosis samples exhibited predominantly spherical, epithelial-like morphology. \nAs the culture progressed, a gradual transition toward an elongated, spindle -\nshaped phenotype— characteristic of mesenchymal cells— was observed, indicat-\ning epithelial-mesenchymal transition (EMT). Notably, by day 5, cultures derived \nfrom severe endometriosis biopsies showed a visibly higher population of mesen-\nchymal-like cells compared to those from mild cases. These observations suggest \na more pronounced EMT progression in cells derived from severe forms of endo-\nmetriosis. Scale bars = 100 µm (\nFigure 2 ). \n \n \nFigure 2. Morphological characterization of cells isolated from mild and severe endome-\ntrial tissue biopsies. Representative images show differences in cellular morphology be-\ntween the two conditions. Images were captured at 10× magnification. Scale bar = 20 µm. \n3.3. Quantification of OXPHOS Gene Expression in Endometrial \nBiopsies \nQuantitative PCR analysis (Figure 3 ) revealed a marked downregulation of genes \nassociated with oxidative phosphorylation (OXPHOS) in endometrial biopsies \nfrom women with severe endometriosis compared to both healthy controls  and \nthose with mild disease (p < 0.0001). In contrast, the expression levels of t hese \ngenes showed no statistically significant differences between the control and mild  \n\n\nR. Rozati et al. \n \n \nDOI: 10.4236/ajmb.2026.161001 8 American Journal of Molecular Biology \n \n \nFigure 3. Relative expression of OXPHOS genes in endometrial biopsies. Gene expression levels were assessed in samples from \nhealthy controls, women with mild endometriosis, and women with severe endometriosis. ( A) Complex I (ND1 and ND6); ( B) \nComplex III (CYT- B); (C) Complex IV ( CO2 and CO3 ); (D) Complex V (ATP6 and ATP8). Note: (***p < 0.0001) represents \nsignificance, ns*-non significant. \n \nendometriosis groups, suggesting that substantial alterations in mitochon drial \ngene expression occur predominantly in advanced stages of the condition.  \nThe genes analyzed are central to mitochondrial bioenergetics. ND1 and ND6 , \ncomponents of Complex I, initiate the electron transport process.  CYTB, part of \nComplex III, facilitates electron transfer from ubiquinol to cytochrome. CO2 and \nCO3, subunits of Complex IV, are involved in the terminal reduction of oxygen \nto water. Finally, ATP6 and ATP8, constituents of Complex V (ATP synth ase), \nare directly responsible for ATP generation. \nThe consistent downregulation of these genes in severe endometriosis points to \nwidespread mitochondrial impairment. This dysfunction likely compromises \nATP production, leading to energy deficits in endometrial cells and potentially \ncontributing to elevated oxidative stress. Such disturbances in mitochondrial ho-\nmeostasis may play a critical role in disease pathophysiology, exacerbating tissue \ndamage and promoting disease progression. \n\n\nR. Rozati et al. \n \n \nDOI: 10.4236/ajmb.2026.161001 9 American Journal of Molecular Biology \n \nThe absence of significant changes in OXPHOS gene expression between con-\ntrol and mild cases suggests that mitochondrial dysfunction may be a hallmark of \nmore advanced disease stages. This pattern of expression could serve as a molec-\nular indicator of disease severity and offers insight into potential therapeutic tar-\ngets aimed at improving mitochondrial function and cellular energy metabolism \nin endometriosis (\nFigure 3 ). \n3.4. Analysis of Mitochondrial Gene Expression in Fission and  \nFusion Genes \nMitochondrial dysfunction appears to intensify with the progression of endome-\ntriosis. In this study, the expression levels of key genes involved in mitochondrial \ndynamics and energy homeostasis— DRP1, OPA1, MFN1, MFN2, and LCLAT1—\nwere evaluated. These genes play critical roles in regulating mitochondrial fission, \nfusion, and lipid remodeling. A significant downregulation, particularly in severe \nstages of the disease, suggests impaired mitochondrial integrity and function.  \nIn the mild stage of endometriosis, the expression profiles of these genes remain \nrelatively stable and comparable to those of healthy controls, indicating that mi-\ntochondrial function is largely preserved. However, as the disease advances, a con-\nsistent and significant reduction in gene expression is observed. This decline likely \ndisrupts mitochondrial morphology and bioenergetics, leading to reduced ATP \nsynthesis, elevated reactive oxygen species (ROS) production, and increased oxi-\ndative stress— all of which contribute to cellular dysfunction and tissue damage. \nThe observed correlation between endometriosis severity and decreased expres-\nsion of mitochondrial-related genes supports the notion that mitochondrial bio-\nenergetic failure is a pivotal factor in disease