The study protocol was approved by the Institutional Ethics Committee with ethics registration number ECR/1609/Inst/TG/2021, and written informed consent was obtained from all participants prior to their enrollment in the study.

A total of 300 women presenting with symptoms suggestive of endometriosis were initially recruited from 1^st March 2023 to 31^st March 2025 in the Department of Obstetrics and Gynecology, MHRT Hospital & Research Centre, Hyderabad, India as per the inclusion and exclusion criteria. Of these, 240 underwent diagnostic laparoscopy. Endometriosis was confirmed in 180 cases based on surgical and histopathological criteria. These patients were stratified into two groups: Stage I-II (minimal to mild disease, n = 90) and Stage III-IV (moderate to severe disease, n = 90) according to the revised American Society for Reproductive Medicine (rASRM) classification. Additionally, 180 healthy women without clinical or laparoscopic evidence of endometriosis were included as controls. Demographic data, reproductive history, body mass index (BMI), and infertility duration were recorded. Serum hormone levels including FSH, LH, AMH, and estradiol were measured using standard immunoassay techniques.

Cell viability of isolated cells from endometriotic tissue biopsies was evaluated using the Trypan Blue Exclusion Assay and Fluorescein Diacetate (FDA) staining. Endometrial biopsy samples were obtained from all participants during the proliferative phase of the menstrual cycle. Single-cell suspensions were prepared from the tissue samples, and cell viability was assessed using FDA staining, while total and viable cell counts were performed using a hemocytometer. Viable cells were subsequently cultured under appropriate conditions to evaluate adherence and morphological characteristics over a five-day period. Phase-contrast microscopy was employed to monitor morphological changes associated with epithelial-mesenchymal transition (EMT).

Peripheral blood samples (4 mL) were collected from all study participants, including individuals with endometriosis and healthy controls, into EDTA-coated tubes. Samples were anonymized using coded identifiers and stored at 4˚C until further processing for RNA extraction.

Total RNA was extracted from endometrial tissues and cultured cells using a commercial RNA isolation kit. Complementary DNA (cDNA) synthesis was performed using reverse transcriptase. Five nanograms of complementary DNA (cDNA) were used for gene expression analysis. Conventional PCR was employed for the detection of selected gene transcripts, while quantitative real-time PCR (RT-qPCR) was performed to quantify transcript levels relative to endogenous reference genes. Quantitative real-time PCR (RT-qPCR) was used to evaluate the expression of genes associated with mitochondrial oxidative phosphorylation (ND1, ND6, CO2, CO3, CYTB, ATP6, ATP8), mitochondrial dynamics (DRP1, OPA1, MFN1, MFN2, LCLAT1), mitophagy (PINK1, PARKIN), hypoxia (HIF-1α), mesenchymal markers (CD73, CD90, CD105), EMT transcription factors (TWIST, SNAIL, SLUG), and cadherins (E-cadherin, N-cadherin). Gene expression levels were quantified relative to those in control subjects, using the endogenous housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference for normalization. Relative gene expression was calculated using the 2^–ΔΔCt method, and fold-change values were determined with the aid of StepOne™ Software (Applied Biosystems).

Mesenchymal stem/stromal cell surface markers CD73, CD90, and CD105 were analyzed using flow cytometry on day 5 of in vitro culture. Cells were stained with fluorochrome-conjugated antibodies and analyzed using a flow cytometer to determine the expression profiles.

Data were analyzed using appropriate statistical software. Group comparisons were performed using one-way ANOVA followed by post hoc tests. A p-value of less than 0.05 was considered statistically significant. Results were expressed as mean ± standard error of the mean (SEM) or percentages where applicable.

Single cells were isolated from endometrial tissue biopsy samples and assessed for viability using fluorescein diacetate (FDA) staining, which selectively labels live cells with intact membranes. Following confirmation of viability, the cells were cultured under appropriate in vitro conditions to support adhesion and proliferation. Over time, the adherent cells exhibited morphological transitions from a spherical to a spindle-shaped phenotype, indicative of epithelial-mesenchymal transition (EMT). These morphological changes provided the basis for subsequent investigations into EMT at both the cellular and molecular levels (Figure 1).

Figure 1. Assessment of membrane integrity. Panel (A) shows unstained cells, while Panel (B) shows cells stained with fluorescein diacetate (FDA), indicating viable cells with intact membranes. Images were captured at 40× magnification. Scale bar = 20 µm.

Morphological evaluation was performed using phase-contrast microscopy from day 0 to day 5 of in vitro culture. Initially, cells from both mild and severe endometriosis samples exhibited predominantly spherical, epithelial-like morphology. As the culture progressed, a gradual transition toward an elongated, spindle-shaped phenotype—characteristic of mesenchymal cells—was observed, indicating epithelial-mesenchymal transition (EMT). Notably, by day 5, cultures derived from severe endometriosis biopsies showed a visibly higher population of mesenchymal-like cells compared to those from mild cases. These observations suggest a more pronounced EMT progression in cells derived from severe forms of endometriosis. Scale bars = 100 µm (Figure 2).