pathophysiology. These findings not \nonly provide insi ght into the molecular mechanisms underlying endometriosis \nprogression but also highlight the potential of mitochondrial dynamics-associated \ngenes as biomarkers for disease severity and as therapeutic targets for restoring \nmitochondrial function and tissue homeostasis (\nFigure 4 ). \n3.5. Relative Expression of Mitophagy Genes PINK1 and PARKIN  \nin Endometrial Tissues \nThe expression of mitophagy-related genes PINK1 and PARKIN was evaluated in \nendometrial tissues from control, mild, and severe endometriosis groups. While \nno significant differences were observed between control and mild cases, a marked \ndownregulation of both genes was evident in severe endometriosis (p < 0.0001). \nPINK1 and PARKIN are essential for mitochondrial quality control, initiating \nthe removal of damaged mitochondria via mitophagy. Their reduced expression \nin severe disease suggests impaired mitophagic activity, potentially leading to mi-\ntochondrial dysfunction, oxidative stress, and inflammation— key features of ad-\nvanced endometriosis. These findings highlight PINK1 and PARKIN as potential \nbiomarkers of disease severity and therapeutic targets to restore mitochondrial \nhomeostasis (\nFigure 5 ). \n\nR. Rozati et al. \n \n \nDOI: 10.4236/ajmb.2026.161001 10 American Journal of Molecular Biology \n \n \nFigure 4.  Relative expression of the fission and fusion gene in endometrial biopsies. Gene expression was quantified in endometrial \ntissues from healthy controls, and from women with mild and severe endometriosis. Note: (***p < 0.0001) represents significan ce, \nns*-non significant. \n \n \nFigure 5. Relative expression of mitophagy-related genes in endometrial biopsies. Expression levels were measured in samples from \nhealthy controls, and from women with mild and severe endometriosis. (A) PINK1; (B) PARKIN. Note: (***p < 0.0001) represents \nsignificance, ns*-non significant. \n3.6. Elevated Expression of HIF-1α in Severe Endometriosis \nThe expression of HIF -1α was evaluated in endometrial biopsies across control, \nmild, and severe endometriosis groups. No significant difference was observed \nbetween control and mild groups, suggesting minimal hypoxic signaling in early \nstages. However, a s ignificant increase in HIF -1α expression was detected in se-\nvere endometriosis (p < 0.0001), indicating activation of hypoxia-associated path-\n\n\nR. Rozati et al. \n \n \nDOI: 10.4236/ajmb.2026.161001 11 American Journal of Molecular Biology \n \nways in advanced disease. \nHIF-1α, a key transcription factor in cellular response to low oxygen, promotes \ngenes involved in angiogenesis, inflammation, and metabolic adaptation. Its up-\nregulation in severe cases suggests a hypoxic microenvironment contributing to \ndisease progression, including abnormal vascularization and fibrosis. These find-\nings underscore the relevance of HIF-1α as a potential biomarker of disease sever-\nity and a therapeutic target in advanced endometriosis (Figure 6 ). \n \n \nFigure 6.  Relative expression of HIF1α in endometrial biopsies. HIF1α \ngene expression was quantified in endometrial tissues from healthy \ncontrols, and from women with mild and severe endometriosis. Note: \n(***p < 0.0001) represents significance, ns*-non significant. \n3.7. Expression of Mesenchymal Markers CD73, CD90, and CD105  \nin Endometrial Stromal Cells \nCD73, CD90, and CD105 are established markers of mesenchymal stem/stromal \ncells (MSCs). Flow cytometry revealed a marked increase in their expression in stro-\nmal cells from severe endometriosis compared to mild cases, indicating a higher \nproportion of MSC-like cells in advanced disease. \nThis enrichment suggests a phenotypic shift toward a mesenchymal state, po-\ntentially driven by epithelial -to-mesenchymal transition (EMT), contributing to \nincreased cellular plasticity, fibrosis, and invasiveness. The elevated expression of \nthese markers reflects enhanced regenerative and invasive capabilities, supporting \nlesion persistence and progression. \nOverall, the data highlight a significant mesenchymal transformation in severe \nendometriosis, underscoring the role of MSC -like stromal cells in disease patho-\ngenesis and their potential as therapeutic targets (Figure 7 ). \n3.8. Upregulation of CD73, CD90, and CD105 Indicates EMT  \nProgression in Severe Endometriosis \nQuantitative RT-PCR analysis was conducted on day 5 of in vitro cultures to assess \nthe expression of key mesenchymal markers— CD73, CD90, and CD105— across \n\n\nR. Rozati et al. \n \n \nDOI: 10.4236/ajmb.2026.161001 12 American Journal of Molecular Biology \n \ncontrol, mild, and severe endometriosis samples. As shown in Figure 8 , expres-\nsion levels of all three markers were markedly elevated in stromal cells from severe \nendometriosis cases compared to both mild forms and healthy controls (p < \n0.0001). A modest but statistically significant increase was also observed in mild \ncases relative to controls (p < 0.01). \n \n \nFigure 7.  