Figure 2.Morphological characterization of cells isolated from mild and severe endometrial tissue biopsies. Representative images show differences in cellular morphology between the two conditions. Images were captured at 10× magnification. Scale bar = 20 µm.

Quantitative PCR analysis (Figure 3) revealed a marked downregulation of genes associated with oxidative phosphorylation (OXPHOS) in endometrial biopsies from women with severe endometriosis compared to both healthy controls and those with mild disease (p < 0.0001). In contrast, the expression levels of these genes showed no statistically significant differences between the control and mild

Figure 3.Relative expression of OXPHOS genes in endometrial biopsies. Gene expression levels were assessed in samples from healthy controls, women with mild endometriosis, and women with severe endometriosis. (A) Complex I (ND1 and ND6); (B) Complex III (CYT-B); (C) Complex IV (CO2 and CO3); (D) Complex V (ATP6 and ATP8). Note: (***p < 0.0001) represents significance, ns*-non significant.

endometriosis groups, suggesting that substantial alterations in mitochondrial gene expression occur predominantly in advanced stages of the condition.

The genes analyzed are central to mitochondrial bioenergetics. ND1 and ND6, components of Complex I, initiate the electron transport process. CYTB, part of Complex III, facilitates electron transfer from ubiquinol to cytochrome. CO2 and CO3, subunits of Complex IV, are involved in the terminal reduction of oxygen to water. Finally, ATP6 and ATP8, constituents of Complex V (ATP synthase), are directly responsible for ATP generation.

The consistent downregulation of these genes in severe endometriosis points to widespread mitochondrial impairment. This dysfunction likely compromises ATP production, leading to energy deficits in endometrial cells and potentially contributing to elevated oxidative stress. Such disturbances in mitochondrial homeostasis may play a critical role in disease pathophysiology, exacerbating tissue damage and promoting disease progression.

The absence of significant changes in OXPHOS gene expression between control and mild cases suggests that mitochondrial dysfunction may be a hallmark of more advanced disease stages. This pattern of expression could serve as a molecular indicator of disease severity and offers insight into potential therapeutic targets aimed at improving mitochondrial function and cellular energy metabolism in endometriosis (Figure 3).

Mitochondrial dysfunction appears to intensify with the progression of endometriosis. In this study, the expression levels of key genes involved in mitochondrial dynamics and energy homeostasis—DRP1, OPA1, MFN1, MFN2, and LCLAT1—were evaluated. These genes play critical roles in regulating mitochondrial fission, fusion, and lipid remodeling. A significant downregulation, particularly in severe stages of the disease, suggests impaired mitochondrial integrity and function.

In the mild stage of endometriosis, the expression profiles of these genes remain relatively stable and comparable to those of healthy controls, indicating that mitochondrial function is largely preserved. However, as the disease advances, a consistent and significant reduction in gene expression is observed. This decline likely disrupts mitochondrial morphology and bioenergetics, leading to reduced ATP synthesis, elevated reactive oxygen species (ROS) production, and increased oxidative stress—all of which contribute to cellular dysfunction and tissue damage.

The observed correlation between endometriosis severity and decreased expression of mitochondrial-related genes supports the notion that mitochondrial bioenergetic failure is a pivotal factor in disease pathophysiology. These findings not only provide insight into the molecular mechanisms underlying endometriosis progression but also highlight the potential of mitochondrial dynamics-associated genes as biomarkers for disease severity and as therapeutic targets for restoring mitochondrial function and tissue homeostasis (Figure 4).

The expression of mitophagy-related genes PINK1 and PARKIN was evaluated in endometrial tissues from control, mild, and severe endometriosis groups. While no significant differences were observed between control and mild cases, a marked downregulation of both genes was evident in severe endometriosis (p < 0.0001).

PINK1 and PARKIN are essential for mitochondrial quality control, initiating the removal of damaged mitochondria via mitophagy. Their reduced expression in severe disease suggests impaired mitophagic activity, potentially leading to mitochondrial dysfunction, oxidative stress, and inflammation—key features of advanced endometriosis. These findings highlight PINK1 and PARKIN as potential biomarkers of disease severity and therapeutic targets to restore mitochondrial homeostasis (Figure 5).

Figure 4. Relative expression of the fission and fusion gene in endometrial biopsies. Gene expression was quantified in endometrial tissues from healthy controls, and from women with mild and severe endometriosis. Note: (***p < 0.0001) represents significance, ns*-non significant.

Figure 5.Relative expression of mitophagy-related genes in endometrial biopsies. Expression levels were measured in samples from healthy controls, and from women with mild and severe endometriosis. (A) PINK1; (B) PARKIN. Note: (***p < 0.0001) represents significance, ns*-non significant.