Immunophenotypic characterization of mesenchymal stem cells (MSCs) isolated from endometrial biopsies. Flow \ncytometric analysis of MSCs derived from women with mild and severe endometriosis, performed on day 5 of in vitro culture. \n \n \nFigure 8.  Expression of mesenchymal stem cell -specific genes in cultured endometrial MSCs. Relative mRNA levels of CD 90 (A), \nCD105 (B), and CD73 (C) were assessed by real-time quantitative PCR (RT-qPCR) on day 5 of in vitro culture. MSCs were isolated \nfrom endometrial biopsies of healthy controls, and women with mild and severe endometriosis. Note: (***p <  0.0001) represents \nsignificance, ns*-non significant. \n \nThis progressive upregulation of mesenchymal markers indicates a phenotypic \n\n\nR. Rozati et al. \n \n \nDOI: 10.4236/ajmb.2026.161001 13 American Journal of Molecular Biology \n \nshift toward a mesenchymal state, suggesting a heightened epithelial -to-mesen-\nchymal transition (EMT) in severe endometriotic conditions. These findings re-\ninforce the morphological observations and support the notion of enhanced EMT \nactivity as a hallmark of disease severity in endometriosis (Figure 8 ). \n3.9. Reduced E-Cadherin and Elevated N-Cadherin Facilitate EMT \nQuantitative real-time PCR analysis demonstrated a marked downregulation of \nE-cadherin and a significant upregulation of N-cadherin in endometrial samples \nfrom individuals with severe endometriosis when compared to both mild and \ncontrol groups (***p < 0.0001). No statistically significant difference in expression \nwas detected between the control and mild groups for either gene. This pattern  \nreflects a cadherin switch, a key feature of the epithelial-mesenchymal transition \n(EMT), and underscores the enhanced activation of EMT in more advanced stages \nof endometriosis, implicating its contribution to disease progression (Figure 9 ). \n \n \nFigure 9.  Expression analysis of epithelial-mesenchymal transition (EMT) markers in endometrial biopsies. Relative mRNA levels \nof E-cadherin (A) and N-cadherin (B) were quantified in endometrial tissues from healthy controls, and from women with mild \nand severe endometriosis to assess EMT progression. Note: (***p < 0.0001) represents significance, ns*-non significant. \n3.10. Stage-Dependent Upregulation of TWIST, SNAIL, and SLUG  \nHighlights EMT Activation in Severe Endometriosis \nTWIST, SNAIL, and SLUG are critical transcription factors that suppress E- cad-\nherin expression and drive the epithelial -mesenchymal transition (EMT). Quan-\ntitative real-time PCR analysis showed a significant increase in the expression of \nthese EMT-inducing factors in severe endometrial samples compared to both mild \nand control groups (***p < 0.0001). In contrast, no significant difference in ex-\npression was observed between the mild and control groups. \nThese results indicate a disease severity-dependent elevation in TWIST, SNAIL, \nand SLUG levels, suggesting their enhanced role in the molecular events underly-\ning EMT activation during advanced endometriosis. When considered alongside \nthe reduced E-cadherin expression, this pattern strongly supports the contribu-\ntion of EMT-related transcriptional regulation to the progression and invasive-\n\n\nR. Rozati et al. \n \n \nDOI: 10.4236/ajmb.2026.161001 14 American Journal of Molecular Biology \n \nness of severe endometrial lesions (Figure 10 ). \n \n \nFigure 10.  Relative expression of EMT-associated transcription factors in endometrial biopsies. Bar graphs show mRNA levels of \nTWIST (A), SNAIL (B), and SLUG (C)  in tissues from healthy controls, women with mild endometriosis, and women with severe \nendometriosis. Note: (***p < 0.0001) represents significance, ns*-non significant. \n4. Discussion \nThe present study highlights the critical role of mitochondrial dysfunction and \nepithelial-to-mesenchymal transition (EMT) in the progression of endometriosis. \nBy evaluating molecular and cellular markers in control, mild, and severe disease \ngroups, we provide evidence that impaired mitochondrial bioenergetics and in-\ncreased mesenchymal characteristics are key features of advanced pathology. \nImportantly, the control group consisted of 180 healthy women without clinical \nsymptoms suggestive of endometriosis, recruited from the same catchment pop-\nulation as patients. All control participants underwent thorough gynecological \nevaluation and pelvic ultrasonography to exclude pelvic pathology. Women with \nany suspicion of endometriosis or other gynecological disorders were excluded. \nAs part of ethical and methodological rigor, controls also had no prior