The expression of HIF-1α was evaluated in endometrial biopsies across control, mild, and severe endometriosis groups. No significant difference was observed between control and mild groups, suggesting minimal hypoxic signaling in early stages. However, a significant increase in HIF-1α expression was detected in severe endometriosis (p < 0.0001), indicating activation of hypoxia-associated pathways in advanced disease.

HIF-1α, a key transcription factor in cellular response to low oxygen, promotes genes involved in angiogenesis, inflammation, and metabolic adaptation. Its upregulation in severe cases suggests a hypoxic microenvironment contributing to disease progression, including abnormal vascularization and fibrosis. These findings underscore the relevance of HIF-1α as a potential biomarker of disease severity and a therapeutic target in advanced endometriosis (Figure 6).

Figure 6. Relative expression of HIF1α in endometrial biopsies. HIF1α gene expression was quantified in endometrial tissues from healthy controls, and from women with mild and severe endometriosis. Note: (***p < 0.0001) represents significance, ns*-non significant.

CD73, CD90, and CD105 are established markers of mesenchymal stem/stromal cells (MSCs). Flow cytometry revealed a marked increase in their expression in stromal cells from severe endometriosis compared to mild cases, indicating a higher proportion of MSC-like cells in advanced disease.

This enrichment suggests a phenotypic shift toward a mesenchymal state, potentially driven by epithelial-to-mesenchymal transition (EMT), contributing to increased cellular plasticity, fibrosis, and invasiveness. The elevated expression of these markers reflects enhanced regenerative and invasive capabilities, supporting lesion persistence and progression.

Overall, the data highlight a significant mesenchymal transformation in severe endometriosis, underscoring the role of MSC-like stromal cells in disease pathogenesis and their potential as therapeutic targets (Figure 7).

Quantitative RT-PCR analysis was conducted on day 5 of in vitro cultures to assess the expression of key mesenchymal markers—CD73, CD90, and CD105—across control, mild, and severe endometriosis samples. As shown in Figure 8, expression levels of all three markers were markedly elevated in stromal cells from severe endometriosis cases compared to both mild forms and healthy controls (p < 0.0001). A modest but statistically significant increase was also observed in mild cases relative to controls (p < 0.01).

Figure 7. Immunophenotypic characterization of mesenchymal stem cells (MSCs) isolated from endometrial biopsies. Flow cytometric analysis of MSCs derived from women with mild and severe endometriosis, performed on day 5 of in vitro culture.

Figure 8. Expression of mesenchymal stem cell-specific genes in cultured endometrial MSCs. Relative mRNA levels of CD90 (A), CD105 (B), and CD73 (C) were assessed by real-time quantitative PCR (RT-qPCR) on day 5 of in vitro culture. MSCs were isolated from endometrial biopsies of healthy controls, and women with mild and severe endometriosis. Note: (***p < 0.0001) represents significance, ns*-non significant.

This progressive upregulation of mesenchymal markers indicates a phenotypic shift toward a mesenchymal state, suggesting a heightened epithelial-to-mesenchymal transition (EMT) in severe endometriotic conditions. These findings reinforce the morphological observations and support the notion of enhanced EMT activity as a hallmark of disease severity in endometriosis (Figure 8).

Quantitative real-time PCR analysis demonstrated a marked downregulation of E-cadherin and a significant upregulation of N-cadherin in endometrial samples from individuals with severe endometriosis when compared to both mild and control groups (***p < 0.0001). No statistically significant difference in expression was detected between the control and mild groups for either gene. This pattern reflects a cadherin switch, a key feature of the epithelial-mesenchymal transition (EMT), and underscores the enhanced activation of EMT in more advanced stages of endometriosis, implicating its contribution to disease progression (Figure 9).

Figure 9. Expression analysis of epithelial-mesenchymal transition (EMT) markers in endometrial biopsies. Relative mRNA levels of E-cadherin (A) and N-cadherin (B) were quantified in endometrial tissues from healthy controls, and from women with mild and severe endometriosis to assess EMT progression. Note: (***p < 0.0001) represents significance, ns*-non significant.

TWIST, SNAIL, and SLUG are critical transcription factors that suppress E-cadherin expression and drive the epithelial-mesenchymal transition (EMT). Quantitative real-time PCR analysis showed a significant increase in the expression of these EMT-inducing factors in severe endometrial samples compared to both mild and control groups (***p < 0.0001). In contrast, no significant difference in expression was observed between the mild and control groups.

These results indicate a disease severity-dependent elevation in TWIST, SNAIL, and SLUG levels, suggesting their enhanced role in the molecular events underlying EMT activation during advanced endometriosis. When considered alongside the reduced E-cadherin expression, this pattern strongly supports the contribution of EMT-related transcriptional regulation to the progression and invasiveness of severe endometrial lesions (Figure 10).

Figure 10. Relative expression of EMT-associated transcription factors in endometrial biopsies. Bar graphs show mRNA levels of TWIST (A), SNAIL (B), and SLUG (C) in tissues from healthy controls, women with mild endometriosis, and women with severe endometriosis. Note: (***p < 0.0001) represents significance, ns*-non significant.