surgical \ndiagnosis of endometriosis, no infertility history linked to pelvic pathology, and \nnormal hormonal profiles. This ensured that the comparison group truly repre-\nsented endometriosis-free individuals, thereby strengthening the validity of ob-\nserved molecular differences. \nQuantitative analysis of oxidative phosphorylation (OXPHOS) genes revealed \nmarked mitochondrial respiratory impairment in severe endometriosis [18], with \nsignificant downregulation of ND1, ND6, CYTB, CO2, CO3, ATP6, and ATP8. \nThis disruption indicates reduced electron transport chain activity and ATP syn-\nthesis, changes largely absent in mild cases, suggesting mitochondrial dysfunction \npredominantly arises in late-stage disease and may serve as a biomarker of pro-\ngression [19] [20]. Reduced ATP levels likely disturb cellular homeostasis, pro-\nmoting chronic inflammation and abnormal cell survival. \nSimilarly,  genes regulating  mitochondrial  dynamics— DRP1, OPA1, MFN1, \n\n\nR. Rozati et al. \n \n \nDOI: 10.4236/ajmb.2026.161001 15 American Journal of Molecular Biology \n \nMFN2, and LCLAT1— were markedly decreased in advanced disease, indicating \ndisrupted fission, fusion, and lipid remodeling. These impairments threaten mi-\ntochondrial integrity and exacerbate metabolic stress [21]-[24]. The parallel down-\nregulation of mitophagy regulators PINK1 and PARKIN further suggests defec-\ntive clearance of damaged mitochondria, potentially leading to excessive ROS ac-\ncumulation and persistent inflammation, consistent with previous reports. \nThe observed upregulation of HIF-1α in severe cases indicates a hypoxic mi-\ncroenvironment within endometriotic lesions, which may drive angiogenesis, fi-\nbrosis, and metabolic reprogramming— hallmarks of invasive disease. \nIn vitro, stromal cells from patients displayed progressive EMT, evident both \nmorphologically and at the molecular level. Severe disease was characterized by \nelevated mesenchymal stem cell markers (CD73, CD90, CD105) and EMT tran-\nscription factors (TWIST, S NAIL, SLUG), coupled with a cadherin switch — de-\ncreased E-cadherin and increased N-cadherin — indicative of enhanced invasive-\nness, loss of cell-cell adhesion, and increased migratory potential [25]-[28]. \nCollectively, these findings support a central hypothesis: advanced endometri-\nosis involves a synergistic interplay between mitochondrial impairment and EMT \nactivation. Mitochondrial stress may trigger EMT programs, while mesenchymal \ntransition can further compromise mitochondrial efficiency, creating a feedback \nloop that sustains lesion persistence and promotes tissue remodeling. \nIn conclusion, mitochondrial bioenergetic failure and EMT emerge as interre-\nlated drivers of severe endometriosis. The identified molecular signatures show \npromise as diagnostic markers and therapeutic targets. Strategies aimed at restor-\ning mitochondrial function or inhibiting EMT could offer novel approaches for \nmanaging progressive, treatment-resistant disease. \n5. Conclusion \nOur study underscores the central role of mitochondrial dysfunction and EMT in \nthe pathophysiology of advanced endometriosis. These processes are tightly inter-\nlinked, and their dysregulation may drive the transition from early-stage to severe \ndisease. The identified molecular markers could serve as valuable tools for diag-\nnosis, prognosis, and targeted intervention, opening new avenues for personalized \ntreatment approaches in endometriosis management. \nConsent to Publication \nConsent to publication was obtained from all those included in the study. \nFunding \nThis study was funded by the Indian Council of Medical Research (ICMR). \nAuthors’ Contributions \nRR designed the study; RR and WT conducted the laboratory work; AAK, WT, \nTNA, AGM, and VAA wrote the manuscript. AAK and WT performed the statis-\n\nR. Rozati et al. \n \n \nDOI: 10.4236/ajmb.2026.161001 16 American Journal of Molecular Biology \n \ntical analysis. All authors reviewed and approved the final manuscript. \nConflicts of Interest \nThe authors declare no conflicts of interest regarding the publication of this paper. \nReferences \n[1] Chantalat, E., Valera, M., Vaysse, C., Noirrit, E., Rusidze, M., Weyl, A., et al. (2020) \nEstrogen Receptors and Endometriosis. International Journal of Molecular Sciences, \n21, Article 2815. https://doi.org/10.3390/ijms21082815 \n[2] Gruber, T.M. and Mechsner, S. (2021) Pathogenesis of Endometriosis: The Origin of \nPain and Subfertility. Cells, 10, Article 1381. https://doi.org/10.3390/cells10061381 \n[3] Hosseinirad, H., Jeong, J. and Barrier, B.F. (2025) Insights into the Molecular Mech-\nanisms and Signaling Pathways of Epithelial to Mesenchymal Transition (EMT) in \nthe Pathophysiology of Endometriosis. 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